BTNL2 promotes colitis-associated tumorigenesis in mice by regulating IL-22 production.

Interleukin 22 (IL-22) has an important role in colorectal tumorigenesis and many colorectal diseases such as inflammatory bowel disease and certain infections. However, the regulation of IL-22 production in the intestinal system is still unclear. Here, we present evidence that butyrophilin-like protein 2 (BTNL2) is required for colorectal IL-22 production, and BTNL2 knockout mice show decreased colonic tumorigenesis and more severe colitis phenotypes than control mice due to defective production of IL-22. Mechanistically, BTNL2 acts on group 3 innate lymphoid cells (ILC3s), CD4+ T cells, and γδ T cells to promote the production of IL-22. Importantly, we find that a monoclonal antibody against BTNL2 attenuates colorectal tumorigenesis in mice and that the mBTNL2-Fc recombinant protein has a therapeutic effect in a dextran sulfate sodium (DSS)-induced colitis model. This study not only identifies a regulatory mechanism of IL-22 production in the colorectal system but also provides a potential therapeutic target for the treatment of human colorectal cancer and inflammatory bowel diseases.

pH and ROS Dual-Sensitive Nanocarriers for the Targeted Co-Delivery and On-Demand Sequential Release of Tofacitinib and Glucosamine for Synergistic Rheumatoid Arthritis Treatment.

Rheumatoid arthritis (RA) progression involves multiple cell types, and sequential drug action on target cells is necessary for RA treatment. Nanocarriers are widely used for RA treatment; however, the targeted delivery and on-demand release of multiple drugs remains challenging. Therefore, in this study, a dual-sensitive polymer is developed using chondroitin sulfate (CS) for the co-delivery of the cartilage repair agent, glucosamine (GlcN), and anti-inflammatory drug, tofacitinib (Tof). In the joint cavity, acidic pH facilitates the cleavage of GlcN from CS polymer to repair the cartilage damage. Subsequently, macrophage uptake via CS-CD44 binding and intracellular reactive oxygen species (ROS) mediate conversion of (methylsulfanyl)propylamine to a hydrophilic segment jointly triggered rapid Tof/GlcN release via micelle disassembly. The combined effects of Tof, GlcN, and ROS depletion promote the M1-to-M2 polarization shift to attenuate inflammation. The synergistic effects of these agents against RA are confirmed in vitro and in vivo. Overall, the dual pH/ROS-sensitive CS nanoplatform simultaneously delivers GlcN and Tof, providing a multifunctional approach for RA treatment with synergistic drug effects.

A real-time deep learning-based system for colorectal polyp size estimation by white-light endoscopy: development and multicenter prospective validation.

BACKGROUND: The choice of polypectomy device and surveillance intervals for colorectal polyps are primarily decided by polyp size. We developed a deep learning-based system (ENDOANGEL-CPS) to estimate colorectal polyp size in real time. METHODS: ENDOANGEL-CPS calculates polyp size by estimating the distance from the endoscope lens to the polyp using the parameters of the lens. The depth estimator network was developed on 7297 images from five virtually produced colon videos and tested on 730 images from seven virtual colon videos. The performance of the system was first evaluated in nine videos of a simulated colon with polyps attached, then tested in 157 real-world prospective videos from three hospitals, with the outcomes compared with that of nine endoscopists over 69 videos. Inappropriate surveillance recommendations caused by incorrect estimation of polyp size were also analyzed. RESULTS: The relative error of depth estimation was 11.3% (SD 6.0%) in successive virtual colon images. The concordance correlation coefficients (CCCs) between system estimation and ground truth were 0.89 and 0.93 in images of a simulated colon and multicenter videos of 157 polyps. The mean CCC of ENDOANGEL-CPS surpassed all endoscopists (0.89 vs. 0.41 [SD 0.29]; P<0.001). The relative accuracy of ENDOANGEL-CPS was significantly higher than that of endoscopists (89.9% vs. 54.7%; P<0.001). Regarding inappropriate surveillance recommendations, the system's error rate is also lower than that of endoscopists (1.5% vs. 16.6%; P<0.001). CONCLUSIONS: ENDOANGEL-CPS could potentially improve the accuracy of colorectal polyp size measurements and size-based surveillance intervals.

H2S-driven chemotherapy and mild photothermal therapy induced mitochondrial reprogramming to promote cuproptosis.

The elevated level of hydrogen sulfide (H2S) in colon cancer hinders complete cure with a single therapy. However, excessive H2S also offers a treatment target. A multifunctional cascade bioreactor based on the H2S-responsive mesoporous Cu2Cl(OH)3-loaded hypoxic prodrug tirapazamine (TPZ), in which the outer layer was coated with hyaluronic acid (HA) to form TPZ@Cu2Cl(OH)3-HA (TCuH) nanoparticles (NPs), demonstrated a synergistic antitumor effect through combining the H2S-driven cuproptosis and mild photothermal therapy. The HA coating endowed the NPs with targeting delivery to enhance drug accumulation in the tumor tissue. The presence of both the high level of H2S and the near-infrared II (NIR II) irradiation achieved the in situ generation of photothermic agent copper sulfide (Cu9S8) from the TCuH, followed with the release of TPZ. The depletion of H2S stimulated consumption of oxygen, resulting in hypoxic state and mitochondrial reprogramming. The hypoxic state activated prodrug TPZ to activated TPZ (TPZ-ed) for chemotherapy in turn. Furthermore, the exacerbated hypoxia inhibited the synthesis of adenosine triphosphate, decreasing expression of heat shock proteins and subsequently improving the photothermal therapy. The enriched Cu2+ induced not only cuproptosis by promoting lipoacylated dihydrolipoamide S-acetyltransferase (DLAT) heteromerization but also performed chemodynamic therapy though catalyzing H2O2 to produce highly toxic hydroxyl radicals ·OH. Therefore, the nanoparticles TCuH offer a versatile platform to exert copper-related synergistic antitumor therapy.

A deep-learning based system using multi-modal data for diagnosing gastric neoplasms in real-time (with video).

BACKGROUND: White light (WL) and weak-magnifying (WM) endoscopy are both important methods for diagnosing gastric neoplasms. This study constructed a deep-learning system named ENDOANGEL-MM (multi-modal) aimed at real-time diagnosing gastric neoplasms using WL and WM data. METHODS: WL and WM images of a same lesion were combined into image-pairs. A total of 4201 images, 7436 image-pairs, and 162 videos were used for model construction and validation. Models 1-5 including two single-modal models (WL, WM) and three multi-modal models (data fusion on task-level, feature-level, and input-level) were constructed. The models were tested on three levels including images, videos, and prospective patients. The best model was selected for constructing ENDOANGEL-MM. We compared the performance between the models and endoscopists and conducted a diagnostic study to explore the ENDOANGEL-MM's assistance ability. RESULTS: Model 4 (ENDOANGEL-MM) showed the best performance among five models. Model 2 performed better in single-modal models. The accuracy of ENDOANGEL-MM was higher than that of Model 2 in still images, real-time videos, and prospective patients. (86.54 vs 78.85%, P = 0.134; 90.00 vs 85.00%, P = 0.179; 93.55 vs 70.97%, P < 0.001). Model 2 and ENDOANGEL-MM outperformed endoscopists on WM data (85.00 vs 71.67%, P = 0.002) and multi-modal data (90.00 vs 76.17%, P = 0.002), significantly. With the assistance of ENDOANGEL-MM, the accuracy of non-experts improved significantly (85.75 vs 70.75%, P = 0.020), and performed no significant difference from experts (85.75 vs 89.00%, P = 0.159). CONCLUSIONS: The multi-modal model constructed by feature-level fusion showed the best performance. ENDOANGEL-MM identified gastric neoplasms with good accuracy and has a potential role in real-clinic.

Unprotonatable and ROS-Sensitive Nanocarrier for NIR Spatially Activated siRNA Therapy with Synergistic Drug Effect.

Although small interfering RNA (siRNA) therapy has achieved great progress, unwanted gene inhibition in normal tissues severely limits its extensive clinical applications due to uncontrolled siRNA biodistribution. Herein, a spatially controlled siRNA activation strategy is developed to achieve tumor-specific siRNA therapy without gene inhibition in the normal tissues. The quaternary ammonium moieties are conjugated to amphiphilic copolymers via reactive oxygen species (ROS)-sensitive thioketal (TK) linkers for co-delivery of siRNA and photosensitizer chlorin e6 (Ce6), showing excellent siRNA complexation capacity and near infrared (NIR)-controlled siRNA release. In the normal tissue, siRNAs are trapped and degraded in the endo-lysosomes due to the unprotonatable property of quaternary ammonium moiety, showing the siRNA activity "off" state. When NIR irradiation is spatially applied to the tumor tissue, the NIR irradiation/Ce6-induced ROS trigger siRNA endo-lysosomal escape and cytosolic release through the photochemical internalization effect and cleavage of TK bonds, respectively, showing the siRNA activity "on" state. The siRNA-mediated glutathione peroxidase 4 gene inhibition enhances ROS accumulation. The synergistic antitumor activity of Ce6 photodynamic therapy and gene inhibition is confirmed in vivo. Spatially controlled tumor-specific siRNA activation and co-delivery with Ce6 using unprotonatable and ROS-sensitive cationic nanocarriers provide a feasible strategy for tumor-specific siRNA therapy with synergistic drug effects.

Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide.

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

Size- and Surface- Dual Engineered Small Polyplexes for Efficiently Targeting Delivery of siRNA.

Though siRNA-based therapy has achieved great progress, efficient siRNA delivery remains a challenge. Here, we synthesized a copolymer PAsp(-N=C-PEG)-PCys-PAsp(DETA) consisting of a poly(aspartate) block grafted with comb-like PEG side chains via a pH-sensitive imine bond (PAsp(-N=C-PEG) block), a poly(l-cysteine) block with a thiol group (PCys block), and a cationic poly(aspartate) block grafted with diethylenetriamine (PAsp(DETA) block). The cationic polymers efficiently complexed siRNA into polyplexes, showing a sandwich-like structure with a PAsp(-N=C-PEG) out-layer, a crosslinked PCys interlayer, and a complexing core of siRNA and PAsp(DETA). Low pH-triggered breakage of pH-sensitive imine bonds caused PEG shedding. The disulfide bond-crosslinking and pH-triggered PEG shedding synergistically decreased the polyplexes' size from 75 nm to 26 nm. To neutralize excessive positive charges and introduce the targeting ligand, the polyplexes without a PEG layer were coated with an anionic copolymer modified with the targeting ligand lauric acid. The resulting polyplexes exhibited high transfection efficiency and lysosomal escape capacity. This study provides a promising strategy to engineer the size and surface of polyplexes, allowing long blood circulation and targeted delivery of siRNA.

Fiber-type distribution and expression of myosin heavy chain isoforms in newborn heterozygous myostatin-knockout pigs.

OBJECTIVES: To explore the effects of heterozygous myostatin-knockout (MSNT+/-) on muscle characteristics, specifically fiber-type distribution and expression of myosin heavy chain isoforms in pigs. RESULTS: The fiber cross-sectional area of the semitendinosus and semimembranosus muscles were much larger in MSTN+/- pigs at birth than in wild-type (WT) pigs. MSTN+/- pigs had a higher proportion of fast-type fibers and lower succinate dehydrogenase activity in muscles than WT pigs. The myosin heavy chain IIB mRNA level in both two muscles was ~ threefold higher in MSTN+/- pigs compared with WT pigs. CONCLUSION: MSTN+/- pigs exhibit a disproportionate increase in muscle mass and can have a higher body weight due to fiber hypertrophy, a change in the fiber-type distribution, and alteration of myosin heavy chain isoforms levels, leading to more fast glycolytic fibers.

RepSox improves viability and regulates gene expression in rhesus monkey-pig interspecies cloned embryos.

OBJECTIVE: To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey-pig interspecies somatic cell nuclear transfer (iSCNT) embryos. RESULTS: Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p < 0.05), but not of Sox2 compared with untreated embryos at the 2-4-cell stage. Expression of the anti-apoptotic gene, Bcl2, and the pro-apoptotic gene Bax was also affected at the 2-4-cell stage. RepSox treatment also increased the immunostaining intensity of Oct4 at the blastocyst stage (p < 0.05). Although the blastocyst developmental rate was higher in the group treated with 25 µM RepSox for 24 h than in the untreated control group (2.4 vs. 1.2%, p > 0.05), this was not significant. CONCLUSION: RepSox can improve the developmental potential of rhesus monkey-pig iSCNT embryos by regulating the expression of pluripotency-related genes.

The developmental competence of oocytes parthenogenetically activated by an electric pulse and anisomycin treatment.

OBJECTIVE: The aim of this study was to investigate the developmental competence of oocytes parthenogenetically activated by an electric pulse (EP) and treated with anisomycin and to determine whether this method is applicable to somatic cell nuclear transfer (SCNT). RESULTS: Embryos derived from porcine oocytes parthenogenetically activated by an EP and treatment with 0.01 µg/mL anisomycin had a significantly improved in vitro developmental capacity. Furthermore, 66.6% of blastocysts derived from these embryos had a diploid karyotype. The blastocyst formation rate of cloned embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 0.01 µg/mL anisomycin for 4 h. The level of maturation-promoting factor was significantly decreased in oocytes activated by an EP and treated with anisomycin. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR. CONCLUSION: Our results demonstrate that porcine oocyte activation via an EP in combination with anisomycin treatment can lead to a high blastocyst formation rate in parthenogenetic activation and SCNT experiments.

Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment.

We examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

Functional Photoacoustic Imaging of Gastric Acid Secretion Using pH-Responsive Polyaniline Nanoprobes.

A stomach functional imaging technique based on photoacoustics achieves noninvasive gastric acid secretory assessment utilizing pH-responsive polyaniline nanoprobes. A testing protocol mimicking clinical practice is established using a mouse model. After imaging, the nanoprobes are excreted outside the body without inducing systematic toxicity. Further optimization and translation of this technology can help alleviate patients' suffering and side effects.

Therapeutic targeting of GSK3ß enhances the Nrf2 antioxidant response and confers hepatic cytoprotection in hepatitis C.

OBJECTIVE: Impaired adaptive response to oxidative injuries is a fundamental mechanism central to the pathogenesis of chronic hepatitis C (CHC). Glycogen synthase kinase (GSK) 3ß is an indispensable regulator of the oxidative stress response. However, the exact role of GSK3ß in CHC is uncertain and was examined. DESIGN: GSK3ß and Nrf2 signalling pathways were examined in JFH1 HCV infected Huh7.5.1 hepatocytes, and also in liver biopsy specimens from CHC patients. RESULTS: HCV infection elicited prominent Nrf2 antioxidant response in hepatocytes, marked by elevated expression of the Nrf2-dependent molecule haem oxygenase-1 and subsequent protection from apoptotic cell death. Inhibitory phosphorylation of GSK3ß seems to be essential and sufficient for HCV-induced Nrf2 response. Mechanistically, GSK3ß associated and physically interacted with Nrf2 in hepatocytes. In silico analysis revealed that Nrf2 encompasses multiple GSK3ß phosphorylation consensus motifs, denoting Nrf2 as a cognate substrate of GSK3ß. In the presence of TGFß1, the HCV-induced GSK3ß phosphorylation was blunted via a protein phosphatase 1-dependent mechanism and the cytoprotective Nrf2 response drastically impaired. This effect was counteracted by lithium, a selective inhibitor of GSK3ß. In liver biopsy specimens from CHC patients, the expression of phosphorylated GSK3ß positively correlated with Nrf2 expression and was inversely associated with the degree of liver injury. Moreover, CHC patients who received long-term lithium carbonate therapy primarily for concomitant psychiatric disorders exhibited much less liver injury, associated with enhanced hepatic expression of Nrf2. CONCLUSIONS: Inhibition of GSK3ß exerts hepatoprotection in CHC possibly through its direct regulation of Nrf2 antioxidant response.

Enhancing extraction of light from metal composite structures for plasmonic emitters using light-coupling effect.

This work demonstrates the efficiency and directionality of a method of extracting light from thin-film emissive devices by near-field evanescent waves in plasmonic emitters used in metal composite grating structures. A near-field evanescent wave can induce a surface plasmon wave on the surface of a metal under resonant conditions. Enhancing the near-field evanescent wave generates strong far-field nonlinear optical effects. This effect is highly efficient in some plasmonic emitter structures. Theoretical and experimental results demonstrate that such a metal composite grating structure exhibits good performance, a high coupling ratio, a small coupling angle, enhanced light extraction and a small FWHM. It also improves luminous efficiency, emitter angle, and directivity.

Hepatitis C virus NS5A drives a PTEN-PI3K/Akt feedback loop to support cell survival.

BACKGROUND & AIMS: Decreased levels of phosphatase and tensin homologue (PTEN) are associated with hepatocellular carcinoma (HCC) pathogenesis and poor prognosis in hepatitis C virus (HCV)-infected HCC patients. The molecular processes governing the reduction in PTEN and outcome of PTEN dysfunction in hepatocytes are poorly understood. METHODS: The levels of proteins and mRNA were assessed by real time PCR and immunoblot. PTEN promoter activity was measured by reporter assay. Signalling pathways were perturbed using siRNAs or pharmacological inhibitors. RESULTS: Here, we report that HCV down-regulates PTEN expression at the transcriptional level by decreasing its promoter activity, mRNA transcription, and protein levels. We further identify NS5A protein as a key determinant of PTEN reduction among HCV proteins. NS5A-mediated down-regulation of PTEN occurs through a cooperation of reactive oxygen species (ROS)-dependent Nuclear Factor- kappa B (NF-κB) and ROS-independent phosphoinositol-3-kinase (PI3K) pathways. Moreover, NS5A protects cells against apoptosis. In addition, we found that down-regulation of PTEN relieves its inhibitory effect on PI3K-Akt pathway and triggers cumulative activation of Akt. This PTEN-PI3K/Akt feedback network mediates the suppression of cell apoptosis caused by NS5A. CONCLUSIONS: These data demonstrate that HCV NS5A down-regulates PTEN expression through a cooperation of ROS-dependent and -independent pathways that subsequently drives a PTEN-PI3K/Akt feedback loop to support cell survival. Our findings provide new insights suggesting that NS5A contributes to HCV-related hepatocarcinogenesis.

Production of Cloned Wuzhishan Miniature Pigs and Application for Alloxan Toxicity Test.

Wuzhishan miniature pig is one of the four most important pig breeds in China and has many major economic characteristics. Herein, we successfully used SCNT to clone Wuzhishan miniature pig. First, ear fibroblasts were isolated from a 2-year-old female Wuzhishan miniature piglet to be used as the donor cell. Second, good-quality COCs were selected from ovaries obtained from pigs at a local slaughterhouse and cultured. Mature eggs with the first polar body and ear fibroblasts were applied SCNT. Lastly, we in total produced 12 piglets with 7 piglets surviving to adults. Next, we used these pigs to test alloxan toxicity and to build T I D diabetes type. We know that diabetes mellitus is a chronic heterogeneous metabolic disease characterized by a high blood glucose level and abnormal insulin secretion. In this study, T I D (type I diabetes) was experimentally induced in cloned Wuzhishan miniature pigs with alloxan. In brief, an intravenous injection of alloxan (group B: 170 mg/kg, n = 3) was administered to pigs weighing between 27 and 39 kg. Sterile saline was administered to control pigs (n = 3). We determined the glycometabolism related index, performed an intravenous glucose tolerance test, and carried out immunohistochemistry experiments. There were no significant differences in body weight, blood glucose, and serum insulin in all groups, before treatment. The level of blood glucose was significantly higher (P < 0.05) in group B (12.18 ± 0.70 mmol/L) than in the control (2.93 ± 0.39 mmol/L). By contrast, the level of serum insulin was lower in group B (5.641 ± 0.573 µIU/mL) than in the control (7.578 ± 0.539 µIU/mL). Histological studies by hematoxylin and eosin (H&E) revealed a loss of ß-cells in the pancreas from pigs treated with 170 mg/kg alloxan. Immunolocalization studies showed a decrease in insulin reactivity in this treatment group as well. To conclude, our model holds promise in future studies of diabetes drug testing and islet xenotransplantation.

Co-delivery of doxorubicin and siRNA with reduction and pH dually sensitive nanocarrier for synergistic cancer therapy.

Drug resistance is the greatest challenge in clinical cancer chemotherapy. Co-delivery of chemotherapeutic drugs and siRNA to tumor cells is a vital means to silence drug resistant genes during the course of cancer chemotherapy for an improved chemotherapeutic effect. This study aims at effective co-delivery of siRNA and anticancer drugs to tumor cells. A ternary block copolymer PEG-PAsp(AED)-PDPA consisting of pH-sensitive poly(2-(diisopropyl amino)ethyl methacrylate) (PDPA), reduction-sensitive poly(N-(2,2'-dithiobis(ethylamine)) aspartamide) PAsp(AED), and poly(ethylene glycol) (PEG) is synthesized and assembled into a core-shell structural micelle which encapsulated doxorubicin (DOX) in its pH-sensitive core and the siRNA-targeting anti-apoptosis BCL-2 gene (BCL-2 siRNA) in a reduction-sensitive interlayer. At the optimized size and zeta potential, the nanocarriers loaded with DOX and BCL-2 siRNA may effectively accumulate in the tumor site via blood circulation. Moreover, the dual stimuli-responsive design of micellar carriers allows microenviroment-specific rapid release of both DOX and BCL-2 siRNA inside acidic lysosomes with enriched reducing agent, glutathione (GSH, up to 10 mM). Consequently, the expression of anti-apoptotic BCL-2 protein induced by DOX treatment is significantly down-regulated, which results in synergistically enhanced apoptosis of human ovarian cancer SKOV-3 cells and thus dramatically inhibited tumor growth.

Downregulation of Wnt causes root resorption.

INTRODUCTION: There are multiple causes of external root resorption, but absent a disease state, it is most often observed when excessive physical force is used during orthodontic treatment. Even without mechanical stimulation, however, root resorption can still occur. The purpose of this study was to test whether Wnt signaling plays a role in pathologic root resorption, by conditionally deleting Wntless (Wls) from odontoblasts and osteoblasts and then evaluating the phenotypic effects on the maintenance of the root surface. METHODS: Ten (age, 1 month) and 20 (age, 3 months) OCN-Cre;Wls(fl/fl) mice and their wild-type littermates were evaluated using microcomputed tomography, histology, and immunohistochemistry. Phenotypic alterations in the alveolar bone, dentin, and cementum were characterized and quantified. RESULTS: In a genetic model of reduced Wnt signaling, we found that RANKL expression is upregulated, and osteoprotegerin expression is downregulated. This molecular disruption results in an increase in osteoclast activity, a decrease in osteoblast activity, and extensive, spontaneous root resorption. A genetic strain of mice in which Wnt signaling is elevated exhibits thicker cementum, whereas, even in the perinatal period, OCN-Cre;Wls(fl/fl) mice exhibit thinner cementum. CONCLUSIONS: Taken together, these data demonstrate that Wnts regulate cementum homeostasis, and that idiopathic cases of root resorption might have as their etiology a reduction in endogenous Wnt signaling.

Functionalized extracellular nanovesicles as advanced CRISPR delivery systems.

The clustered regularly interspaced short palindromic repeat (CRISPR) system, an emerging tool for genome editing, has garnered significant public interest for its potential in treating genetic diseases. Despite the rapid advancements in CRISPR technology, the progress in developing effective delivery strategies lags, impeding its clinical application. Extracellular nanovesicles (EVs), either in their endogenous forms or with engineered modifications, have emerged as a promising solution for CRISPR delivery. These EVs offer several advantages, including high biocompatibility, biological permeability, negligible immunogenicity, and straightforward production. Herein, we first summarize various types of functional EVs for CRISPR delivery, such as unmodified, modified, engineered virus-like particles (VLPs), and exosome-liposome hybrid vesicles, and examine their distinct intracellular pathways. Then, we outline the cutting-edge techniques for functionalizing extracellular vesicles, involving producer cell engineering, vesicle engineering, and virus-like particle engineering, emphasizing the diverse CRISPR delivery capabilities of these nanovesicles. Lastly, we address the current challenges and propose rational design strategies for their clinical translation, offering future perspectives on the development of functionalized EVs.