Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2.

Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22phox are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple ß-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation.

Endoplasmic Reticulum-Targeting Self-Assembly Nanosheets Promote Autophagy and Regulate Immunosuppressive Tumor Microenvironment for Efficient Photodynamic Immunotherapy.

The poor efficiency and low immunogenicity of photodynamic therapy (PDT), and the immunosuppressive tumor microenvironment (ITM) lead to tumor recurrence and metastasis. In this work, TCPP-TER-Zn@RSV nanosheets (TZR NSs) that co-assembled from the endoplasmic reticulum (ER)-targeting photosensitizer TCPP-TER-Zn nanosheets (TZ NSs for short) and the autophagy promoting and indoleamine-(2, 3)-dioxygenase (IDO) inhibitor-like resveratrol (RSV) are fabricated to enhance antitumor PDT. TZR NSs exhibit improved therapeutic efficiency and amplified immunogenic cancer cell death (ICD) by ER targeting PDT and ER autophagy promotion. TZR NSs reversed the ITM with an increase of CD8+ T cells and reduce of immunosuppressive Foxp3 regulatory T cells, which effectively burst antitumor immunity thus clearing residual tumor cells. The ER-targeting TZR NSs developed in this paper presents a simple but valuable reference for high-efficiency tumor photodynamic immunotherapy.

Impaired p47phox phosphorylation in neutrophils from patients with p67phox-deficient chronic granulomatous disease.

Superoxide production by the phagocyte reduced NAD phosphate (NADPH) oxidase is essential for innate immunity as shown in chronic granulomatous disease (CGD), an immunodeficiency disease resulting from mutations in 1 of its genes. The NADPH oxidase is composed of 2 membrane proteins (gp91phox/NOX2 and p22phox) and 4 cytosolic proteins (p47phox, p67phox, p40phox, and Rac1/2). The phosphorylation of p47phox is required for NADPH oxidase activation in cells. As p47phox and p67phox can form a tight complex in cells, we hypothesized that p67phox could regulate p47phox phosphorylation. To investigate this hypothesis, we used phospho-specific antibodies against 5 major p47phox-phosphorylated sites (Ser304, Ser315, Ser320, Ser328, and Ser345) and neutrophils from healthy donors and from p67phox-/- CGD patients. Results showed that formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate induced a time- and a concentration-dependent phosphorylation of p47phox on Ser304, Ser315, Ser320, and Ser328 in healthy human neutrophils. Interestingly, in neutrophils and Epstein-Barr virus-transformed B lymphocytes from p67phox-/- CGD patients, phosphorylation of p47phox on serine residues was dramatically reduced. In COSphox cells, the presence of p67phox led to increased phosphorylation of p47phox. In vitro studies showed that recombinant p47phox was phosphorylated on Ser304, Ser315, Ser320, and Ser328 by different PKC isoforms and the addition of recombinant p67phox alone or in combination with p40phox potentiated this process. Thus, p67phox and p40phox are required for optimal p47phox phosphorylation on Ser304, Ser315, Ser320, and Ser328 in intact cells. Therefore, p67phox and p40phox are novel regulators of p47phox-phosphorylation.

Statistical properties for an optically injected chaotic semiconductor laser with a high-pass filter.

Chaotic waveforms with Gaussian distributions are significant for laser-chaos-based applications such as random number generation. By exploring the injection parameter space of the optical injection semiconductor lasers, we numerically investigate the associated probability density functions of the generated chaotic waveforms when different high-pass filters with different cutoff frequencies are used. Our results demonstrate that the chaotic waveforms with Gaussian probability density functions can be obtained once the cutoff frequency of the high-pass filter is larger than the laser relaxation resonance frequency. Especially, we find that the Gaussian probability density function can reach a superhigh coefficient of determination R2 ≥ 99.5% and an ultralow skewness |S|<0.1 in a large parameter space by jointly controlling the injection parameter and cutoff frequency.

Self-Delivery Nanomedicines Reverse Thermal Resistance to Enhance Tumor Mild-Temperature Photothermal Therapy.

Tumoral thermal defense mechanisms considerably attenuate the therapeutic outcomes of mild-temperature photothermal therapy (PTT). Thus, developing a simple, efficient, and universal therapeutic strategy to sensitize mild-temperature PTT is desirable. Herein, we report self-delivery nanomedicines ACy NPs comprising a near-infrared (NIR) photothermal agent (Cypate), mitochondrial oxidative phosphorylation inhibitor (ATO), and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), which have a high drug-loading efficiency that can reverse tumoral thermal resistance, thereby increasing mild-temperature PTT efficacy. ACy NPs achieved targeted tumor accumulation and performed NIR fluorescence imaging capability in vivo to guide tumor PTT for optimized therapeutic outcomes. The released ATO reduced intracellular ATP levels to downregulate multiple heat shock proteins (including HSP70 and HSP90) before PTT, which reversed the thermal resistance of tumor cells, contributing to the excellent results of mild-temperature PTT in vitro and in vivo. Therefore, this study provides a simple, biosafe, advanced, and universal heat shock protein-blocking strategy for tumor PTT.

Relationship between glycaemic control and frailty in older Chinese patients: The mediating role of diabetes distress.

PURPOSE: This study aimed to investigate the effects of glycaemic control and diabetes distress on frailty in older Chinese patients with diabetes, and to explore the mediating role of diabetes distress between glycaemic control and frailty. DESIGN: This is a descriptive, cross-sectional study. A total of 209 older patients with diabetes were recruited from a teaching hospital in Zhejiang Province. Data were collected from February to September 2022. METHODS: A self-designed questionnaire was used to collect demographic and disease-related data. The Fried Scale and Diabetes Distress Scale were employed to assess frailty and diabetes distress, respectively The bootstrap method was used to examine the mediating effects of diabetes distress on glycaemic control and frailty. The STROBE checklist was adhered to in the reporting of this study (see details in File S1). RESULTS: The findings indicated a positive correlation between the level of glycaemic control and frailty, as well as between diabetes distress and frailty. Furthermore, diabetes distress was found to play a complete mediating role between glycaemic control and frailty. CONCLUSIONS: The study findings highlight the relationship between glycaemic control, diabetes distress and frailty offering a valuable reference for enhancing the management of frailty in older patients with diabetes. RELEVANCE TO CLINICAL PRACTICE: This study emphasizes the significance of managing glycaemic control and diabetes distress in older patients with diabetes to prevent frailty, and may contribute for healthcare professionals to developing effective measures to improve the frailty of older diabetic patients in clinical settings. PATIENT OR PUBLIC CONTRIBUTION: This study was conducted with the participation of older patients with diabetes who contributed data by completing study questionnaires and undergoing physical assessments.

Screening for post-intensive care syndrome: Validation of the Healthy Aging Brain Care Monitor Self-Report Chinese version.

BACKGROUND: The Healthy Aging Brain Care Monitor Self-Report (HABC-M SR) is a tool to evaluate physical, mental and cognitive impairments. It has been validated in several countries for post-intensive care syndrome (PICS) assessment, but there is no Chinese version yet. AIM: The aim of this study is to translate and cross-cultural adapt HABC-M SR, and validate its psychological properties among Chinese ICU survivors. STUDY DESIGN: This is a cross-sectional study. the HABC-M SR was translated into Chinese and validated in intensive care unit survivors (n = 145). Measures of internal consistency, construct validity, concurrent validity, and content validity were evaluated. RESULTS: A 19-item Chinese version of HABC-M SR was yielded, with good reliability (Cronbach's α = 0.92) and validity (the variance was 64.4%, overall content validity was 0.91, and correlation coefficients were 0.62-0.90). CONCLUSIONS: The 19-item Chinese version HABC-M SR is a reliable and valid tool for PICS assessing and may be regarded as a standard measurement. RELEVANCE TO CLINICAL PRACTICE: The Chinese version HABC-M SR may help in selecting PICS high-risk survivors for ICU follow-up interventions. The HABC-M SR can also be regarded as a standard specific PICS measurement, thus promote the comparability between studies and transformation of the clinical evidence.

RANKL Reduces Body Weight and Food Intake via the Modulation of Hypothalamic NPY/CART Expression.

The receptor activator of nuclear factor-κB ligand (RANKL) modulates energy metabolism. However, how RANKL regulates energy homeostasis is still not clear. This study aims to investigate the central mechanisms by which central administration of RANKL inhibits food intake and causes weight loss in mice. We carried out a systematic and in-depth analysis of the neuronal pathways by which RANKL mediates catabolic effects. After intracerebroventricle (i.c.v.) injection of RANKL, the expression of neuropeptide Y (NPY) mRNA in the Arc was significantly decreased, while the CART mRNA expression dramatically increased in the Arc and DMH. However, the agouti-related protein (AgRP) and pro-opiomelanocortin (POMC) mRNA had no significant changes compared with control groups. Together, the results suggest that central administration of RANKL reduces food intake and causes weight loss via modulating the hypothalamic NPY/CART pathways.

Different effects of neuropeptide Y on proliferation of vascular smooth muscle cells via regulation of Geminin.

The proliferation-promoting effect of neuropeptide Y (NPY) always functions in low-serum-cultured vascular smooth muscle cells (VSMCs), and the phenotypic switch of VSMCs is regulated by concentrations of serum. Whether the property of the NPY proliferative effect in VSMCs relies on phenotype of VSMCs is unclear. We aimed to explore the role of NPY on proliferation of different VSMC phenotypes in the pathogenesis of atherosclerosis. By stimulating A10 cells with 200 nM NPY in 0.5 or 10% serum, 3H-thymidine and 5-ethynyl-2'-deoxyuridine (EdU) and CCK8 measurements were used to detect VSMC proliferation. RT-PCR and Flow cytometry were performed to detect the factors involved in different properties of the NPY proliferative effect in VSMCs. Instead of facilitating proliferation, NPY had no significant effect on the growth of VSMCs when cultured in 10% serum (VSMCs stayed at synthetic states). The underlying mechanism may be involved in down-regulation of Y1 receptor (P < 0.05 vs. Vehicle) and up-regulation of Geminin (P < 0.05 vs. Vehicle) in 10% serum-cultured VSMCs co-incubated with 200 nM NPY. Besides, modulation of Geminin was effectively blocked by the Y1 receptor antagonist. The stimulation of NPY on proliferation of VSMCs could be a double-edged sword in the development of atherosclerosis and thus provides new knowledge for therapy of atherosclerosis.

Prioritization of potential candidate disease genes by topological similarity of protein-protein interaction network and phenotype data.

Identifying candidate disease genes is important to improve medical care. However, this task is challenging in the post-genomic era. Several computational approaches have been proposed to prioritize potential candidate genes relying on protein-protein interaction (PPI) networks. However, the experimental PPI network is usually liable to contain a number of spurious interactions. In this paper, we construct a reliable heterogeneous network by fusing multiple networks, a PPI network reconstructed by topological similarity, a phenotype similarity network and known associations between diseases and genes. We then devise a random walk-based algorithm on the reliable heterogeneous network called RWRHN to prioritize potential candidate genes for inherited diseases. The results of leave-one-out cross-validation experiments show that the RWRHN algorithm has better performance than the RWRH and CIPHER methods in inferring disease genes. Furthermore, RWRHN is used to predict novel causal genes for 16 diseases, including breast cancer, diabetes mellitus type 2, and prostate cancer, as well as to detect disease-related protein complexes. The top predictions are supported by literature evidence.

Neuronal conversion from glia to replenish the lost neurons.

ABSTRACT: Neuronal injury, aging, and cerebrovascular and neurodegenerative diseases such as cerebral infarction, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, and Huntington's disease are characterized by significant neuronal loss. Unfortunately, the neurons of most mammals including humans do not possess the ability to self-regenerate. Replenishment of lost neurons becomes an appealing therapeutic strategy to reverse the disease phenotype. Transplantation of pluripotent neural stem cells can supplement the missing neurons in the brain, but it carries the risk of causing gene mutation, tumorigenesis, severe inflammation, and obstructive hydrocephalus induced by brain edema. Conversion of neural or non-neural lineage cells into functional neurons is a promising strategy for the diseases involving neuron loss, which may overcome the above-mentioned disadvantages of neural stem cell therapy. Thus far, many strategies to transform astrocytes, fibroblasts, microglia, Müller glia, NG2 cells, and other glial cells to mature and functional neurons, or for the conversion between neuronal subtypes have been developed through the regulation of transcription factors, polypyrimidine tract binding protein 1 (PTBP1), and small chemical molecules or are based on a combination of several factors and the location in the central nervous system. However, some recent papers did not obtain expected results, and discrepancies exist. Therefore, in this review, we discuss the history of neuronal transdifferentiation, summarize the strategies for neuronal replenishment and conversion from glia, especially astrocytes, and point out that biosafety, new strategies, and the accurate origin of the truly converted neurons in vivo should be focused upon in future studies. It also arises the attention of replenishing the lost neurons from glia by gene therapies such as up-regulation of some transcription factors or down-regulation of PTBP1 or drug interference therapies.

Efficient Photon Beam Diffusion for Directional Subsurface Scattering.

Real-time subsurface scattering techniques are widely used in translucent material rendering. Among advanced methods that rely on the bidirectional scattering-surface reflectance distribution function (BSSRDF), screen space algorithms exhibit limited translucency, while existing large-distance methods are inefficient and yield poor illumination details. To address these limitations for better large-distance scattering, we develop a novel algorithm by extending the photon beam diffusion (PBD) model within the light view and screen space. Unlike surface irradiance in prior methods, we incorporate the refracted beam in the medium into real-time scattering estimation, presenting a new consideration for photon beam utilization. Concretely, we store all photon beam samples in light view textures and utilize an adaptive sampling pattern for beam sample selection in large filtering kernel sizes. This can reduce the sample count based on surface attributes. In screen space, virtual sources are derived from samples to estimate PBD contributions, with an approximation that preserves boundary conditions. To avoid possible overestimation, we implement correction factors that scale contributions, effectively aligning our results with path-tracing references. Through these reformulations, our efficient PBD generates results closest to references among existing methods. The experiments accurately represent better front-face illumination details and backlit translucency effects, while significantly accelerating performance compared to previous large-distance methods.

Nanodrugs mediate TAMs-related arginine metabolism interference to boost photodynamic immunotherapy.

As a potential treatment strategy for low immunogenic triple negative breast cancer (TNBC), photodynamic therapy (PDT) induced antitumor immunotherapy is greatly limited by the immunosuppressive tumor microenvironment (ITM), especially the M2 phenotype tumor-associated macrophages (TAMs). The balance of arginine metabolism plays an important role in TAMs polarization. Herein, a multifunctional nanoplatform (defined as HN-HFPA) was employed to burst the anti-tumor immunity of TNBC post PDT by reeducating TAMs through interfering the TAMs-associated arginine metabolism. The L-arginine (L-Arg) was loaded in the hollow cavity of HN-HFPA, which could not only generate nitric oxide (NO) for tumor therapy, but also serve as a substrate of arginine metabolism pathway. As an inhibitor of arginases-1 (Arg-1) of M2 TAMs, L-norvaline (L-Nor) was modified to the hyaluronic acid (HA), and coated in the surface of HFPA. After degradation of HA by hyaluronidase in tumor tissue and GSH-mediated disintegration, HN-HFPA depleted intracellular GSH, produced remarkable reactive oxygen species (ROS) under light irradiation and released L-Arg to generate NO, which induced tumor immunogenic cell death (ICD). Real-time ultrasound imaging of tumor was realized taking advantage of the gas feature of NO. The L-Nor suppressed the Arg-1 overexpressed in M2, which skewed the balance of arginine metabolism and reversed the ITM with increased ratios of M1 and CD8+ T cells, finally resulted in amplified antitumor immune response and apparent tumor metastasis inhibition. This study remodeled ITM to strengthen immune response post PDT, which provided a promising treatment strategy for TNBC.

Combined effects of nitrogen limitation and overexpression of malic enzyme gene on lipid accumulation in Dunaliella parva.

Overexpression of Dunaliella parva (D. parva) malic enzyme (ME) gene (DpME) significantly increased DpME expression and ME enzyme activity in transgenic D. parva. Nitrogen limitation had an inhibitory effect on protein content, and DpME overexpression could improve protein content. Nitrogen limitation increased carbohydrate content, and Dunaliella parva overexpressing malic enzyme gene under nitrogen limitation (DpME-N-) group showed the lowest starch content among all groups. Dunaliella parva overexpressing malic enzyme gene under nitrogen sufficient condition (DpME) and DpME-N- groups showed considerably high mRNA levels of DpME. ME activity was significantly enhanced by DpME overexpression, and nitrogen limitation caused a smaller increase. DpME overexpression and nitrogen limitation obviously enhanced lipid accumulation, and DpME overexpression had more obvious effect. Compared with control (wild type), lipid content (68.97%) obviously increased in DpME-N- group. This study indicated that the combination of DpME overexpression and nitrogen limitation was favorable to the production of microalgae biodiesel.

Mouse serum albumin induces neuronal apoptosis and tauopathies.

The elderly frequently present impaired blood-brain barrier which is closely associated with various neurodegenerative diseases. However, how the albumin, the most abundant protein in the plasma, leaking through the disrupted BBB, contributes to the neuropathology remains poorly understood. We here demonstrated that mouse serum albumin-activated microglia induced astrocytes to A1 phenotype to remarkably increase levels of Elovl1, an astrocytic synthase for very long-chain saturated fatty acids, significantly promoting VLSFAs secretion and causing neuronal lippoapoptosis through endoplasmic reticulum stress response pathway. Moreover, MSA-activated microglia triggered remarkable tau phosphorylation at multiple sites through NLRP3 inflammasome pathway. Intracerebroventricular injection of MSA into the brains of C57BL/6J mice to a similar concentration as in patient brains induced neuronal apoptosis, neuroinflammation, increased tau phosphorylation, and decreased the spatial learning and memory abilities, while Elovl1 knockdown significantly prevented the deleterious effect of MSA. Overall, our study here revealed that MSA induced tau phosphorylation and neuron apoptosis based on MSA-activated microglia and astrocytes, respectively, showing the critical roles of MSA in initiating the occurrence of tauopathies and cognitive decline, and providing potential therapeutic targets for MSA-induced neuropathology in multiple neurodegenerative disorders.

Thrombomodulin reduces α-synuclein generation and ameliorates neuropathology in a mouse model of Parkinson's disease.

The neurotoxic α-synuclein (α-syn) oligomers play an important role in the occurrence and development of Parkinson's disease (PD), but the factors affecting α-syn generation and neurotoxicity remain unclear. We here first found that thrombomodulin (TM) significantly decreased in the plasma of PD patients and brains of A53T α-syn mice, and the increased TM in primary neurons reduced α-syn generation by inhibiting transcription factor p-c-jun production through Erk1/2 signaling pathway. Moreover, TM decreased α-syn neurotoxicity by reducing the levels of oxidative stress and inhibiting PAR1-p53-Bax signaling pathway. In contrast, TM downregulation increased the expression and neurotoxicity of α-syn in primary neurons. When TM plasmids were specifically delivered to neurons in the brains of A53T α-syn mice by adeno-associated virus (AAV), TM significantly reduced α-syn expression and deposition, and ameliorated the neuronal apoptosis, oxidative stress, gliosis and motor deficits in the mouse models, whereas TM knockdown exacerbated these neuropathology and motor dysfunction. Our present findings demonstrate that TM plays a neuroprotective role in PD pathology and symptoms, and it could be a novel therapeutic target in efforts to combat PD. Schematic representation of signaling pathways of TM involved in the expression and neurotoxicity of α-syn. A TM decreased RAGE, and resulting in the lowered production of p-Erk1/2 and p-c-Jun, and finally reduce α-syn generation. α-syn oligomers which formed from monomers increase the expression of p-p38, p53, C-caspase9, C-caspase3 and Bax, decrease the level of Bcl-2, cause mitochondrial damage and lead to oxidative stress, thus inducing neuronal apoptosis. TM can reduce intracellular oxidative stress and inhibit p53-Bax signaling by activating APC and PAR-1. B The binding of α-syn oligomers to TLR4 may induce the expression of IL-1ß, which is subsequently secreted into the extracellular space. This secreted IL-1ß then binds to its receptor, prompting p65 to translocate from the cytoplasm into the nucleus. This translocation downregulates the expression of KLF2, ultimately leading to the suppression of TM expression. By Figdraw.

Neuronal double-stranded DNA accumulation induced by DNase II deficiency drives tau phosphorylation and neurodegeneration.

BACKGROUND: Deoxyribonuclease 2 (DNase II) plays a key role in clearing cytoplasmic double-stranded DNA (dsDNA). Deficiency of DNase II leads to DNA accumulation in the cytoplasm. Persistent dsDNA in neurons is an early pathological hallmark of senescence and neurodegenerative diseases including Alzheimer's disease (AD). However, it is not clear how DNase II and neuronal cytoplasmic dsDNA influence neuropathogenesis. Tau hyperphosphorylation is a key factor for the pathogenesis of AD. The effect of DNase II and neuronal cytoplasmic dsDNA on neuronal tau hyperphosphorylation remains unclarified. METHODS: The levels of neuronal DNase II and dsDNA in WT and Tau-P301S mice of different ages were measured by immunohistochemistry and immunolabeling, and the levels of DNase II in the plasma of AD patients were measured by ELISA. To investigate the impact of DNase II on tauopathy, the levels of phosphorylated tau, phosphokinase, phosphatase, synaptic proteins, gliosis and proinflammatory cytokines in the brains of neuronal DNase II-deficient WT mice, neuronal DNase II-deficient Tau-P301S mice and neuronal DNase II-overexpressing Tau-P301S mice were evaluated by immunolabeling, immunoblotting or ELISA. Cognitive performance was determined using the Morris water maze test, Y-maze test, novel object recognition test and open field test. RESULTS: The levels of DNase II were significantly decreased in the brains and the plasma of AD patients. DNase II also decreased age-dependently in the neurons of WT and Tau-P301S mice, along with increased dsDNA accumulation in the cytoplasm. The DNA accumulation induced by neuronal DNase II deficiency drove tau phosphorylation by upregulating cyclin-dependent-like kinase-5 (CDK5) and calcium/calmodulin activated protein kinase II (CaMKII) and downregulating phosphatase protein phosphatase 2A (PP2A). Moreover, DNase II knockdown induced and significantly exacerbated neuron loss, neuroinflammation and cognitive deficits in WT and Tau-P301S mice, respectively, while overexpression of neuronal DNase II exhibited therapeutic benefits. CONCLUSIONS: DNase II deficiency and cytoplasmic dsDNA accumulation can initiate tau phosphorylation, suggesting DNase II as a potential therapeutic target for tau-associated disorders.

Osteoblast-specific down-regulation of NLRP3 inflammasome by aptamer-functionalized liposome nanoparticles improves bone quality in postmenopausal osteoporosis rats.

Rationale: NLRP3 inflammasome is critical in the development and progression of many metabolic diseases driven by chronic inflammation, but its effect on the pathology of postmenopausal osteoporosis (PMOP) remains poorly understood. Methods: We here firstly examined the levels of NLRP3 inflammasome in PMOP patients by ELISA. Then we investigated the possible mechanisms underlying the effect of NLRP3 inflammasome on PMOP by RNA sequencing of osteoblasts treated with NLRP3 siRNA and qPCR. Lastly, we accessed the effect of decreased NLRP3 levels on ovariectomized (OVX) rats. To specifically deliver NLRP3 siRNA to osteoblasts, we constructed NLRP3 siRNA wrapping osteoblast-specific aptamer (CH6)-functionalized lipid nanoparticles (termed as CH6-LNPs-siNLRP3). Results: We found that the levels of NLRP3 inflammasome were significantly increased in patients with PMOP, and were negatively correlated with estradiol levels. NLRP3 knock-down influenced signal pathways including immune system process, interferon signal pathway. Notably, of the top ten up-regulated genes in NLRP3-reduced osteoblasts, nine genes (except Mx2) were enriched in immune system process, and five genes were related to interferon signal pathway. The in vitro results showed that CH6-LNPs-siNLRP3 was relatively uniform with a dimeter of 96.64 ± 16.83 nm and zeta potential of 38.37 ± 1.86 mV. CH6-LNPs-siNLRP3 did not show obvious cytotoxicity and selectively delivered siRNA to bone tissue. Moreover, CH6-LNPs-siNLRP3 stimulated osteoblast differentiation by activating ALP and enhancing osteoblast matrix mineralization. When administrated to OVX rats, CH6-LNPs-siNLRP3 promoted bone formation and bone mass, improved bone microarchitecture and mechanical properties by decreasing the levels of NLRP3, IL-1ß and IL-18 and increasing the levels of OCN and Runx2. Conclusion: NLRP3 inflammasome may be a new biomarker for PMOP diagnosis and plays a key role in the pathology of PMOP. CH6-LNPs-siNLRP3 has potential application for the treatment of PMOP.

Consumer Perceived Brand Innovativeness and Authenticity of Chinese Time-Honored Brand Restaurants: The Moderated Mediation Effect of Personal Traits.

Purpose: Numerous time-honored brand restaurants are gradually losing their authenticity in the development process. Brand authenticity serves as a symbol of China's unique culinary culture, and consistency lies at the core of preserving its authenticity. Failure to integrate innovative elements into the original components can potentially erode the brand's consistent image, leading to a negative impact on perceived authenticity and purchase intention (PI). However, existing research has largely neglected to investigate the influence of consumer perceived brand innovativeness (CPBI) and consumer perceived brand authenticity (CPBA) specifically within the context of time-honored brand restaurants. Additionally, there is a lack of research examining the individual differences of consumers and how these intersect with time-honored brands. Therefore, our research aims to address these research gaps. Methods: The list of Chinese time-honored brands issued by the Ministry of Commerce of China served as the basis for the study's choice of time-honored restaurant brands. 689 relevant consumers were obtained through convenience sampling within China and the self-report method was adopted for data collection. Using the partial least squares structural equation modeling method and the SmartPLS software, the data was analyzed and the hypotheses were tested. Results: CPBI positively influences PI. CPBA mediates the relationship between CPBI and PI. In contrast to personal innovativeness, which positively moderates the mediating relationship between CPBI and CPBA, nostalgia proneness moderates this relationship negatively. Conclusion: Our results revealed that both CPBI and CPBA have a positive impact on PI within the domain of consumption in Chinese time-honored brand restaurants. This study addresses the research gap in brand innovativeness and authenticity in these restaurants. Furthermore, we identified the influence of consumer traits in this context. Our results can assist time-honored brand restaurants to effectively innovate and preserve their traditions, which will ultimately contribute to a more authentic service experience.

Secretory expression of ß-1,3-glucomannanase in the oleaginous yeast Rhodosporidium toruloides for improved lipid extraction.

Lipids produced by oleaginous yeasts are considered as sustainable sources for the production of biofuels and oleochemicals. The red yeast Rhodosporidium toruloides can accumulate lipids to over 70% of its dry cell mass. To facilitate lipid extraction, a recombinant ß-1,3-glucomannanase, MAN5C, has been applied to partially breakdown R. toruloides cell wall. In this study, R. toruloides NP11 was engineered for secretory expression of MAN5C to simplify the lipid extraction process. Specifically, a cassette contained a codon-optimized gene MAN5C was integrated into the genome of R. toruloides by Agrobacterium-mediated transformation. The engineered strain NP11-MAN5C was found with proper expression and secretion of active MAN5C, yet no notable compromise in terms of cell growth and lipid production. When NP11-MAN5C cell cultures were extracted with ethyl acetate without any pretreatment, 20% of total lipids were recovered, 4.3-fold higher than that of the parental strain NP11. When the cells were heat-treated followed by extraction with ethyl acetate in the presence of the culture broth supernatants, up to 93% of total lipids were recovered, confirming beneficial effects of MAN5C produced in situ. This study provides a new strategy to engineer oleaginous yeasts for more viable lipid extraction and down-stream processes.