Nrf2 differentially regulates osteoclast and osteoblast differentiation for bone homeostasis.

Nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) is a master regulator of antioxidant response and protects cells from excessive oxidative stress. Nrf2 emerges as a prospective therapeutic target for metabolic bone disorders, in which the balance between osteoblastic bone formation and osteoclastic bone resorption is disrupted. However, the molecular mechanism through which Nrf2 modulates bone homeostasis remains unclear. In this study, we compared the differences in Nrf2-mediated antioxidant response and ROS regulation in osteoblasts and osteoclasts, both in vitro and in vivo. Findings indicated a close connection between the Nrf2 expression and its related antioxidant response with osteoclasts than osteoblasts. We next pharmacologically manipulated the Nrf2-mediated antioxidant response during osteoclast or osteoblast differentiation. Nrf2 inhibition enhanced osteoclastogenesis, while its activation suppressed it. In contrast, osteogenesis decreased irrespective of whether Nrf2 was inhibited or activated. These findings highlight the distinct ways in which the Nrf2-mediated antioxidant response regulates osteoclast and osteoblast differentiation, thereby contributing to the development of Nrf2 targeted therapies for metabolic bone diseases.

Traumatic temporomandibular joint bony ankylosis in growing rats.

BACKGROUND: The pathogenesis of traumatic temporomandibular joint (TMJ) bony ankylosis remains unknown. This study aimed to explore the pathogenesis of traumatic TMJ bony ankylosis in a rat model. METHODS: Twenty-four 3-week-old male Sprague-Dawley rats were used in this study. Excision of the whole disc, the fibrocartilage damage of the condyle and glenoid fossa, and narrowed joint space were performed in the left TMJ of the operation group to induce TMJ bony ankylosis (experimental side). The right TMJ underwent a sham operation (sham side). The control group did not undergo any operations. At 1, 4, and 8 weeks postoperatively, rats of the operation group were sacrificed and TMJ complexes were evaluated by gross observation, Micro-CT, histological examinations, and immunofluorescence microscopy. Total RNA of TMJ complexes in the operation group were analyzed using RNA-seq. RESULTS: Gross observations revealed TMJ bony ankylosis on the experimental side. Micro-CT analysis demonstrated that compared to the sham side, the experimental side showed a larger volume of growth, and a considerable calcified bone callus formation in the narrowed joint space and on the rougher articular surfaces. Histological examinations indicated that endochondral ossification was observed on the experimental side, but not on the sham side. RNA-seq analysis and immunofluorescence revealed that Matrix metallopeptidase 13 (MMP13) and Runt-related transcription factor 2 (RUNX2) genes of endochondral ossification were significantly more downregulated on the experimental side than on the sham side. The primary pathways related to endochondral ossification were Parathyroid hormone synthesis, secretion and action, Relaxin signaling pathway, and IL-17 signaling pathway. CONCLUSIONS: The present study provided an innovative and reliable rat model of TMJ bony ankylosis by compound trauma and narrowed joint space. Furthermore, we demonstrated the downregulation of MMP13 and RUNX2 in the process of endochondral ossification in TMJ bony ankylosis.

Enhanced Photothermal Therapy through the In Situ Activation of a Temperature and Redox Dual-Sensitive Nanoreservoir of Triptolide.

Photothermal therapy (PTT) has attracted tremendous attention due to its noninvasiveness and localized treatment advantages. However, heat shock proteins (HSPs) associated self-preservation mechanisms bestow cancer cells thermoresistance to protect them from the damage of PTT. To minimize the thermoresistance of cancer cells and improve the efficacy of PTT, an integrated on-demand nanoplatform composed of a photothermal conversion core (gold nanorod, GNR), a cargo of a HSPs inhibitor (triptolide, TPL), a mesoporous silica based nanoreservoir, and a photothermal and redox di-responsive polymer shell is developed. The nanoplatform can be enriched in the tumor site, and internalized into cancer cells, releasing the encapsulated TPL under the trigger of intracellular elevated glutathione and near-infrared laser irradiation. Ultimately, the liberated TPL could diminish thermoresistance of cancer cells by antagonizing the PTT induced heat shock response via multiple mechanisms to maximize the PTT effect for cancer treatment.

CDDO-Me, Sulforaphane and tBHQ attenuate the RANKL-induced osteoclast differentiation via activating the NRF2-mediated antioxidant response.

Metabolic bone diseases are global public health concerns and are primarily caused by uncontrolled osteoclast (OC) formation and activation. During OC differentiation, intracellular reactive oxygen species (ROS) stimulated by receptor activator of nuclear factor kappa-B ligand (RANKL) can serve as the signaling molecules to promote osteoclastic genes expression. Nuclear factor erythroid-2 related factor 2 (NRF2), a master mediator of cellular antioxidant response, also plays a critical role in OC differentiation through the regulation of redox homeostasis. In this study, we investigated the effects of three NRF2 inducers on osteoclastogenesis, including Bardoxolone methyl (CDDO-Me), Sulforaphane (SFN), and tert-butylhydroquinone (tBHQ). By treating RAW cells with three compounds, we found that NRF2 was activated and its downstream antioxidant genes were upregulated, and the RANKL-induced intracellular ROS production and osteoclastogenesis were impaired. Additionally, the expression of nuclear factor of activated T cells c1 (NFATC1), C-FOS and tumor necrosis factor alpha (TNFα) were inhibited after acute exposures (6 h) to the three compounds. Furthermore, suppressed the expression of osteoclast differentiation-associated genes, tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), matrix metalloproteinase-9 (MMP-9) and dendritic cell-specific transmembrane protein (DC-STAMP) were observed after prolonged exposures (5 days) to the compounds. Taken together, these results suggest that CDDO-Me, SFN and tBHQ attenuate RANKL-induced osteoclastogenesis via activation of NRF2-mediated antioxidant response. Among these compounds, relatively low concentrations of CDDO-Me showed stronger active and inhibitory effects on antioxidant response and osteoclastogenesis, respectively.

Human apolipoprotein A-I exerts a prophylactic effect on high-fat diet-induced atherosclerosis via inflammation inhibition in a rabbit model.

Apolipoprotein A-I (apoA-I) is the major functional protein fraction of high-density lipoprotein. The prophylactic effect and mechanism of human apoA-I on atherosclerosis (AS) were investigated in a high-fat diet-induced AS rabbit model. The rabbits were injected with apoA-I once a week while fed high-fat diet for 20 weeks. Our results showed that apoA-I could raise the serum level of high-density lipoprotein-cholesterol and reduce those of lipid total cholesterol, triglyceride, and low-density lipoprotein-cholesterol in AS rabbits. Decreased aortic plaque area and aortic injury degree were also observed by Oil Red O staining and HE staining in apoA-I-treated high-fat diet-induced AS rabbits. Further study elucidated that apoA-I could down-regulate the expression of some inflammatory mediators including intercellular adhesion molecule type 1, vascular adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1, tumor necrosis factor-α, interleukin-6 (IL-6), and C-reactive protein in serum and aorta of AS rabbits. In addition, real-time quantitative RT-PCR analyses showed that the apoA-I infusions decreased the mRNA levels of two pro-inflammatory molecules, i.e. nuclear factor kappa B (NF-κB) and cyclooxygenase-2 (COX-2), in aorta of AS rabbits, which was associated with a concomitant reduction in endothelial VCAM-1 and IL-6 mRNA transcription. Together, our results support the atheroprotective and prophylactic role of apoA-I in vivo, and this activity may be correlated with its anti-inflammatory effect.

The impact of digital transformation of infrastructure on carbon emissions: Based on a "local-neighborhood" perspective.

In light of the recent worldwide scientific and technological revolution, it is imperative that urban infrastructure undergo a digital transformation in order to lower carbon emissions and support sustainable urban growth. However, to date, there is a lack of empirical research on carbon emissions based on the digital transformation of urban infrastructure. This paper uses data from 178 prefecture-level cities in China from 2005 to 2020 to study the impact of digital transformation of urban infrastructure on carbon emissions based on the "local-neighbourhood" perspective using a spatial difference-in-differences model. The results show that the digital transformation of urban infrastructure reduces the intensity of local carbon emissions while also reducing the carbon emissions of neighbouring cities, with a spatial spillover effect, and the boundary of this spatial spillover is 600 km. Mechanistic analyses suggest that digital transformation of urban infrastructure can reduce carbon emissions locally as well as in nearby areas by promoting green technological innovations. In light of this, this study has important policy implications for maximising the contribution of digital transformation of infrastructure to reducing carbon emissions.

Penicillium polonicum-mediated green synthesis of silver nanoparticles: Unveiling antimicrobial and seed germination advancements.

Silver nanoparticles (AgNPs), widely recognized for their nanoscale geometric size and unique properties, such as large specific surface area, high permeability, and high safety, were synthesized using the endophytic fungus Penicillium polonicum PG21 through a green approach. Four key synthesis factors-48 h, 45 °C, pH 9.0, and 80 mM AgNPs concentration-were optimized. Characterization via ultraviolet-visible spectroscopy, transmission electron microscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction revealed the AgNPs as approximately 3-25 nm spherical particles with numerous functional groups ensuring stability. AgNPs were tested against various fungal and bacterial plant pathogens, including Botrytis cinerea (EB-1), Alternaria alternata (EB-2, EB-3), Fusarium solani (RG-1), Williamsia serinedens (SL-1), Sphingopyxis macrogoltabida (SL-2), Bacillus velezensis (SL-3), and Pseudomonas mediterranea (SL-4), causing agricultural challenges. PG21-synthesized AgNPs exhibited inhibition rates against all tested fungi, with 60 µg/mL AgNPs demonstrating optimal inhibition rates. Notably, EB-1 experienced a significant growth inhibition, reaching an inhibition rate reached of 74.22 ± 1.54%. Conversely, RG-1 exhibited the smallest inhibitory effect at 48.13 ± 0.92%. The effect of AgNPs on safflower seed germination and growth revealed notable increases in shoot length, fresh weight, stem length, and number of lateral roots-1.4, 1.4, 1.33, and 10.67 times higher than the control, respectively, at an AgNPs concentration of 80 µg/mL. In conclusion, green-synthesized AgNPs demonstrate pathogen toxicity, showcasing potential applications in disease management for industrial crops and promoting plant growth.

Peptide targeting improves the delivery and therapeutic index of glucocorticoids to treat rheumatoid arthritis.

Rheumatoid arthritis (RA) is a prevalent autoimmune disease characterized by excessive inflammation in the joints. Glucocorticoid drugs are used clinically to manage RA symptoms, while their dosage and duration need to be tightly controlled due to severe adverse effects. Using dexamethasone (DEX) as a model drug, we explored here whether peptide-guided delivery could increase the safety and therapeutic index of glucocorticoids for RA treatment. Using multiple murine RA models such as collagen-induced arthritis (CIA), we found that CRV, a macrophage-targeting peptide, can selectively home to the inflammatory synovium of RA joints upon intravenous injection. The expression of the CRV receptor, retinoid X receptor beta (RXRB), was also elevated in the inflammatory synovium, likely being the basis of CRV targeting. CRV-conjugated DEX increased the accumulation of DEX in the inflamed synovium but not in healthy organs of CIA mice. Therefore, CRV-DEX demonstrated a stronger efficacy to suppress synovial inflammation and alleviate cartilage/bone destruction. Meanwhile, CRV conjugation reduced immune-related adverse effects of DEX even after a long-term use. Last, we found that RXRB expression was significantly elevated in human patient samples, demonstrating the potential of clinical translation. Taken together, we provide a novel, peptide-targeted strategy to improve the therapeutic efficacy and safety of glucocorticoids for RA treatment.

Polymer/copper nanocomplex-induced lysosomal cell death promotes tumor lymphocyte infiltration and synergizes anti-PD-L1 immunotherapy for triple-negative breast cancer.

Our previous research discovered that combining the PDA-PEG polymer with copper ions can selectively kill cancer cells. However, the precise mechanism by which this combination functions was not fully understood. This study revealed that the PDA-PEG polymer and copper ions form complementary PDA-PEG/copper (Poly/Cu) nanocomplexes by facilitating copper ion uptake and lysosomal escape. An in vitro study found that Poly/Cu killed 4T1 cells through a lysosome cell death pathway. Furthermore, Poly/Cu inhibited both the proteasome function and autophagy pathway and induced immunogenic cell death (ICD) in 4T1 cells. The Poly/Cu induced ICD coupled with the checkpoint blockade effect of the anti-PD-L1 antibody (aPD-L1) synergistically promoted immune cell penetration into the tumor mass. Benefiting from the tumor-targeting effect and cancer cell-selective killing effect of Poly/Cu complexes, the combinatory treatment of aPD-L1 and Poly/Cu effectively suppressed the progression of triple-negative breast cancer without inducing systemic side effects.

Optineurin regulates osteoblast function in an age-dependent fashion in a mouse model of Paget's disease of bone.

Paget's disease of bone (PDB) is a degenerative disorder affecting the skull and bones. Hyperactive osteoclasts (OCs) initiate bone degradation in the early stage, followed by increased bone formation by osteoblasts (OBs) in trabecular bones during the advanced stage. This OB-OC uncoupling results in bone deformations and irregular trabecular bone patterns. Current mouse models poorly replicate the advanced-stage characteristics of PDB. Optineurin (Gene: OPTN in humans, Optn in mice, protein: OPTN) has been implicated in PDB by genetic analyses. We identified PDB-like cortical lesions associated with OC hyperactivation in an Optn knockout (Optn-/-) mouse model. However, the effects of OPTN dysfunction on OBs and trabecular bone in advanced PDB remain unclear. In this study, we used the Optn-/- mouse model to investigate trabecular bone abnormalities and OB activity in PDB. Micro-computed tomography analysis revealed severe pagetic alterations in craniofacial bones and femurs of aged Optn-/- mice, resembling clinical manifestations of PDB. Altered OB activity was observed in aged Optn-/- mice, implicating compensatory OB response in trabecular bone anomalies. To elucidate the role of OC-OB interactions in PDB, we conducted in vitro experiments using OC conditioned media (CM) to examine the effects on OB osteogenic potential. We found OC CM restored compromised osteogenic induction of Optn-/- bone marrow stromal cells (BMSCs) from young mice, suggesting OCs maintain OB activity through secreted factors. Strikingly, OC CM from aged Optn-/- mice significantly enhanced osteogenic capability of Optn-/- BMSCs, providing evidence for increased OB activity in advanced stages of PDB. We further identified TGF-ß/BMP signaling pathway in mediating the effects of OC CM on OBs. Our findings provide insights into Optn's role in trabecular bone abnormalities and OB activity in PDB. This enhances understanding of PDB pathogenesis and may contribute to potential therapeutic strategies for PDB and related skeletal disorders.

Trim-Away in adult animals through Nano-ERASER and its application in cancer therapy.

Trim-Away is a versatile intracellular protein degradation pathway that has been extensively explored in vitro. However, the in vivo application of Trim-Away is limited at oocyte and zygote stages due to the lack of an in vivo practical approach for intracellular antibody delivery. To broaden the application of Trim-Away, especially for clinical use, we developed a nanogel-based Nano-ERASER system. Here, we demonstrated that the intracellular delivery of anti-programmed cell death ligand 1 (PD-L1) antibody through Nano-ERASER could effectively deplete PD-L1 in triple negative breast cancer (TNBC) cells and induce cancer cell death. Furthermore, with the help of a tumor tissue-targeted nanogel, anti-PD-L1 antibody-loaded Nano-ERASER effectively inhibited tumor progression in a TNBC mouse model. These results confirmed that Nano-ERASER realized Trim-Away in adult animals for the first time, which could be an effective tool for disease treatment and studying gene/protein function both in vitro and in vivo.

Optineurin regulates NRF2-mediated antioxidant response in a mouse model of Paget's disease of bone.

Degenerative diseases affecting the nervous and skeletal systems affect the health of millions of elderly people. Optineurin (OPTN) has been associated with numerous neurodegenerative diseases and Paget's disease of bone (PDB), a degenerative bone disease initiated by hyperactive osteoclastogenesis. In this study, we found age-related increase in OPTN and nuclear factor E2-related factor 2 (NRF2) in vivo. At the molecular level, OPTN could directly interact with both NRF2 and its negative regulator Kelch-like ECH-associated protein 1 (KEAP1) for up-regulating antioxidant response. At the cellular level, deletion of OPTN resulted in increased intracellular reactive oxygen species and increased osteoclastogenic potential. At the tissue level, deletion of OPTN resulted in substantially increased oxidative stress derived from leukocytes that further stimulate osteoclastogenesis. Last, curcumin attenuated hyperactive osteoclastogenesis induced by OPTN deficiency in aged mice. Collectively, our findings reveal an OPTN-NRF2 axis maintaining bone homeostasis and suggest that antioxidants have therapeutic potential for PDB.

The impact of economic policy uncertainty on firms' investment in innovation: Evidence from Chinese listed firms.

This paper uses data of Chinese listed enterprises and economic policy uncertainty index for empirical analysis, and conducts a study through three channels of monetary policy uncertainty affecting enterprise innovation investment, and finds that economic policy uncertainty has a positive promotion effect on enterprise R&D investment, and its increase in tension is instead a clear signal that can effectively increase enterprise R&D investment, this promotion effect seems unexpected, this paper Through theoretical analysis and combined with the actual practice, this incentive effect is found to be in line with reality. However, in the subsequent heterogeneity analysis, this paper finds that it positively promotes R&D investment when economic policy uncertainty is low and may have a suppressive effect on R&D investment when monetary policy uncertainty is high.

A Therapeutic Sheep in Metastatic Wolf's Clothing: Trojan Horse Approach for Cancer Brain Metastases Treatment.

Early-stage brain metastasis of breast cancer (BMBC), due to the existence of an intact blood-brain barrier (BBB), is one of the deadliest neurologic complications. To improve the efficacy of chemotherapy for BMBC, a Trojan horse strategy-based nanocarrier has been developed by integrating the cell membrane of a brain-homing cancer cell and a polymeric drug depot. With the camouflage of a MDA-MB-231/Br cell membrane, doxorubicin-loaded poly (D, L-lactic-co-glycolic acid) nanoparticle (DOX-PLGA@CM) shows enhanced cellular uptake and boosted killing potency for MDA-MB-231/Br cells. Furthermore, DOX-PLGA@CM is equipped with naturally selected molecules for BBB penetration, as evidenced by its boosted capacity in entering the brain of both healthy and early-stage BMBC mouse models. Consequently, DOX-PLGA@CM effectively reaches the metastatic tumor lesions in the brain, slows down cancer progression, reduces tumor burden, and extends the survival time for the BMBC animal. Furthermore, the simplicity and easy scale-up of the design opens a new window for the treatment of BMBC and other brain metastatic cancers.

The spatial characteristics of embodied carbon emission flow in Chinese provinces: a network-based perspective.

Combining the multi-regional input-output model with complex network technology, based on the provincial scale, this paper systematically describes the characteristics of the spatial network flow and its changing track of embodied carbon emissions among provinces in China from three dimensions of network structure, flow distribution characteristics, and spatial flow characteristics. Furthermore, the maximum spanning tree method is used to identify the spatial flow path of embodied carbon emissions between provinces and to distinguish the hierarchical status and roles of different provinces on the path. The research results show that the embodied carbon emission flow network among provinces has obvious small-world characteristics during the study, and the roles of different provinces in the network have significant heterogeneity. In general, Hebei and Inner Mongolia mainly act the role of suppliers of embodied carbon emissions, Guangdong and Zhejiang mainly act the role of consumers of embodied carbon emissions, while Zhejiang and Jiangsu mainly act as the media in the network. The spatial distribution characteristics of embodied carbon emission flow have a significant long tail effect; about 6% of the embodied carbon emission flow relationship among provinces accounts for 30% of that on the country level. The spatial flow direction of embodied carbon emissions was mainly concentrated in north China, central China, and southeast coastal region in the early stage and gradually shifted to inter-regional flows, presenting a divergent state of multiple regions and multiple centers. According to the flow path identification, it is found that Jiangsu, Guangdong, Hebei, Zhejiang, and other provinces are the key nodes on the spatial flow path of embodied carbon emissions in China, and the local center in a space is prominent. Based on the analysis and conclusions, the paper finally puts forward the corresponding countermeasures and suggestions in carbon reduction.

Dopamine suppresses osteoclast differentiation via cAMP/PKA/CREB pathway.

How the nervous system regulates bone remodeling is an exciting area of emerging research in bone biology. Accumulating evidence suggest that neurotransmitter-mediated inputs from neurons may act directly on osteoclasts. Dopamine is a neurotransmitter that can be released by hypothalamic neurons to regulate bone metabolism through the hypothalamic-pituitary-gonadal axis. Dopamine is also present in sympathetic nerves that penetrate skeletal structures throughout the body. It has been shown that dopamine suppresses osteoclast differentiation via a D2-like receptors (D2R)-dependent manner, but the intracellular secondary signaling pathway has not been elucidated. In this study, we found that cAMP-response element binding protein (CREB) activity responds to dopamine treatment during osteoclastogenesis. Considering the critical role of CREB in osteoclastogenesis, we hypothesize that CREB may be a critical target in dopamine's regulation of osteoclast differentiation. We confirmed that D2R is also present in RAW cells and activated by dopamine. Binding of dopamine to D2R inhibits the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway which ultimately decreases CREB phosphorylation during osteoclastogenesis. This was also associated with diminished expression of osteoclast markers that are downstream of CREB. Pharmacological activation of adenylate cyclase (to increase cAMP production) and PKA reverses the effect of dopamine on CREB activity and osteoclastogenesis. Therefore, we have identified D2R/cAMP/PKA/CREB as a candidate pathway that mediates dopamine's inhibition of osteoclast differentiation. These findings will contribute to our understanding of how the nervous and skeletal systems interact to regulate bone remodeling. This will enable future work toward elucidating the role of the nervous system in bone development, repair, aging, and degenerative disease.

Biomimetic polydopamine-laced hydroxyapatite collagen material orients osteoclast behavior to an anti-resorptive pattern without compromising osteoclasts' coupling to osteoblasts.

Polydopamine-assisted modification for bone substitute materials has recently shown great application potential in bone tissue engineering due to its excellent biocompatibility and adhesive properties. A scaffold material's impact on osteoclasts is equally as important as its impact on osteoblasts when considering tissue engineering for bone defect repair, as healthy bone regeneration requires an orchestrated coupling between osteoclasts and osteoblasts. How polydopamine-functionalized bone substitute materials modulate the activity of osteoblast lineage cells has been extensively investigated, but much less is known about their impact on osteoclasts. Moreover, most of the polydopamine-functionalized materials would need to additionally load a biomolecule to exert the modulation on osteoclast activity. Herein, we demonstrated that our biomimetic polydopamine-laced hydroxyapatite collagen (PDHC) scaffold material, which does not need to load additional bioactive agent, is sufficiently able to modulate osteoclast activity in vitro. First, PDHC showed an anti-resorptive potential, characterized by decreased osteoclast differentiation and resorption capacity and changes in osteoclasts' transcriptome profile. Next, cAMP response element-binding protein (CREB) activity was found to mediate PDHC's anti-osteoclastogenic effect. Finally, although PDHC altered clastokines expression pattern of osteoclasts, as revealed by transcriptomic and secretomic analysis, osteoclasts' coupling to osteoblasts was not compromised by PDHC. Collectively, this study demonstrated the PDHC material orients osteoclast behavior to an anti-resorptive pattern without compromising osteoclasts' coupling to osteoblasts. Such a feature is favorable for the net increase of bone mass, which endows the PDHC material with great application potential in preclinical/clinical bone defect repair.

Deficiency of optineurin enhances osteoclast differentiation by attenuating the NRF2-mediated antioxidant response.

Abnormally increased resorption contributes to bone degenerative diseases such as Paget's disease of bone (PDB) through unclear mechanisms. Recently, the optineurin (OPTN) gene has been implicated in PDB, and global OPTN knockout mice (Optn-/-) were shown to exhibit increased formation of osteoclasts (osteoclastogenesis). Growing evidence, including our own, has demonstrated that intracellular reactive oxygen species (ROS) stimulated by receptor activator of nuclear factor kappa-B ligand (RANKL) can act as signaling molecules to promote osteoclastogenesis. Here, we report that OPTN interacts with nuclear factor erythroid-derived factor 2-related factor 2 (NRF2), the master regulator of the antioxidant response, defining a pathway through which RANKL-induced ROS could be regulated for osteoclastogenesis. In this study, monocytes from Optn-/- and wild-type (Optn+/+) mice were utilized to differentiate into osteoclasts, and both qRT-PCR and tartrate-resistant acid phosphatase (TRAP) staining showed that the Optn-/- monocytes exhibited enhanced osteoclastogenesis compared to the Optn+/+ cells. CellROX® staining, qRT-PCR, and Western blotting indicated that OPTN deficiency reduced the basal expression of Nrf2, inhibited the expression of NRF2-responsive antioxidants, and increased basal and RANKL-induced intracellular ROS levels, leading to enhanced osteoclastogenesis. Coimmunoprecipitation (co-IP) showed direct interaction, and immunofluorescence staining showed perinuclear colocalization of the OPTN-NRF2 granular structures during differentiation. Finally, curcumin and the other NRF2 activators attenuated the hyperactive osteoclastogenesis induced by OPTN deficiency. Collectively, our findings reveal a novel OPTN-mediated mechanism for regulating the NRF2-mediated antioxidant response in osteoclasts and extend the therapeutic potential of OPTN in the aging process resulting from ROS-triggered oxidative stress, which is associated with PDB and many other degenerative diseases.

Application of bioluminescence resonance energy transfer-based cell tracking approach in bone tissue engineering.

Bioluminescent imaging (BLI) has emerged as a popular in vivo tracking modality in bone regeneration studies stemming from its clear advantages: non-invasive, real-time, and inexpensive. We recently adopted bioluminescence resonance energy transfer (BRET) principle to improve BLI cell tracking and generated the brightest bioluminescent signal known to date, which thus enables more sensitive real-time cell tracking at deep tissue level. In the present study, we brought BRET-based cell tracking strategy into the field of bone tissue engineering for the first time. We labeled rat mesenchymal stem cells (rMSCs) with our in-house BRET-based GpNLuc reporter and evaluated the cell tracking efficacy both in vitro and in vivo. In scaffold-free spheroid 3D culture system, using BRET-based GpNLuc labeling resulted in significantly better correlation to cell numbers than a fluorescence based approach. In scaffold-based 3D culture system, GpNLuc-rMSCs displayed robust bioluminescence signals with minimal background noise. Furthermore, a tight correlation between BLI signal and cell number highlighted the robust reliability of using BRET-based BLI. In calvarial critical sized defect model, robust signal and the consistency in cell survival evaluation collectively supported BRET-based GpNLuc labeling as a reliable approach for non-invasively tracking MSC. In summary, BRET-based GpNLuc labeling is a robust, reliable, and inexpensive real-time cell tracking method, which offers a promising direction for the technological innovation of BLI and even non-invasive tracking systems, in the field of bone tissue engineering.

Carrier-Free Nanoassembly of Curcumin-Erlotinib Conjugate for Cancer Targeted Therapy.

Anticancer drug-loaded nanoparticles have been explored extensively to decrease side effects while improving their therapeutic efficacy. However, due to the low drug loading content, premature drug release, nonstandardized carrier structure, and difficulty in predicting the fate of the carrier, only a few nanomedicines have been approved for clincial use. Herein, a carrier-free nanoparticle based on the self-assembly of the curcumin-erlotinib conjugate (EPC) is developed. The EPC nanoassembly exhibits more potent cell killing, better antimigration, and anti-invasion effects for BxPC-3 pancreatic cancer cells than the combination of free curcumin and erlotinib. Furthermore, benefiting from both passive and active tumor targeting effect, EPC nanoassembly can effectively accumulate in the tumor tissue in a xenograft pancreatic tumor mouse model. Consequently, EPC effectively reduces the growth of pancreatic tumors and extends the median survival time of the tumor-bearing mice from 22 to 68 days. In addition, no systemic toxicity is detected in the mice receiving EPC treatment. Attributed to the uniformity of the curcumin-erlotinib conjugate and easiness of scaling up, it is expected that the EPC can be translated into a powerful tool in fighting against pancreatic cancer and other epidermal growth factor receptor positive cancers.