Engineered exosomes with a photoinducible protein delivery system enable CRISPR-Cas-based epigenome editing in Alzheimer's disease.

Effective intracellular delivery of therapeutic proteins can potentially treat a wide array of diseases. However, efficient delivery of functional proteins across the cell membrane remains challenging. Exosomes are nanosized vesicles naturally secreted by various types of cells and may serve as promising nanocarriers for therapeutic biomolecules. Here, we engineered exosomes equipped with a photoinducible cargo protein release system, termed mMaple3-mediated protein loading into and release from exosome (MAPLEX), in which cargo proteins can be loaded into the exosomes by fusing them with photocleavable protein (mMaple3)-conjugated exosomal membrane markers and subsequently released from the exosomal membrane by inducing photocleavage with blue light illumination. Using this system, we first induced transcriptional regulation by delivering octamer-binding transcription factor 4 and SRY-box transcription factor 2 to fibroblasts in vitro. Second, we induced in vivo gene recombination in Cre reporter mice by delivering Cre recombinase. Last, we achieved targeted epigenome editing in the brains of 5xFAD and 3xTg-AD mice, two models of Alzheimer's disease. Administration of MAPLEXs loaded with ß-site amyloid precursor protein cleaving enzyme 1 (Bace1)-targeting single guide RNA-incorporated dCas9 ribonucleoprotein complexes, coupled with the catalytic domain of DNA methyltransferase 3A, resulted in successful methylation of the targeted CpG sites within the Bace1 promoter. This approach led to a significant reduction in Bace1 expression, improved recognition memory impairment, and reduced amyloid pathology in 5xFAD and 3xTg-AD mice. These results suggest that MAPLEX is an efficient intracellular protein delivery system that can deliver diverse therapeutic proteins for multiple diseases.

Discovery of TCP-(MP)-caffeic acid analogs as a new class of agents for treatment of osteoclastic bone loss.

Inhibition of LSD1 was proposed as promising and attractive therapies for treating osteoporosis. Here, we synthesized a series of novel TCP-(MP)-Caffeic acid analogs as potential LSD1 inhibitors to assess their inhibitory effects on osteoclastogenesis by using TRAP-staining assay and try to explore the preliminary SAR. Among them, TCP-MP-CA (11a) demonstrated osteoclastic bone loss both in vitro and in vivo, showing a significant improvement in the in vivo effects compared to the LSD1 inhibitor GSK-LSD1. Additionally, we elucidated a mechanism that 11a and its precursor that 11e directly bind to LSD1/CoREST complex through FAD to inhibit LSD1 demethylation activity and influence its downstream IκB/NF-κB signaling pathway, and thus regulate osteoclastic bone loss. These findings suggested 11a or 11e as potential novel candidates for treating osteoclastic bone loss, and a concept for further development of TCP-(MP)-Caffeic acid analogs for therapeutic use in osteoporosis clinics.

Novel inhibitor N-cyclopropyl-4-((4-((4-(trifluoromethyl)phenyl)sulfonyl)piperazin-1-yl)methyl)benzamide attenuates RANKL-mediated osteoclast differentiation in vitro.

Both cyclopropyl amide and piperazine sulfonamide functional groups are known for their various biological properties used for drug development. Herein, we synthesized nine new derivatives with different substituent groups incorporating these moieties and screened them for their anti-osteoclast differentiation activity. After analyzing the structure-activity relationship (SAR), the inhibitory effect against osteoclastogenesis was determined to be dependent on the lipophilicity of the compound. Derivative 5b emerged as the most effective dose-dependent inhibitor after TRAP staining with an IC50 of 0.64 µM against RANKL-induced osteoclast cells. 5b was also able to suppress F-acting ring formation and bone resorption activity of osteoclasts in vitro. Finally, well-acknowledged gene and protein osteoclast-specific marker expression levels were decreased after 5b administration on primary murine osteoclast cells.

Developing a Framework for Self-regulatory Governance in Healthcare AI Research: Insights from South Korea.

This paper elucidates and rationalizes the ethical governance system for healthcare AI research, as outlined in the 'Research Ethics Guidelines for AI Researchers in Healthcare' published by the South Korean government in August 2023. In developing the guidelines, a four-phase clinical trial process was expanded to six stages for healthcare AI research: preliminary ethics review (stage 1); creating datasets (stage 2); model development (stage 3); training, validation, and evaluation (stage 4); application (stage 5); and post-deployment monitoring (stage 6). Researchers identified similarities between clinical trials and healthcare AI research, particularly in research subjects, management and regulations, and application of research results. In the step-by-step articulation of ethical requirements, this similarity benefits from a reliable and flexible use of existing research ethics governance resources, research management, and regulatory functions. In contrast to clinical trials, this procedural approach to healthcare AI research governance effectively highlights the distinct characteristics of healthcare AI research in research and development process, evaluation of results, and modifiability of findings. The model exhibits limitations, primarily in its reliance on self-regulation and lack of clear delineation of responsibilities. While formulated through multidisciplinary deliberations, its application in the research field remains untested. To overcome the limitations, the researchers' ongoing efforts for educating AI researchers and public and the revision of the guidelines are expected to contribute to establish an ethical research governance framework for healthcare AI research in the South Korean context in the future.

Chrysophanol inhibits of colorectal cancer cell motility and energy metabolism by targeting the KITENIN/ErbB4 oncogenic complex.

BACKGROUND: Expression of the KITENIN/ErbB4 oncogenic complex is associated with metastasis of colorectal cancer to distant organs and lymph nodes and is linked with poor prognosis and poor survival. METHODS: Here, we used in vitro and in silico methods to test the ability of chrysophanol, a molecule of natural origin, to suppress the progression of colorectal cancer by targeting the KITENIN/ErbB4 complex. RESULTS: Chrysophanol binds to ErbB4, disrupting the ErbB4/KITENIN complex and causing autophagic degradation of KITENIN. We demonstrated that chrysophanol binds to ErbB4 according to a molecular docking model. Chrysophanol reversed KITENIN-mediated effects on cell motility, aerobic glycolysis, and expression of downstream effector genes. Moreover, under conditions of KITENIN overexpression, chrysophanol suppressed the production of onco-metabolites. CONCLUSION: Chrysophanol suppresses oncogenic activities by targeting the KITENIN/ErbB4 complex.

Extracellular Vesicles Derived from Adipose Stem Cells Alleviate Systemic Sclerosis by Inhibiting TGF-ß Pathway.

Systemic sclerosis is an autoimmune disease characterized by inflammatory reactions and fibrosis. Myofibroblasts are considered therapeutic targets for preventing and reversing the pathogenesis of fibrosis in systemic sclerosis. Although the mechanisms that differentiate into myofibroblasts are diverse, transforming growth factor ß (TGF-ß) is known to be a key mediator of fibrosis in systemic sclerosis. This study investigated the effects of extracellular vesicles derived from human adipose stem cells (ASC-EVs) in an in vivo systemic sclerosis model and in vitro TGF-ß1-induced dermal fibroblasts. The therapeutic effects of ASC-EVs on the in vivo systemic sclerosis model were evaluated based on dermal thickness and the number of α-smooth muscle actin (α-SMA)-expressing cells using hematoxylin and eosin staining and immunohistochemistry. Administration of ASC-EVs decreased both the dermal thickness and α-SMA expressing cell number as well as the mRNA levels of fibrotic genes, such as Acta2, Ccn2, Col1a1 and Comp. Additionally, we discovered that ASC-EVs can decrease the expression of α-SMA and CTGF and suppress the TGF-ß pathway by inhibiting the activation of SMAD2 in dermal fibroblasts induced by TGF-ß1. Finally, TGF-ß1-induced dermal fibroblasts underwent selective death through ASC-EVs treatment. These results indicate that ASC-EVs could provide a therapeutic approach for preventing and reversing systemic sclerosis.

Usnic Acid Targets 14-3-3 Proteins and Suppresses Cancer Progression by Blocking Substrate Interaction.

The anticancer therapeutic effects of usnic acid (UA), a lichen secondary metabolite, have been demonstrated in vitro and in vivo. However, the mechanism underlying the anticancer effect of UA remains to be clarified. In this study, the target protein of UA was identified using a UA-linker-Affi-Gel molecule, which showed that UA binds to the 14-3-3 protein. UA binds to 14-3-3, causing the degradation of proteasomal and autophagosomal proteins. The interaction of UA with 14-3-3 isoforms modulated cell invasion, cell cycle progression, aerobic glycolysis, mitochondrial biogenesis, and the Akt/mTOR, JNK, STAT3, NF-κB, and AP-1 signaling pathways in colorectal cancer. A peptide inhibitor of 14-3-3 blocked or regressed the activity of UA and inhibited its effects. The results suggest that UA binds to 14-3-3 isoforms and suppresses cancer progression by affecting 14-3-3 targets and phosphorylated proteins.

Long non-coding RNA SOX2OT in tamoxifen-resistant breast cancer.

Hormone receptor (HR)-positive breast cancer can become aggressive after developing hormone-treatment resistance. This study elucidated the role of long non-coding RNA (lncRNA) SOX2OT in tamoxifen-resistant (TAMR) breast cancer and its potential interplay with the tumor microenvironment (TME). TAMR breast cancer cell lines TAMR-V and TAMR-H were compared with the luminal type A cell line (MCF-7). LncRNA expression was assessed via next-generation sequencing, RNA extraction, lncRNA profiling, and quantitative RT-qPCR. SOX2OT overexpression effects on cell proliferation, migration, and invasion were evaluated using various assays. SOX2OT was consistently downregulated in TAMR cell lines and TAMR breast cancer tissue. Overexpression of SOX2OT in TAMR cells increased cell proliferation and cell invasion. However, SOX2OT overexpression did not significantly alter SOX2 levels, suggesting an independent interaction within TAMR cells. Kaplan-Meier plot analysis revealed an inverse relationship between SOX2OT expression and prognosis in luminal A and B breast cancers. Our findings highlight the potential role of SOX2OT in TAMR breast cancer progression. The downregulation of SOX2OT in TAMR breast cancer indicates its involvement in resistance mechanisms. Further studies should explore the intricate interactions between SOX2OT, SOX2, and TME in breast cancer subtypes.

Discovery of a novel homoisoflavonoid derivative 5g for anti-osteoclastic bone loss via targeting FGFR1.

Several flavonoids have been shown to exert anti-osteoporosis activity. However, the structure-activity relationship and the mechanism of anti-osteoporosis activity of flavonoids remain unknown. In this study, we prepared a series of novel homoisoflavonoid (HIF) derivatives to evaluate their inhibitory effects on osteoclastogenesis using TRAP-activity in vitro assay. Then, the preliminary structure-activity relationship was studied. Among the evaluated novel flavonoids, derivative 5g exerted the most inhibitory bioactivity on primary osteoclast differentiation without interfering with osteogenesis. It was hence selected for further in vitro, in vivo and mechanism of action investigation. Results show that 5g likely directly binds to the fibroblast growth factor receptor 1 (FGFR1), decreasing the activation of ERK1/2 and IκBα/NF-κB signaling pathways, which in turn blocks osteoclastogenesis in vitro and osteoclastic bone loss in vivo. Our study shows that homoisoflavonoid (HIF) derivatives 5g can serve as a potential novel candidate for treating osteoporosis via inhibition of FGFR1.

Computational Probing of the Folding Mechanism of Human Telomeric G-Quadruplex DNA.

The human telomeric (htel) sequences in the terminal regions of human telomeres form diverse G-quadruplex (GQ) structures. Despite much experimental efforts to elucidate the folding pathways of htel GQ, no comprehensive model of htel GQ folding has been presented. Here, we describe folding pathways of the htel GQ determined by state-of-the-art enhanced sampling molecular dynamics simulation at the all-atom level. Briefly, GQ folding is initiated by the formation of a single-hairpin and then followed by the formation of double-hairpins, which then branch via distinct folding pathways to produce different GQ topologies (antiparallel chair, antiparallel basket, hybrids 1 and 2, and parallel propeller). In addition to these double-hairpin states, three-triad and two-tetrad structures in antiparallel backbone alignment serve as key intermediates that connect the GQ folding and transition between two different GQs.

Macrosphelides from Antarctic fungus Pseudogymnoascus sp. (strain SF-7351) and their neuroprotective effects on BV2 and HT22 cells.

Strategies for reducing inflammation in neurodegenerative diseases have attracted increasing attention. Herein, we discovered and evaluated the neuroprotective potential of fungal metabolites isolated from the Antarctic fungus Pseudogymnoascus sp. (strain SF-7351). The chemical investigation of the EtOAc extract of the fungal strain isolate revealed a novel naturally occurring epi-macrosphelide J (1), a novel secondary metabolite macrosphelide N (2), and three known compounds, namely macrosphelide A (3), macrosphelide B (4), and macrosphelide J (5). Their structures were established unambiguously using spectroscopic methods, such as one-dimensional and two-dimensional nuclear magnetic resonance (1D and 2D-NMR) spectroscopy, high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), and gauge-including atomic orbital (GIAO) NMR chemical shift calculations, with the support of the advanced statistical method DP4+. Among the isolated metabolites, the absolute configuration of epi-macrosphelide J (1) was further confirmed using single-crystal X-ray diffraction analysis. The neuroprotective effects of the isolated metabolites were evaluated in lipopolysaccharide (LPS)-induced BV2 and glutamate-stimulated HT22 cells. Only macrosphelide B (4) displayed substantial protective effects in both BV2 and HT22 cells. Molecular mechanisms underlying this activity were investigated using western blotting and molecular docking studies. Macrosphelide B (4) inhibited the inflammatory response by reducing the nuclear translocation of NF-κB (p65) in LPS-induced BV2 cells and induced the Nrf2/HO-1 signaling pathway in both BV2 and HT22 cells. The neuroprotective effect of macrosphelide B (4) is related to the interaction between Keap1 and p65. These results suggest that macrosphelide B (4), present in the fungus Pseudogymnoascus sp. (strain SF-7351), may serve as a candidate for the treatment of neurodegenerative diseases.

Comparing the Effectiveness of Two New CPR Training Methods in Korea: Medical Virtual Reality Simulation and Flipped Learning.

Background: We compared the educational effects of two training methods that have gained momentum: medical virtual reality (medi-VR) simulation and flipped learning. Methods: Firefighters (n=128; 116 men and 12 women; mean age=28 years) in training from the Emergency Educational Simulation Center of Korea National Fire Service Academy, Gongju-si, Korea, were randomly assigned to two groups: medi-VR simulation and flipped learning in 2022. The participants were trained to perform cardiopulmonary resuscitation (CPR) using medi-VR simulation and the flipped learning methods. CPR self-efficacy, knowledge, performance, class immersion, and class satisfaction were compared between the groups. To analyze educational effects, paired and independent t-tests were performed. Results: The post-education scores for CPR performance knowledge and CPR performance were significantly higher in the medi-VR simulation group compared to the flipped learning counterparts (P<0.001). Moreover, despite the lack of a significant difference between the groups, post-education scores for self-efficacy, class immersion, and class satisfaction showed a positive effect on learning. Conclusion: Medi-VR simulation can be utilized as effective educational intervention, while providing a new direction for teaching methods.

Synthesis and evaluation of 2-NMPA derivatives as potential agents for prevention of osteoporosis in vitro and in vivo.

Abnormal osteoclast differentiation causes various bone disorders such as osteoporosis. Targeting the formation and activation of osteoclasts has been recognized as an effective approach for preventing osteoporosis. Herein, we synthesized eleven 2-NMPA derivatives which are (2-(2-chlorophenoxy)-N-(4-alkoxy-2-morpholinophenyl) acetamides, and evaluated their suppression effects on osteoclastogenesis in vitro by using TRAP-staining assay. Among the synthesized eleven novel 2-NMPAs, 4-(2-(2-chlorophenoxy)acetamido)-3-morpholinophenyl trifluoromethanesulfonate (11b), 4-(2-(2-chlorophenoxy) acetamido)-3-morpholinophenyl-3-(N-(2-oxo-2-((2-(phenylthio) phenyl) amino) ethyl)methylsulfonamido)benzoate (11d), and 4-(2-(2-chlorophenoxy) acetamido)-3-morpholinophenyl 4-acetamidobenzenesulfonate (11h) displayed highly inhibitory bioactivity on the differentiation of primary osteoclasts. 11h was selected for further investigation of the inhibitory effects and potential mechanism involved in the suppression of osteoclastogenesis. In vitro analysis suggested that 11h inhibited osteoclastogenesis with an IC50 of 358.29 nM, decreased the formation of F-action belts and bone resorption, without interfering cell viability and osteoblast differentiation. Furthermore, the mRNA expressions of osteoclast-specific genes such as Acp5, Nfatc1, Dc-stamp, Atp6v0d2, Mmp9, and Ctsk significantly decreased following 11h treatment. RANKL-induced osteoclast-specific proteins analysis demonstrated that 11h suppressed osteoclast differentiation by downregulating of RANKL-mediated TRAF6 expression, followed by inactivation of PI3K/AKT and IκBα/NF-κB signaling pathways. Finally, 11h inhibited ovariectomy-induced bone loss in vivo. Therefore, the current work highlighted the therapeutic potential of 11h as an anti-osteoporosis lead compound.

Discovery of PMSA Derivative 11 as a Novel Lead Compound for Therapeutic Treatment of Osteoporosis In Vitro and In Vivo.

To discover a potent candidate for suppressing mature osteoclasts formation in vitro using a TRAP staining assay. A series of PMSA derivatives were synthesized and evaluated for their bioactivity in our current study. Our results showed that PMSA derivative 11 exhibited the most promising bioactivity, with an IC50 value of 322.9 nM, which was ∼15-fold better than PMSA-3-Ac in suppressing osteoclastogenesis in vitro. Additionally, 11 blocked the formation of F-action belts and bone resorption in a concentration-dependent manner. Mechanistically, 11 decreased the expression of genes required for osteoclastogenesis by blocking NFATc1 translocation from the cytoplasm to nucleus. Furthermore, 11 demonstrated a therapeutic inhibitory effect on the differentiation of human iPSC-derived primary osteoclasts. In vivo investigation showed that 11 prevented excessive osteoclastogenesis-mediated bone loss in ovariectomized osteoporosis mimic mice. These findings highlighted the therapeutic potential of 11 as a lead compound for anti-osteoporosis by targeting NFATc1 translocation.

Polygenic risk score for tumor aggressiveness and early-onset prostate cancer in Asians.

We attempted to assess the performance of an ethnic-specific polygenic risk score (PRS) designed from a Korean population to predict aggressive prostate cancer (PCa) and early-onset (age < 60). A PRS score comprised of 22 SNPs was computed in 3695 patients gathered from one of 4 tertiary centers in Korea. Males with biopsy or radical prostatectomy-proven PCa were included for analysis, collecting additional clinical parameters such as age, BMI, PSA, Gleason Group (GG), and staging. Patients were divided into 4 groups of PRS quartiles. Intergroup differences were assessed, as well as risk ratio and predictive performance based on GG using logistic regression analysis and AUC. No significant intergroup differences were observed for BMI, PSA, and rate of ≥ T3a tumors on pathology. Rate of GG ≥ 2, GG ≥ 3, and GG ≥ 4 showed a significant pattern of increase by PRS quartile (p < 0.001, < 0.001, and 0.039, respectively). With the lowest PRS quartile as reference, higher PRS groups showed sequentially escalating risk for GG ≥ 2 and GG ≥ 3 pathology, with a 4.6-fold rise in GG ≥ 2 (p < 0.001) and 2.0-fold rise in GG ≥ 3 (p < 0.001) for the highest PRS quartiles. Combining PRS with PSA improved prediction of early onset csPCa (AUC 0.759) compared to PRS (AUC 0.627) and PSA alone (AUC 0.736). To conclude, an ethnic-specific PRS was found to predict susceptibility of aggressive PCa in addition to improving detection of csPCa when combined with PSA in early onset populations. PRS may have a role as a risk-stratification model in actual practice. Large scale, multi-ethnic trials are required to validate our results.

Potential Functional Role of Phenethylamine Derivatives in Inhibiting Dopamine Reuptake: Structure-Activity Relationship.

Numerous psychotropic and addictive substances possess structural features similar to those of ß-phenethylamine (ß-PEA). In this study, we selected 29 ß-PEA derivatives and determined their structure-activity relationship (SAR) to their ability to inhibit dopamine (DA) reuptake; conducted docking simulation for two selected compounds; and identified their potential functionals. The compounds were subdivided into arylethylamines, 2-(alkyl amino)-1-arylalkan-1-one derivatives and alkyl 2-phenyl-2-(piperidin-2-yl)acetate derivatives. An aromatic group, alkyl group, and alkylamine derivative were attached to the arylethylamine and 2-(alkyl amino)-1-arylalkan-1-one derivatives. The inhibitory effect of the compounds on dopamine reuptake increased in the order of the compounds substituted with phenyl, thiophenyl, and substituted phenyl groups in the aromatic position; compounds with longer alkyl groups and smaller ring-sized compounds at the alkylamine position showed stronger inhibitory activities. Docking simulation conducted for two compounds, 9 and 28, showed that the (S)-form of compound 9 was more stable than the (R)-form, with a good fit into the binding site covered by helices 1, 3, and 6 of human dopamine transporter (hDAT). In contrast, the (R, S)-configuration of compound 28 was more stable than that of other isomers and was firmly placed in the binding pocket of DAT bound to DA. DA-induced endocytosis of dopamine D2 receptors was inhibited when they were co-expressed with DAT, which lowered extracellular DA levels, and uninhibited when they were pretreated with compound 9 or 28. In summary, this study revealed critical structural features responsible for the inhibition of DA reuptake and the functional role of DA reuptake inhibitors in regulating D2 receptor function.

Integrative epigenomic and transcriptomic analyses reveal metabolic switching by intermittent fasting in brain.

Intermittent fasting (IF) remains the most effective intervention to achieve robust anti-aging effects and attenuation of age-related diseases in various species. Epigenetic modifications mediate the biological effects of several environmental factors on gene expression; however, no information is available on the effects of IF on the epigenome. Here, we first found that IF for 3 months caused modulation of H3K9 trimethylation (H3K9me3) in the cerebellum, which in turn orchestrated a plethora of transcriptomic changes involved in robust metabolic switching processes commonly observed during IF. Second, a portion of both the epigenomic and transcriptomic modulations induced by IF was remarkably preserved for at least 3 months post-IF refeeding, indicating that memory of IF-induced epigenetic changes was maintained. Notably, though, we found that termination of IF resulted in a loss of H3K9me3 regulation of the transcriptome. Collectively, our study characterizes the novel effects of IF on the epigenetic-transcriptomic axis, which controls myriad metabolic processes. The comprehensive analyses undertaken in this study reveal a molecular framework for understanding how IF impacts the metabolo-epigenetic axis of the brain and will serve as a valuable resource for future research.

Synthesis of DPIE [2-(1,2-Diphenyl-1H-indol-3-yl)ethanamine] Derivatives and Their Regulatory Effects on Pro-Inflammatory Cytokine Production in IL-1ß-Stimulated Primary Human Oral Cells.

Interleukin-1 beta (IL-1ß) has diverse physiological functions and plays important roles in health and disease. In this report, we focus on its function in the production of pro-inflammatory cytokines, including IL-6 and IL-8, which are implicated in several autoimmune diseases and host defense against infection. IL-1ß activity is markedly dependent on the binding affinity toward IL-1 receptors (IL-1Rs). Several studies have been conducted to identify suitable small molecules that can modulate the interactions between 1L-1ß and 1L-1R1. Based on our previous report, where DPIE [2-(1,2-Diphenyl-1H-indol-3-yl)ethanamine] exhibited such modulatory activity, three types of DPIE derivatives were synthesized by introducing various substituents at the 1, 2, and 3 positions of the indole group in DPIE. To predict a possible binding pose in complex with IL-1R1, a docking simulation was performed. The effect of the chemicals was determined in human gingival fibroblasts (GFs) following IL-1ß induction. The DPIE derivatives affected different aspects of cytokine production. Further, a group of the derivatives enabled synergistic pro-inflammatory cytokine production, while another group caused diminished cytokine production compared to DPIE stimulation. Some groups displayed no significant difference after stimulation. These findings indicate that the modification of the indole site could modulate IL-1ß:IL1R1 binding affinity to reduce or enhance pro-inflammatory cytokine production.

Neferine increases sensitivities to multiple anticancer drugs via downregulation of Bcl-2 expression in renal cancer cells.

BACKGROUND: Neferine is the major alkaloid extracted from a seed embryo of Nelumbo nucifera and shows cytotoxic effects in various human cancer cells. However, no detailed studies have been reported on its antitumor efficacy of a combinational treatment in human renal cancer cells. OBJECTIVE: This study evaluated the antitumor effects of a combination therapy of neferine and various drugs on renal cancer Caki-1 cells. METHODS: Flow cytometry analysis was performed to evaluate the cell cycle analysis and apoptosis, respectively. Western blotting and reverse transcription polymerase chain reaction were performed to analyze the effect of neferine on the expression of apoptosis-related genes in Caki-1 cells. In addition, reactive oxygen species (ROS) generation was evaluated using flow cytometry. RESULTS: Treatment with neferine dose-dependently induces apoptosis and Bcl-2 downregulation in Caki-1 cells. In addition, neferine triggers cell cycle arrest at the G2/M phase in Caki-1 cells. The neferine-induced apoptosis was mediated by ROS generation, and neferine-facilitated Bcl-2 downregulation was regulated at the transcriptional level through the suppression of p65 expression, resulting in inactivation of the NF-κB pathway in Caki-1 cells. The ROS scavenger, N-acetyl-l-cysteine (NAC), intensely reversed the effects of neferine on apoptosis and Bcl-2 downregulation. We determined that neferine markedly potentiates the antitumor effects of multiple anticancer drugs (cisplatin, silybin, and thapsigargin), and those effects can be reversed by Bcl-2 overexpression or NAC pretreatment in Caki-1 cells. CONCLUSION: These results suggest that neferine can increase chemosensitivities to anticancer drugs via downregulation of Bcl-2 expression through ROS-dependent suppression of the NF-κB signaling pathway in human renal cancer cells.

Engineered small extracellular vesicles displaying ACE2 variants on the surface protect against SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry is mediated by the interaction of the viral spike (S) protein with angiotensin-converting enzyme 2 (ACE2) on the host cell surface. Although a clinical trial testing soluble ACE2 (sACE2) for COVID-19 is currently ongoing, our understanding of the delivery of sACE2 via small extracellular vesicles (sEVs) is still rudimentary. With excellent biocompatibility allowing for the effective delivery of molecular cargos, sEVs are broadly studied as nanoscale protein carriers. In order to exploit the potential of sEVs, we design truncated CD9 scaffolds to display sACE2 on the sEV surface as a decoy receptor for the S protein of SARS-CoV-2. Moreover, to enhance the sACE2-S binding interaction, we employ sACE2 variants. sACE2-loaded sEVs exhibit typical sEVs characteristics and bind to the S protein. Furthermore, engineered sEVs inhibit the entry of wild-type (WT), the globally dominant D614G variant, Beta (K417N-E484K-N501Y) variant, and Delta (L452R-T478K-D614G) variant SARS-CoV-2 pseudovirus, and protect against authentic SARS-CoV-2 and Delta variant infection. Of note, sACE2 variants harbouring sEVs show superior antiviral efficacy than WT sACE2 loaded sEVs. Therapeutic efficacy of the engineered sEVs against SARS-CoV-2 challenge was confirmed using K18-hACE2 mice. The current findings provide opportunities for the development of new sEVs-based antiviral therapeutics.