Identifying the protective effects of miR-874-3p/ATF3 axis in intervertebral disc degeneration by single-cell RNA sequencing and validation.

Intervertebral disc degeneration (IVDD) severely affects the work and the quality of life of people. We previously demonstrated that silencing activation transcription factor 3 (ATF3) blocked the IVDD pathological process by regulating nucleus pulposus cell (NPC) ferroptosis, apoptosis, inflammation, and extracellular matrix (ECM) metabolism. Nevertheless, whether miR-874-3p mediated the IVDD pathological process by targeting ATF3 remains unclear. We performed single-cell RNA sequencing (scRNA-seq) and bioinformatics analysis to identify ATF3 as a key ferroptosis gene in IVDD. Then, Western blotting, flow cytometry, ELISA, and animal experiments were performed to validate the roles and regulatory mechanisms of miR-874-3p/ATF3 signalling axis in IVDD. ATF3 was highly expressed in IVDD patients and multiple cell types of IVDD rat, as revealed by scRNA-seq and bioinformatics analysis. GO analysis unveiled the involvement of ATF3 in regulating cell apoptosis and ECM metabolism. Furthermore, we verified that miR-874-3p might protect against IVDD by inhibiting NPC ferroptosis, apoptosis, ECM degradation, and inflammatory response by targeting ATF3. In vivo experiments displayed the protective effect of miR-874-3p/ATF3 axis on IVDD. These findings propose the potential of miR-874-3p and ATF3 as biomarkers of IVDD and suggest that targeting the miR-874-3p/ATF3 axis may be a therapeutic target for IVDD.

A Disconnection for Rapid Access to Heterocyclic Benzylic Amines with Fully Substituted α-Carbons.

2- or 4-Pyridyl benzylic amines represent a privileged motif in drug discovery. However, the formation of heterocyclic benzylic amines with fully substituted α-carbons can require the execution of lengthy synthetic routes, which limit their application. Addition of various nucleophilic agents to Ellman's imines has been well established; however, there is no precedented literature reported for pyridyl-type nucleophiles, which are very important for medicinal chemistry. In this letter, we disclose the development of a one-step synthesis of heterocyclic benzylic amines with fully substituted α-carbons from heteroaryl halides and sulfinyl imines. Starting from 2,4-dibromopyridine, regioselective synthesis of 2- or 4-pyridyl benzylic amines could be achieved by choosing toluene or MTBE as a solvent.

Toward the next generation of vascularized human neural organoids.

Thanks to progress in the development of three-dimensional (3D) culture technologies, human central nervous system (CNS) development and diseases have been gradually deciphered by using organoids derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). Selforganized neural organoids (NOs) have been used to mimic morphogenesis and functions of specific organs in vitro. Many NOs have been reproduced in vitro, such as those mimicking the human brain, retina, and spinal cord. However, NOs fail to capitulate to the maturation and complexity of in vivo neural tissues. The persistent issues with current NO cultivation protocols are inadequate oxygen supply and nutrient diffusion due to the absence of vascular networks. In vivo, the developing CNS is interpenetrated by vasculature that not only supplies oxygen and nutrients but also provides a structural template for neuronal growth. To address these deficiencies, recent studies have begun to couple NO culture with bioengineering techniques and methodologies, including genetic engineering, coculture, multidifferentiation, microfluidics and 3D bioprinting, and transplantation, which might promote NO maturation and create more functional NOs. These cutting-edge methods could generate an ever more reliable NO model in vitro for deciphering the codes of human CNS development, disease progression, and translational application. In this review, we will summarize recent technological advances in culture strategies to generate vascularized NOs (vNOs), with a special focus on cerebral- and retinal-organoid models.

Alleviation of Photoreceptor Degeneration Based on Fullerenols in rd1 Mice by Reversing Mitochondrial Dysfunction via Modulation of Mitochondrial DNA Transcription and Leakage.

Poor therapeutic outcomes of antioxidants in ophthalmologic clinical applications, including glutathione during photoreceptor degeneration in retinitis pigmentosa (RP), are caused by limited anti-oxidative capacity. In this study, fullerenols are synthesized and proven to be highly efficient in vitro radical scavengers. Fullerenol-based intravitreal injections significantly improve the flash electroretinogram and light/dark transition tests performed for 28 days on rd1 mice, reduce the thinning of retinal outer nuclear layers, and preserve the Rhodopsin, Gnat-1, and Arrestin expressions of photoreceptors. RNA-sequencing, RT-qPCR, and Western blotting validate that mitochondrial DNA (mt-DNA)-encoded genes of the electron transport chain (ETC), such as mt-Nd4l, mt-Co1, mt-Cytb, and mt-Atp6, are drastically downregulated in the retinas of rd1 mice, whereas nuclear DNA (n-DNA)-encoded genes, such as Ndufa1 and Atp5g3, are abnormally upregulated. Fullerenols thoroughly reverse the abnormal mt-DNA and n-DNA expression patterns of the ETC and restore mitochondrial function in degenerating photoreceptors. Additionally, fullerenols simultaneously repress Flap endonuclease 1 (FEN1)-mediated mt-DNA cleavage and mt-DNA leakage via voltage-dependent anion channel (VDAC) pores by downregulating the transcription of Fen1 and Vdac1, thereby inactivating the downstream pro-inflammatory cGAS-STING pathway. These findings demonstrate that fullerenols can effectively alleviate photoreceptor degeneration in rd1 mice and serve as a viable treatment for RP.

Application of self-anchored lateral lumbar interbody fusion in lumbar degenerative diseases.

STUDY DESIGN: This is a retrospective study. OBJECTIVE: The aim of the study was to evaluate the efficacy of self-anchored lateral lumbar interbody fusion (SA-LLIF) in lumbar degenerative diseases. METHODS: Forty-eight patients with lumbar degenerative disease between January 2019 and June 2020 were enrolled in this study. All patients complained of low back and leg pain, which were aggravated during standing activities and alleviated or disappeared during lying. After general anesthesia, the patient was placed in the right decubitus position. The anterior edge of the psoas major muscle was exposed through an oblique incision of approximately 6 cm, using an extraperitoneal approach. The psoas major muscle was then properly retracted dorsally to expose the disc. After discectomy, a suitable cage filled with autogenous bone graft from the ilium was implanted. Two anchoring plates were inserted separately into the caudal and cranial vertebral bodies to lock the cage. Clinical efficacy was evaluated using the visual analog scale (VAS) and Oswestry Disability Index (ODI). Lumbar lordosis, intervertebral disc height, spondylolisthesis rate, cage subsidence and fusion rate were also recorded. RESULTS: A total of 48 patients were enrolled in this study, including 20 males and 28 females, aged 61.4 ± 7.3 (range 49-78) years old. Surgery was successfully performed in all patients. Lumbar stenosis and instability were observed in 22 cases, disc degenerative disease in eight cases, degenerative spondylolisthesis in nine cases, degenerative scoliosis in six cases, and postoperative revision in three cases. In addition, five patients were diagnosed with osteoporosis. The index levels included L2-3 in three patients, L3-4 in 13 patients, L4-5 in 23 patients, L2-4 in three patients, and L3-5 in six patients. The operation time was 81.1 ± 6.4 (range 65-102) min. Intraoperative blood loss was 39.9 ± 8.5 (range 15-72) mL. No severe complications occurred, such as nerve or blood vessel injuries. The patients were followed up for 11.7 ± 2.3 (range 4-18) months. At the last follow-up, the VAS decreased from 6.2 ± 2.3 to 1.7 ± 1.1, and the ODI decreased from 48.4% ± 11.2% to 10.9% ± 5.5%. Radiography showed satisfactory postoperative spine alignment. No cage displacement was found, but cage subsidence 2-3 mm was found in five patients without obvious symptoms, except transient low back pain in an obese patient. The lumbar lordosis recovered from 36.8° ± 7.9° to 47.7° ± 6.8°, and intervertebral disc height recovered from 8.2 ± 2.0 mm to 11.4 ± 2.5 mm. The spondylolisthesis rate decreased from 19.9% ± 4.9% to 9.4% ± 3.2%. The difference between preoperative and last follow-up was statistically significant (P<0.05). CONCLUSION: SA-LLIF can provide immediate stability and good results for lumbar degenerative diseases with a standalone anchored cage without posterior internal fixation.

Transplanted OECs Protect Visual Function by Regulating the Glutamate Metabolic Microenvironment in the Glaucoma Model.

BACKGROUND: Glaucoma is the leading cause of irreversible blindness, and the loss of retinal ganglion cells (RGCs) is the most important pathological feature. During the progression of glaucoma, glutamate content in the optic nerve increases, and glutamate-induced excitotoxicity will aggregate the damage and death of RGCs. We have previously reported that olfactory ensheathing cells (OECs) transplantation preserved the visual function of the glaucoma model but the mechanism is unknown. METHODS: Adult Long-Evans rats were used in the present study and injecting magnetic microspheres was used to establish a glaucoma model in rats. Optokinetic response test and Pattern electroretinogram recording were used to assess the visual functions of rats. RT-PCR, immunofluorescence, and co-culture experiments were performed to investigate the therapeutic effects and mechanisms of OECs for glaucoma. RESULTS: In the glaucoma model, increased glutamate content and the damage of astrocytes (AC) and RGCs were observed. OECs transplantation reduced the glutamate concentration in the optic nerve, alleviated the apoptosis of AC and RGCs, and protected the visual function of the glaucoma model. Furthermore, we found that OECs possessed a stronger capacity to metabolize excessive glutamate compared with AC and Müller glia. OECs could improve the glutamate microenvironment of the optic nerve to prevent AC and RGCs from glutamate-induced excitotoxicity in glaucoma. And the recovery of AC function further supported the survival of RGCs. CONCLUSIONS: We demonstrate that OECs transplantation could play a neuroprotective role by regulating the glutamate microenvironment in glaucoma.

Multisensor and Multiscale Data Integration Method of TLS and GPR for Three-Dimensional Detailed Virtual Reconstruction.

Integrated TLS and GPR data can provide multisensor and multiscale spatial data for the comprehensive identification and analysis of surficial and subsurface information, but a reliable systematic methodology associated with data integration of TLS and GPR is still scarce. The aim of this research is to develop a methodology for the data integration of TLS and GPR for detailed, three-dimensional (3D) virtual reconstruction. GPR data and high-precision geographical coordinates at the centimeter level were simultaneously gathered using the GPR system and the Global Navigation Satellite System (GNSS) signal receiver. A time synchronization algorithm was proposed to combine each trace of the GPR data with its position information. In view of the improved propagation model of electromagnetic waves, the GPR data were transformed into dense point clouds in the geodetic coordinate system. Finally, the TLS-based and GPR-derived point clouds were merged into a single point cloud dataset using coordinate transformation. In addition, TLS and GPR (250 MHz and 500 MHz antenna) surveys were conducted in the Litang fault to assess the feasibility and overall accuracy of the proposed methodology. The 3D realistic surface and subsurface geometry of the fault scarp were displayed using the integration data of TLS and GPR. A total of 40 common points between the TLS-based and GPR-derived point clouds were implemented to assess the data fusion accuracy. The difference values in the x and y directions were relatively stable within 2 cm, while the difference values in the z direction had an abrupt fluctuation and the maximum values could be up to 5 cm. The standard deviations (STD) of the common points between the TLS-based and GPR-derived point clouds were 0.9 cm, 0.8 cm, and 2.9 cm. Based on the difference values and the STD in the x, y, and z directions, the field experimental results demonstrate that the GPR-derived point clouds exhibit good consistency with the TLS-based point clouds. Furthermore, this study offers a good future prospect for the integration method of TLS and GPR for comprehensive interpretation and analysis of the surficial and subsurface information in many fields, such as archaeology, urban infrastructure detection, geological investigation, and other fields.

SCF/SCFR signaling plays an important role in the early morphogenesis and neurogenesis of human embryonic neural retina.

The stem cell factor receptor (SCFR) has been demonstrated to be expressed in the neural retina of mice, rat and human for decades. Previous reports indicated that the SCFR correlates with glia differentiation of late retinal progenitor cells (RPCs), retinal vasculogenesis and homeostasis of the blood-retinal barrier. However, the role of SCF/SCFR signaling in the growth and development of the neural retina (NR), especially in the early embryonic stage, remains poorly understood. Here, we show that SCF/SCFR signaling orchestrates invagination of the human embryonic stem cell (hESC)-derived NR via regulation of cell cycle progression, cytoskeleton dynamic and apical constriction of RPCs in the ciliary marginal zone (CMZ). Furthermore, activation of SCF/SCFR signaling promotes neurogenesis in the central-most NR via acceleration of the migration of immature ganglion cells and repressing apoptosis. Our study reveals an unreported role for SCF/SCFR signaling in controlling ciliary marginal cellular behaviors during early morphogenesis and neurogenesis of the human embryonic NR, providing a new potential therapeutic target for human congenital eye diseases such as anophthalmia, microphthalmia and congenital high myopia.

Discovery and pharmacodynamic evaluation of the novel butene lactone derivative M355 against influenza A virus in vitro and in vivo.

A new series of butene lactone derivatives were designed according to an influenza neuraminidase target and their antiviral activities against H1N1 infection of Madin-Darby canine kidney cells were evaluated. Among them, a compound that was given the name M355 was identified as the most potent against H1N1 (EC50 = 14.7 µM) with low toxicity (CC50 = 538.13 µM). It also visibly reduced the virus-induced cytopathic effect. Time-of-addition analysis indicated that H1N1 was mostly suppressed by M355 at the late stage of its infectious cycle. M355 inhibited neuraminidase in a dose-dependent fashion to a similar extent as oseltamivir, which was also indicated by a computer modeling experiment. In a mouse model, lung lesions and virus load were reduced and the expression of nucleoprotein was moderated by M355. The enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction analyses revealed that the levels of interferon-γ, interferon regulatory factor-3, Toll-like receptor-3, tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and IL-8 were downregulated in the M355-treated groups, whereas the levels of IL-10 and IL-13 were upregulated. Similarly, IgG was found to be increased in infected mice plasma. These results demonstrate that M355 inhibit the expression of H1N1 in both cellular and animal models. Thus, M355 has the potential to be effective in the treatment of influenza A virus infection.

Loss of liver X receptor ß in astrocytes leads to anxiety-like behaviors via regulating synaptic transmission in the medial prefrontal cortex in mice.

Astrocytes are integral components of synaptic transmission, and their dysfunction leads to neuropsychiatric disorders such as anxiety and depression. Liver X receptor ß (LXRß) is expressed in astrocytes, and LXRß global knockout mice shows impaired synaptic formation. In order to define the role of LXRß in astrocytes, we used a conditional Cre-loxP system to specifically remove LXRß from astrocytes. We found that this deletion caused anxiety-like but not depressive-like behaviors in adult male mice. This behavioral phenotype could be completely reproduced by selective deletion of LXRß in astrocytes in the medial prefrontal cortex (mPFC). Pyramidal neurons in layer V of mPFC are involved in mood behaviors. We found that there was an increased spontaneous excitatory synaptic transmission in layer V pyramidal neurons of the mPFC of these mice. This was concurrent with increased dendritic complexity, despite normal appearance and number of dendritic spines. In addition, gene ontology analysis of RNA sequencing revealed that deletion of astrocytic LXRß led to the enrichment of the process of synaptic transmission in mPFC. Finally, we also confirmed that renormalized excitatory synaptic transmission in layer V pyramidal neurons alleviated the anxiety in mice with astrocytic LXRß deletion in mPFC. Together, our findings reveal that astrocytic LXRß in mPFC is critical in the regulation of synaptic transmission, and this provides a potential new target for treatment of anxiety-like behavior.

Circ_0008365 Suppresses Apoptosis, Inflammation and Extracellular Matrix Degradation of IL-1ß-treated Chondrocytes in Osteoarthritis by Regulating miR-324-5p/BMPR2/NF-κB Signaling Axis.

BACKGROUND: Recent studies have revealed that circular RNAs (circRNAs) play crucial roles in the progression of osteoarthritis (OA). This study aimed to investigate the biological function and regulatory mechanism of circ_0008365 in OA. METHODS: OA cell model in vitro was established in chondrocytes by treatment with Interleukin-1ß (IL-1ß). The levels of inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The expression levels of circ_0008365, microRNA-324-5p (miR-324-5p) and bone morphogenetic protein type 2 receptor (BMPR2) were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was assessed using flow cytometry and caspase3 activity assays. The protein expression was determined via a western blot assay. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) and RNA pull-down assays were used to analyze the correlation between targets. RESULTS: IL-1ß level and miR-324-5p expression were increased, while circ_0008365 was downregulated in OA patients. IL-1ß treatment-induced cell apoptosis, inflammation and extracellular matrix (ECM) degradation in chondrocytes. Besides, circ_0008365 overexpression partly relieved IL-1ß-induced cell damage in chondrocytes. Circ_0008365 could interact with miR-324-5p, and BMPR2 was a downstream target of miR-324-5p. Overexpression of miR-324-5p or BMPR2 knockdown partly overturned the inhibiting effect of circ_0008365 on cell damage in IL-1ß-induced chondrocytes. In addition, circ_0008365 inactivated NF-κB pathway via regulating miR-324-5p/BMPR2 axis. CONCLUSION: Circ_0008365 reduced IL-1ß-induced cell damage in chondrocytes via inactivating NF-κB signaling pathway and regulating miR-324-5p/BMPR2 axis.Abbreviations OA: osteoarthritis; BMPR2: bone morphogenetic protein type 2 receptor.

Activatable fluorescence molecular imaging and anti-tumor effects investigation of GSH-sensitive BRD4 ligands.

Overexpression of bromodomain 4 (BRD4) is closely correlated with a variety of human cancers by regulating the histone post-translational modifications, which renders BRD4 a promising target for pharmacological discoveries of novel therapeutic agents for cancer therapy. We herein present the design, chemical synthesis, cellular imaging and biological assessment of a novel tumor-sensitive BRD4 ligand (compound 4) by introducing anticancer BRD4 inhibitor into naphthalimide moiety (fluorescent reporter) via a sulfonamide unit as glutathione (GSH)-specific cleavable linker. Upon reaction with abundant intramolecular GSH in cancer cells or free GSH in aqueous solution (pH = 7.4), sulfonamide cleavage of 4 occurs, leading to the release of BRD4 inhibitor and concomitant fluorescence-on. This activatable fluorescence molecular imaging was demonstrated to preferentially occur in tumor cells. Moreover, towards cancer cell lines MGC-803 cells and THP-1, compound 4 was identified to show better antitumor efficacy than net BRD4 inhibitor. Collectively, this study presents a drug delivery strategy, wherein the drug release can be directly monitored in the cellular content by fluorescence imaging, and provides a valuable compound 4 as a potential antitumor agent. Compound 4 may represent a useful tool for explorative studies of BRD4 inhibition, such as an improved understanding of BRD4 inhibitor release-related information.

The multifaceted roles of peptides in "always-on" near-infrared fluorescent probes for tumor imaging.

Tumor-targeted near-infrared (NIR) fluorescence imaging by using NIR fluorescent probes has attracted extensive attentions in the field of tumor imaging and is becoming an attractive strategy for early cancer diagnosis and surgical guidance for the past few decades. Especially, due to the high affinity and low toxicity, the peptide-based NIR fluorescent probes play an important role in the field of tumor-targeted imaging and therapy. Extensive attempts have been made to develop a set of nontoxic and efficacious "always-on" peptide-based NIR fluorescent probes. In this review, we give a comprehensive analysis of the "always-on" peptide-based NIR fluorescent probes for tumor-targeting for the past 5 years, highlighting the design strategy, chemical synthesis, therapeutical applications, spectroscopic and pharmacological characterization, as well as the multifaceted roles of peptides. A comprehensive understanding of the tumor-targeted, "always-on" peptide-based NIR fluorescent probes will increase our knowledge of cancer diagnosis and benefit clinical cancer therapeutics.

Advanced human developmental toxicity and teratogenicity assessment using human organoid models.

Tremendous progress has been made in the field of toxicology leading to the advance of developmental toxicity assessment. Conventional animal models and in vitro two-dimensional models cannot accurately describe toxic effects and predict actual in vivo responses due to obvious inter-species differences between humans and animals, as well as the lack of a physiologically relevant tissue microenvironment. Human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived three-dimensional organoids are ideal complex and multicellular organotypic models, which are indispensable in recapitulating morphogenesis, cellular interactions, and molecular processes of early human organ development. Recently, human organoids have been used for drug discovery, chemical toxicity and safety in vitro assessment. This review discusses the recent advances in the use of human organoid models, (i.e., brain, retinal, cardiac, liver, kidney, lung, and intestinal organoid models) for developmental toxicity and teratogenicity assessment of distinct tissues/organs following exposure to pharmaceutical compounds, heavy metals, persistent organic pollutants, nanomaterials, and ambient air pollutants. Combining next-generation organoid models with innovative engineering technologies generates novel and powerful tools for developmental toxicity and teratogenicity assessment, and the rapid progress in this field is expected to continue.

Development of human retinal organoid models for bisphenol toxicity assessment.

Bisphenols, including Bisphenol A (BPA), Tetrabromobisphenol A (TBBPA), and Tetrabromobisphenol S (TBBPS), have been widely applied in the production of polycarbonate plastics and epoxy resins and have been detected in the environment worldwide. The frequent detection of bisphenols in maternal and fetal samples has raised concerns about their toxic effects on human embryonic development, especially on the development of the central nervous system. However, the effect of bisphenols on human retinal development is still unknown. In this study, to evaluate the toxicity of bisphenols on early retinal development, human embryonic stem cells were induced to differentiate into retinal organoids that responded to BPA, TBBPA, and TBBPS, at human exposure relevant concentrations. The global gene expression of retinal organoids was analyzed by RNA sequencing (RNA-seq). A set of retinal development-related biological processes, including neuron differentiation, phototransduction, axon guidance, and retina layer formation, were identified in retinal organoids corresponding to different developmental stages. The RNA-seq data also showed that BPA, TBBPA, and TBBPS influenced retinal development by interfering with the Cytokine-cytokine receptor interaction pathway. HSPA6, HIF1A-AS3, CDC20B, IL19, OAS1, HSPA7, and RN7SK were dysregulated by these chemicals. Additionally, BPA, TBBPA, and TBBPS exhibited different toxic effects on neural retina development, with TBBPA appearing to exert more toxicity than BPA and TBBPS. Furthermore, three bisphenols exhibited different effects at different stages of neural retina development. The sensitivity of retinal development to bisphenols depends on their developmental stage. This study provides new insights into the deep dissection of retinotoxicity after prenatal bisphenol exposure.

Chemical molecular-based approach to overcome multidrug resistance in cancer by targeting P-glycoprotein (P-gp).

Multidrug resistance (MDR) remains one of the major impediments for efficacious cancer chemotherapy. Increased efflux of multiple chemotherapeutic drugs by transmembrane ATP-binding cassette (ABC) transporter superfamily is considered one of the primary causes for cancer MDR, in which the role of P-glycoprotein (P-gp/ABCB1) has been most well-established. The clinical co-administration of P-gp drug efflux inhibitors, in combination with anticancer drugs which are P-gp transport substrates, was considered to be a treatment modality to surmount MDR in anticancer therapy by blocking P-gp-mediated multidrug efflux. Extensive attempts have been carried out to screen for sets of nontoxic, selective, and efficacious P-gp efflux inhibitors. In this review, we highlight the recent achievements in drug design, characterization, structure-activity relationship (SAR) studies, and mechanisms of action of the newly synthetic, potent small molecules P-gp inhibitors in the past 5 years. The development of P-gp inhibitors will increase our knowledge of the mechanisms and functions of P-gp-mediated drug efflux which will benefit drug discovery and clinical cancer therapeutics where P-gp transporter overexpression has been implicated in MDR.

Design and Application of Receptor-Targeted Fluorescent Probes Based on Small Molecular Fluorescent Dyes.

In recent years, a variety of receptor-targeted fluorescent probes have been developed and widely used to realize the visualization of certain receptors, which facilitates the early diagnosis and treatment of diseases. In this Review, we focus on the recent achievements in design, chemical structure, imaging characterization, and potential applications of receptor-targeted fluorescent probes from the past 10 years. The development and application of receptor-targeted fluorescent probes will expand our knowledge of the distribution and function of disease-related receptors, shed light on the drug discovery for clinical diseases where receptors are implicated, and feed into the diagnosis and treatment of a plethora of diseases, including tumors.

Functional assessment of cryopreserved clinical grade hESC-RPE cells as a qualified cell source for stem cell therapy of retinal degenerative diseases.

The biosafety and efficiency of transplanting retinal pigment epithelial (RPE) cells derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been evaluated in phase I and phase II clinical trials. For further large-scale application, cryopreserved RPE cells must be used; thus, it is highly important to investigate the influence of cryopreservation and thawing on the biological characteristics of hESC-RPE cells and their post-transplantation vision-restoring function. Here, via immunofluorescence, qPCR, transmission electron microscopy, transepithelial electrical resistance, and enzyme-linked immunosorbent assays (ELISAs), we showed that cryopreserved hESC-RPE cells retained the specific gene expression profile, morphology, ultrastructure, and maturity-related functions of induced RPE cells. Additionally, cryopreserved hESC-RPE cells exhibited a polarized monolayer, tight junction, and gap junction structure and an in vitro nanoparticle phagocytosis capability similar to those of induced hESC-RPE cells. However, the level of pigment epithelium-derived factor (PEDF) secretion was significantly decreased in cryopreserved hESC-RPE cells. Royal College of Surgeons rats with cryopreserved hESC-RPE cells engrafted into the subretinal space exhibited a significant decrease in the b-wave amplitude compared with rats engrafted with induced hESC-RPE cells at 4 weeks post transplantation. However, the difference disappeared at 8 weeks and 12 weeks post operation. No significant difference in the outer nuclear layer (ONL) thickness was observed between the two groups. Our data showed that even after cryopreservation and thawing, cryopreserved hESC-RPE cells are still qualified as a donor cell source for cell-based therapy of retinal degenerative diseases.

The design, synthesis and cellular imaging of a tumor-anchored, potent and cell-permeable BRD4-targeted fluorescent ligands.

Bromodomain 4 (BRD4) proteins play an important role in histone post-translational modifications and facilitate several important physiological and pathological processes, including cancers. The inhibition of BRD4 by small molecule inhibitors shows promise as a therapeutic strategy for cancer treatment. However, their clinical applications were limited, which is largely hampered by off-target effects-induced toxicity. We herein report the design, synthesis, and cellular imaging of a set of tumor-anchored and BRD4-targeted fluorescent ligands by introducing selective and potent BRD4 inhibitor into different fluorophores via variable linkers. One of the fluorescent conjugates (compound 6) was demonstrated to be cell-permeable and low cytotoxic, preferentially accumulated in cancer cells, and display pronounced fluorescent signal. More importantly, 6 was identified to show specific BRD4 engagement in the cellular content. Collectively, this study provides a pathway for developing labeled BRD4 ligands and highlights that compound 6 may represent a valuable tool for explorative learning and target delivery study of BRD4.

Recent advances on the intervention sites targeting USP7-MDM2-p53 in cancer therapy.

The ubiquitin-specific protease 7 (USP7)-murine double minute 2 (MDM2)-p53 network plays an important role in the regulation of p53, a tumor suppressor which plays critical roles in regulating cell growth, proliferation, cell cycle progression, apoptosis and immune response. The overexpression of USP7 and MDM2 in human cancers contributes to cancer initiation and progression, and their inhibition reactivates p53 signalings and causes cell cycle arrest and apoptosis. Herein, the current state of pharmacological characterization, potential applications in cancer treatment and mechanism of action of small molecules used to target and inhibit MDM2 and USP7 proteins are highlighted, along with the outcomes in clinical and preclinical settings. Moreover, challenges and advantages of these strategies, as well as perspectives in USP7-MDM2-p53 field are analyzed in detail. The investigation and application of MDM2 and USP7 inhibitors will deepen our understanding of the function of USP7-MDM2-p53 network, and feed in the development of effective and safe cancer therapies where USP7-MDM2-p53 network is implicated.