Near infrared light fluorescence imaging-guided biomimetic nanoparticles of extracellular vesicles deliver indocyanine green and paclitaxel for hyperthermia combined with chemotherapy against glioma.

BACKGROUND: We investigated the therapeutic effect of targeting extracellular vesicles (EVs) loaded with indocyanine green (ICG) and paclitaxel (PTX) on glioma. METHODS: Raw264.7 cells were harvested to extract EVs for the preparation of ICG/PTX@RGE-EV by electroporation and click chemistry. We evaluated the success of modifying Neuropilin-1 targeting peptide (RGE) on the EV membrane of ICG/PTX@RGE-EV using super-resolution fluorescence microscopy and flow cytometry. Spectrophotometry and high performance liquid chromatography (HPLC) were implemented for qualitative and quantitative analysis of the ICG and PTX loaded in EVs. Photothermal properties of the vesicles were evaluated by exposing to 808-nm laser light. Western blot analysis, cell counting kit 8 (CCK-8), Calcein Acetoxymethyl Ester/propidium iodide (Calcein-AM/PI) staining, and flow cytometry were utilized for assessing effects of vesicle treatment on cellular behaviors. A nude mouse model bearing glioma was established to test the targeting ability and anti-tumor action of ICG/PTX@RGE-EV in vivo. RESULTS: Under exposure to 808-nm laser light, ICG/PTX@RGE-EV showed good photothermal properties and promotion of PTX release from EVs. ICG/PTX@RGE-EV effectively targeted U251 cells, with activation of the Caspase-3 pathway and elevated apoptosis in U251 cells through chemotherapy combined with hyperthermia. The anti-tumor function of ICG/PTX@RGE-EV was confirmed in the glioma mice via increased accumulation of PTX in the ICG/PTX@RGE-EV group and an increased median survival of 48 days in the ICG/PTX@RGE-EV group as compared to 25 days in the PBS group. CONCLUSION: ICG/PTX@RGE-EV might actively target glioma to repress tumor growth by accelerating glioma cell apoptosis through combined chemotherapy-hyperthermia.

Glioma exosomal microRNA-148a-3p promotes tumor angiogenesis through activating the EGFR/MAPK signaling pathway via inhibiting ERRFI1.

BACKGROUND: Glioma is the most frequent and lethal primary brain malignancy. Amounting evidence has highlighted the importance of exosomal microRNAs (miRNAs or miRs) in this malignancy. This study aimed to investigate the regulatory role of exosomal miR-148a-3p in glioma. METHODS: Bioinformatics analysis was firstly used to predict the target genes of miR-148a-3p. Exosomes were then extracted from normal human astrocytes and glioma cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was applied to determine the expression patterns of miR-148a-3p and ERBB receptor feedback inhibitor 1 (ERRFI1). Dual-luciferase reporter gene assay was applied to verify the direct binding between miR-148a-3p and ERRFI1. Cell counting kit-8 and tube formation assays were further conducted to assess the proliferation and angiogenic properties of human umbilical vein endothelial cells (HUVECs) in the co-culture system with exosomes. Lastly, glioma tumor models were established in BALB/c nude mice to study the role of exosomal miR-148a-3p in vivo. RESULTS: miR-148a-3p was highly expressed, while ERRFI1 was poorly expressed in glioma. miR-148a-3p was found to be enriched in glioma cells-derived exosomes and could be transferred to HUVECs via exosomes to promote their proliferation and angiogenesis. ERRFI1 was identified as a target gene of miR-148a-3p. In addition, miR-148a-3p activated the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signaling pathway by inhibiting ERRFI1. In the co-culture system, our data demonstrated that glioma cells-derived exosomal miR-148a-3p down-regulated ERRFI1 and activated the EGFR/MAPK signaling pathway, so as to promote cell proliferation and angiogenesis. In vivo experimentation further demonstrated that this mechanism was responsible for the promotive role of exosomal miR-148a-3p in tumorigenesis and angiogenesis. CONCLUSION: Taken together, glioma-derived exosomal miR-148a-3p promoted tumor angiogenesis through activation of the EGFR/MAPK signaling pathway by ERRFI1 inhibition.

Generation of induced pluripotent stem cells (ZZUCSBi001-A) from skin fibroblasts of a healthy donor.

Fibroblasts were extracted from the scalp of a healthy 55-year-old male and subsequently transformed into pluripotent stem cells by introducing episomal plasmids harboring essential reprogramming factors. These induced pluripotent stem cells exhibited a normal karyotype and demonstrated the capacity to differentiate into all three germ layers, as confirmed through teratoma assays. This specific cell line serves as a valuable reference for comparative investigations alongside other induced pluripotent stem cell lines generated from somatic cells of patients afflicted by genetic neurodegenerative disorders.

Neurotransmitter accumulation and Parkinson's disease-like phenotype caused by anion channelrhodopsin opto-controlled astrocytic mitochondrial depolarization in substantia nigra pars compacta.

Parkinson's disease (PD) is a mitochondria-related neurodegenerative disease characterized by locomotor deficits and loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). Majority of PD research primarily focused on neuronal dysfunction, while the roles of astrocytes and their mitochondria remain largely unexplored. To bridge the gap and investigate the roles of astrocytic mitochondria in PD progression, we constructed a specialized optogenetic tool, mitochondrial-targeted anion channelrhodopsin, to manipulate mitochondrial membrane potential in astrocytes. Utilizing this tool, the depolarization of astrocytic mitochondria within the SNc in vivo led to the accumulation of γ-aminobutyric acid (GABA) and glutamate in SNc, subsequently resulting in excitatory/inhibitory imbalance and locomotor deficits. Consequently, in vivo calcium imaging and interventions of neurotransmitter antagonists demonstrated that GABA accumulation mediated movement deficits of mice. Furthermore, 1 h/day intermittent astrocytic mitochondrial depolarization for 2 weeks triggered spontaneous locomotor dysfunction, α-synuclein aggregation, and the loss of DA neurons, suggesting that astrocytic mitochondrial depolarization was sufficient to induce a PD-like phenotype. In summary, our findings suggest the maintenance of proper astrocytic mitochondrial function and the reinstatement of a balanced neurotransmitter profile may provide a new angle for mitigating neuronal dysfunction during the initial phases of PD.

Progress in pH-Sensitive sensors: essential tools for organelle pH detection, spotlighting mitochondrion and diverse applications.

pH-sensitive fluorescent proteins have revolutionized the field of cellular imaging and physiology, offering insight into the dynamic pH changes that underlie fundamental cellular processes. This comprehensive review explores the diverse applications and recent advances in the use of pH-sensitive fluorescent proteins. These remarkable tools enable researchers to visualize and monitor pH variations within subcellular compartments, especially mitochondria, shedding light on organelle-specific pH regulation. They play pivotal roles in visualizing exocytosis and endocytosis events in synaptic transmission, monitoring cell death and apoptosis, and understanding drug effects and disease progression. Recent advancements have led to improved photostability, pH specificity, and subcellular targeting, enhancing their utility. Techniques for multiplexed imaging, three-dimensional visualization, and super-resolution microscopy are expanding the horizon of pH-sensitive protein applications. The future holds promise for their integration into optogenetics and drug discovery. With their ever-evolving capabilities, pH-sensitive fluorescent proteins remain indispensable tools for unravelling cellular dynamics and driving breakthroughs in biological research. This review serves as a comprehensive resource for researchers seeking to harness the potential of pH-sensitive fluorescent proteins.

A sensitive mitochondrial thermometry 2.0 and the availability of thermogenic capacity of brown adipocyte.

The temperature of a living cell is a crucial parameter for cellular events, such as cell division, gene expressions, enzyme activities and metabolism. We previously developed a quantifiable mitochondrial thermometry 1.0 based on rhodamine B methyl ester (RhB-ME) and rhodamine 800 (Rh800), and the theory for mitochondrial thermogenesis. Given that the synthesized RhB-ME is not readily available, thus, a convenient mitochondrial thermometry 2.0 based on tetra-methyl rhodamine methyl ester (TMRM) and Rh800 for the thermogenic study of brown adipocyte was further evolved. The fluorescence of TMRM is more sensitive (∼1.4 times) to temperature than that of RhB-ME, then the TMRM-based mito-thermometry 2.0 was validated and used for the qualitatively dynamic profiles for mitochondrial thermogenic responses and mitochondrial membrane potential in living cells simultaneously. Furthermore, our results demonstrated that the heterogenous thermogenesis evoked by ß3 adrenoceptor agonist only used overall up to ∼46% of the thermogenic capacity evoked by CCCP stimulation. On the other hand, the results demonstrated that the maximum thermogenesis evoked by NE and oligomycin A used up to ∼79% of the thermogenic capacity, which suggested the maximum thermogenic capacity under physiological conditions by inhibiting the proton-ATPase function of the mitochondrial complex V, such as under the cold activation of sympathetic nerve and the co-release of sympathetic transmitters.

The in cis compound EGFR mutations in Chinese advanced non-small cell lung cancer patients.

Literatures regarding the prevalence and clinical significance of compound EGFR mutations are limited. Until now, none of retrospective or prospective research has focused on in cis compound EGFR mutations except case reports. In this study, we screened a cohort of 3,000 treatment-naïve Chinese advanced NSCLC patients using capture-based ultra-deep targeted sequencing to evaluate the prevalence of EGFR in cis compound mutations and the efficacy of EGFR-TKI in this population. Of the 3,000 patients screened, 1,266 (42.2%) had EGFR mutation; among them, 15 patients (1.2%) harboring in cis compound EGFR mutations, with 10 patients carrying EGFR L858R in combination with a rare mutation and five patients carrying two rare EGFR mutations. No patient with EGFR 19del was observed. Interestingly, no in trans configuration was identified in this cohort. All of the patients harboring in cis compound EGFR mutations were non-smokers, histologically diagnosed with adenocarcinoma and received first-generation EGFR-TKI. Furthermore, our data also revealed that patients with in cis compound EGFR mutations exhibit comparable PFS to first generation EGFR-TKI comparing to patients with single activating EGFR mutation. This observation was further supported by in silico molecular modeling analyses which demonstrated in cis compound mutations do not alter the ATP-binding pocket of EGFR, thus having no effect on the interaction between gefitinib and EGFR.

Mechanism of microtubule stabilization by taccalonolide AJ.

As a major component of the cytoskeleton, microtubules consist of αß-tubulin heterodimers and have been recognized as attractive targets for cancer chemotherapy. Microtubule-stabilizing agents (MSAs) promote polymerization of tubulin and stabilize the polymer, preventing depolymerization. The molecular mechanisms by which MSAs stabilize microtubules remain elusive. Here we report a 2.05 Å crystal structure of tubulin complexed with taccalonolide AJ, a newly identified taxane-site MSA. Taccalonolide AJ covalently binds to ß-tubulin D226. On AJ binding, the M-loop undergoes a conformational shift to facilitate tubulin polymerization. In this tubulin-AJ complex, the E-site of tubulin is occupied by GTP rather than GDP. Biochemical analyses confirm that AJ inhibits the hydrolysis of the E-site GTP. Thus, we propose that the ß-tubulin E-site is locked into a GTP-preferred status by AJ binding. Our results provide experimental evidence for the connection between MSA binding and tubulin nucleotide state, and will help design new MSAs to overcome taxane resistance.

Structural basis of Dscam1 homodimerization: Insights into context constraint for protein recognition.

The Drosophila neural receptor Dscam1 (Down syndrome cell adhesion molecule 1) plays an essential role in neuronal wiring and self-avoidance. Dscam1 potentially encodes 19,008 ectodomains through alternative RNA splicing and exhibits exquisite isoform-specific homophilic binding, which makes it an exceptional example for studying protein binding specificity. However, structural information on Dscam1 is limited, which hinders illumination of the mechanism of Dscam1 isoform-specific recognition. Whether different Dscam1 isoforms adopt the same dimerization mode remains a subject of debate. We present 12 Dscam1 crystal structures, provide direct evidence indicating that all isoforms adopt a conserved homodimer geometry in a modular fashion, identify two mechanisms for the Ig2 binding domain to dispel electrostatic repulsion during dimerization, decode Ig2 binding specificity by a central motif at its symmetry center, uncover the role of glycosylation in Dscam1 homodimerization, and find electrostatic potential complementarity to help define the binding region and the antiparallel binding mode. We then propose a concept that the context of a protein may set restrictions to regulate its binding specificity, which provides a better understanding of protein recognition.

Protein production, crystallization and preliminary crystallographic analysis of the four N-terminal immunoglobulin domains of Down syndrome cell adhesion molecule 1. Corrigendum.

An extra author is added to the article by Chen et al. [(2015), Acta Cryst. F71, 775-778].

Protein production, crystallization and preliminary X-ray analysis of two isoforms of the Dscam1 Ig7 domain.

Drosophila Down syndrome cell adhesion molecule 1 (Dscam1) plays a critical role in neural development. It can potentially form 38 016 isoforms through alternative RNA splicing, and exhibits isoform-specific homophilic interaction through three variable Ig domains (Ig2, Ig3 and Ig7). The diversity and homophilic interaction are essential for its functions. Ig7 has 33 isoforms and is the most variable among the three variable Ig domains. However, only one isoform of Ig7 (isoform 30) has been structurally determined to date. Here, two isoforms of Dscam1 Ig7 (isoforms 5 and 9; Ig75 and Ig79) were produced and crystallized. Diffraction data from Ig75 and Ig79 crystals were processed to resolutions of 1.95 and 2.37 Å, respectively. Comparison of different Dscam1 Ig7 isoforms will provide insight into the mechanism of its binding specificity.

Protein production, crystallization and preliminary crystallographic analysis of the four N-terminal immunoglobulin domains of Down syndrome cell adhesion molecule 1.

Down syndrome cell adhesion molecule 1 (Dscam1), a member of the immunoglobulin (Ig) superfamily, plays important roles in both the nervous and the immune systems. Via alternative RNA splicing, Drosophila Dscam1 encodes a vast family of Ig-containing proteins that exhibit isoform-specific homophilic binding. Whether different Dscam1 isoforms adopt the same dimerization mode is under debate, and the detailed mechanism of Dscam1 specificity remains unclear. In this study, eight different isforms of Dscam1 Ig1-4 have been cloned, overexpressed, purified to homogeneity and crystallized. X-ray data were collected to 1.9-4.0 Å resolution. These structures will provide the opportunity to perform extensive structural comparisons of different Dscam1 isoforms and provide insight into its specificity.