Glioma stem cell-derived exosomes induce the transformation of astrocytes via the miR-3065-5p/DLG2 signaling axis.

Tumor-associated astrocytes (TAAs) in the glioblastoma microenvironment play an important role in tumor development and malignant progression initiated by glioma stem cells (GSCs). In the current study, normal human astrocytes (NHAs) were cultured and continuously treated with GSC-derived exosomes (GSC-EXOs) induction to explore the mechanism by which GSCs affect astrocyte remodeling. This study revealed that GSC-EXOs can induce the transformation of NHAs into TAAs, with relatively swollen cell bodies and multiple extended processes. In addition, high proliferation, elevated resistance to temozolomide (TMZ), and increased expression of TAA-related markers (TGF-ß, CD44, and tenascin-C) were observed in the TAAs. Furthermore, GSC-derived exosomal miR-3065-5p could be delivered to NHAs, and miR-3065-5p levels increased significantly in TAAs, as verified by miRNA expression profile sequencing and Reverse transcription polymerase chain reaction. Overexpression of miR-3065-5p also enhanced NHA proliferation, elevated resistance to TMZ, and increased the expression levels of TAA-related markers. In addition, both GSC-EXO-induced and miR-3065-5p-overexpressing NHAs promoted tumorigenesis of GSCs in vivo. Discs Large Homolog 2 (DLG2, downregulated in glioblastoma) is a direct downstream target of miR-3065-5p in TAAs, and DLG2 overexpression could partially reverse the transformation of NHAs into TAAs. Collectively, these data demonstrate that GSC-EXOs induce the transformation of NHAs into TAAs via the miR-3065-5p/DLG2 signaling axis and that TAAs can further promote the tumorigenesis of GSCs. Thus, precisely blocking the interactions between astrocytes and GSCs via exosomes may be a novel strategy to inhibit glioblastoma development, but more in-depth mechanistic studies are still needed.

Sequences in the stalk domain regulate auto-inhibition and ciliary tip localization of the immotile kinesin-4 KIF7.

The kinesin-4 member KIF7 plays critical roles in Hedgehog signaling in vertebrate cells. KIF7 is an atypical kinesin as it binds to microtubules but is immotile. We demonstrate that, like conventional kinesins, KIF7 is regulated by auto-inhibition, as the full-length protein is inactive for microtubule binding in cells. We identify a segment, the inhibitory coiled coil (inhCC), that is required for auto-inhibition of KIF7, whereas the adjacent regulatory coiled coil (rCC) that contributes to auto-inhibition of the motile kinesin-4s KIF21A and KIF21B is not sufficient for KIF7 auto-inhibition. Disease-associated mutations in the inhCC relieve auto-inhibition and result in strong microtubule binding. Surprisingly, uninhibited KIF7 proteins did not bind preferentially to or track the plus ends of growing microtubules in cells, as suggested by previous in vitro work, but rather bound along cytosolic and axonemal microtubules. Localization to the tip of the primary cilium also required the inhCC, and could be increased by disease-associated mutations regardless of the auto-inhibition state of the protein. These findings suggest that loss of KIF7 auto-inhibition and/or altered cilium tip localization can contribute to the pathogenesis of human disease.

One-Pot Synthesis of Seven-Membered Heterocyclic Derivatives of Diazepines Involving Copper-Catalyzed Rearrangement Cascade Allyl-Amination.

A novel and efficient method has been proposed for the synthesis of 1,4-benzodiazepine-5-ones from o-nitrobenzoic N-allylamides by using molybdenyl acetylacetonate and copper(II) trifluoromethanesulfonate as catalysts in the presence of triphenylphosphine. This synthesis process involves nitrene formation, C-H bond insertion, C═C bond rearrangement, and C-N bond formation cascade reactions via copper- and molybdenum-catalyzed mediation. The method features a wide substrate scope and a moderate to high yield (up to 90%), exhibiting the possibility for practical applications.

Mitochondria Penetrating Peptide-Conjugated TAMRA for Live-Cell Long-Term Tracking.

Mitochondria are essential targets for treatment of diseases with mitochondrial disorders such as diabetes, cancer, and cardiovascular and neurodegenerative diseases. Mitochondria penetrating peptides (MPPs) are composed of cationic and hydrophobic amino acids that can target and permeate the mitochondrial membrane. Herein, a novel d-argine-phenylalanine-d-argine-phenylalanine-d-argine-phenylalanine-NH2 (rFrFrF) was tagged with a rhodamine-based fluorescent chromophore (TAMRA). This probe (TAMRA-rFrFrF) exhibited advantageous properties for long-term mitochondria tracking as demonstrated by fluorescence microscopy. Cell viability assays and oxygen consumption rates indicate low cytotoxicity and high biocompatibility of the new contrast agent. Colocalization studies suggest that TAMRA-rFrFrF is a promising candidate for continuous mitochondrial tracking for up to 3 days.

A Highly Selective Fluorescence Turn-On Sensor for Extracellular Calcium Ion Detection.

A highly water-soluble, fluorescence turn-on sensor for Ca(2+) is reported. The sensor affords high selectivity in sensing Ca(2+) over other biologically important metal cations. The dissociation constant of the sensor in binding Ca(2+) is 0.92 mm. Fluorescence microscopy experiments demonstrate that the sensor is cell-impermeable and capable of detecting extracellular Ca(2+) .

Insect Cell-Based Expression of Cytoskeletal Motor Proteins for Single-Molecule Studies.

Cytoskeletal motor proteins are essential molecular machines that hydrolyze ATP to generate force and motion along cytoskeletal filaments. Members of the dynein and kinesin superfamilies play critical roles in transporting biological payloads (such as proteins, organelles, and vesicles) along microtubule pathways, cause the beating of flagella and cilia, and act within the mitotic and meiotic spindles to segregate replicated chromosomes to progeny cells. Understanding the underlying mechanisms and behaviors of motor proteins is critical to provide better strategies for the treatment of motor protein-related diseases. Here, we provide detailed protocols for the recombinant expression of the Kinesin-1 motor KIF5C using a baculovirus/insect cell system and provide updated protocols for performing single-molecule studies using total internal reflection fluorescence microscopy and optical tweezers to study the motility and force generation of the purified motor.

SENP1-Mediated deSUMOylation Regulates the Tumor Remodeling of Glioma Stem Cells Under Hypoxic Stress.

Objectives: This study aimed to investigate the effect of specific small ubiquitin-like modifier (SUMO) proteases 1 (SENP1)-mediated deSUMOylation on the malignant behavior of glioma stem cells (GSCs) under hypoxia conditions and evaluate the clinical value of prevention in glioma patients. Introductions: Under hypoxic conditions, upregulated hypoxia-inducible factor 1α (HIF1α) expression in GSCs activates Wnt/ß-catenin signaling pathways, which provide rich nutritional support for glioblastoma (GBM). SENP1-mediated deSUMOylation stabilizes the expression of HIF1α and ß-catenin, leading to the occurrence of GSCs-initiated tumorigenesis. Targeting SENP1-mediated deSUMOylation may suppress the malignancy of GSCs and disrupt GBM progression. Methods: The expression of SENP1 in different World Health Organization grades was observed by immunohistochemistry and western blot. Lentivirus-packaged SENP1shRNA downregulated the expression of SENP1 in GSCs, and the downregulated results were verified by western blotting and polymerase chain reaction. The effects of LV-SENP1shRNA on the migration and proliferation of GSCs were detected by scratch and cloning experiments. The effect of LV-SENP1shRNA on the tumor formation ability of GSCs was observed in nude mice. Immunoprecipitation clarified the mechanism of SENP1 regulating the malignant behavior of GSCs under hypoxia. The correlation between the expression level of SENP1 and the survival of glioma patients was determined by statistical analysis. Results: SENP1 expression in GSCs derived from clinical samples was upregulated in GBM. SUMOylation was observed in GSCs in vitro, and deSUMOylation, accompanied by an increase in SENP1 expression, was induced by hypoxia. SENP1 expression was downregulated in GSCs with lentivirus-mediated stable transfection, which attenuated the proliferation and differentiation of GSCs, thus diminishing tumorigenesis. Mechanistically, HIF1α induced activation of Wnt/ß-catenin, which depended on SENP1-mediated deSUMOylation, promoting GSC-driven GBM growth under the hypoxia microenvironment. Conclusion: Our findings indicate that SENP1-mediated deSUMOylation as a feature of GSCs is essential for GBM maintenance, suggesting that targeting SENP1 against GSCs may effectively improve GBM therapeutic efficacy.

Kinesin-14 HSET and KlpA are non-processive microtubule motors with load-dependent power strokes.

Accurate chromosome segregation during cell division relies on coordinated actions of microtubule (MT)-based motor proteins in the mitotic spindle. Kinesin-14 motors play vital roles in spindle assembly and maintenance by crosslinking antiparallel MTs at the spindle midzone and anchoring spindle MTs' minus ends at the poles. In this study, we investigate the force generation and motility of the Kinesin-14 motors HSET and KlpA. Our findings reveal that both motors are non-processive, producing single load-dependent power strokes per MT encounter, with estimated load-free power strokes of ~30 and ~35 nm, respectively. Each homodimeric motor generates forces of ~0.5 pN, but when assembled in teams, they cooperate to generate forces of 1 pN or more. Notably, the cooperative activity among multiple motors leads to increased MT-sliding velocities. These results quantitatively elucidate the structure-function relationship of Kinesin-14 motors and underscore the significance of cooperative behavior in their cellular functions.

Hsa_circ_0021205 enhances lipolysis via regulating miR-195-5p/HSL axis and drives malignant progression of glioblastoma.

Abnormal lipid metabolism is an essential hallmark of glioblastoma. Hormone sensitive lipase (HSL), an important rate-limiting enzyme contributed to lipolysis, which was involved in aberrant lipolysis of glioblastoma, however, its definite roles and the relevant regulatory pathway have not been fully elucidated. Our investigations disclosed high expression of HSL in glioblastoma. Knock-down of HSL restrained proliferation, migration, and invasion of glioblastoma cells while adding to FAs could significantly rescue the inhibitory effect of si-HSL on tumor cells. Overexpression of HSL further promoted tumor cell proliferation and invasion. Bioinformatics analysis and dual-luciferase reporter assay were performed to predict and verify the regulatory role of ncRNAs on HSL. Mechanistically, hsa_circ_0021205 regulated HSL expression by sponging miR-195-5p, which further promoted lipolysis and drove the malignant progression of glioblastoma. Besides, hsa_circ_0021205/miR-195-5p/HSL axis activated the epithelial-mesenchymal transition (EMT) signaling pathway. These findings suggested that hsa_circ_0021205 promoted tumorigenesis of glioblastoma through regulation of HSL, and targeting hsa_circ_0021205/miR-195-5p/HSL axis can serve as a promising new strategy against glioblastoma.

TBX15 facilitates malignant progression of glioma by transcriptional activation of TXDNC5.

T-box transcription factor 15 (TBX15) plays important role in various cancers; however, its expression and role in glioma is still unclear. In this study, our findings indicated that TBX15 was increased in gliomas compared to normal brain tissues, and high levels of TBX15 were related to poor survival. Furthermore, TBX15 silencing in glioma cells not only inhibited their proliferation, migration, and invasion in vitro, but also weakened their ability to recruit macrophages and polarize the latter to the M2 subtype. Mechanism study indicated that thioredoxin domain containing 5 (TXNDC5) lies downstream of TBX15. Furthermore, rescue assays verified that the role of TBX15 in glioma cells is dependent on TXNDC5. Moreover, sh-TBX15 loaded into DNA origami nanocarrier suppressed the malignant phenotype of glioma in vitro and in vivo. Taken together, the TBX15/TXNDC5 axis is involved in the genesis and progression of glioma, and is a potential therapeutic target.