The protocol of tagging endogenous proteins with fluorescent tags using CRISPR-Cas9 genome editing.

The currently widely used CRISPR-Cas9 genome editing technology enables the editing of target genes (knock-out or knock-in) with high accuracy and efficiency. Guided by the small guide RNA, the Cas9 nuclease induces a DNA double-strand break at the targeted genomic locus. The DNA double-strand break can be repaired by the homology-directed repair pathway in the presence of a repair template. With the repair template containing the coding sequence of a fluorescent tag, the targeted gene can be inserted with the sequence of a fluorescent tag at the designed position. The genome editing mediated labeling of endogenous proteins with fluorescent tags avoids the potential artifacts caused by gene overexpression and substantially improves the reproductivity of imaging experiments. This protocol focuses on creating mammalian cell lines with endogenous proteins tagged with fluorescent proteins or self-labeling protein tags using CRISPR-Cas9 genome editing.

Mitochondria are transported along microtubules in membrane nanotubes to rescue distressed cardiomyocytes from apoptosis.

Membrane nanotubes (MNTs) act as "highways" between cells to facilitate the transfer of multiple signals and play an important role in many diseases. Our previous work reported on the transfer of mitochondria via MNTs between cardiomyocytes (CMs) and cardiac myofibroblasts (MFs); however, the elucidation of the underlying mechanism and pathophysiological significance of this transfer requires additional study. In this study, we determined that the mean movement velocity of mitochondria in MNTs between CMs and MFs was approximately 17.5 ± 2.1 nm/s. Meanwhile, treatment with microtubule polymerisation inhibitors nocodazole or colcemid in cell culture decreased mitochondrial velocity, and knockdown of the microtubule motor protein kinesin family member 5B (KIF5B) led to a similar effect, indicating that mitochondrial movement was dependent on microtubules and the motor protein KIF5B. Furthermore, we showed that hypoxia/reoxygenation-induced CM apoptosis was attenuated by coculture with intact or hypoxia/reoxygenation-treated MFs, which transferred mitochondria to CMs. This rescue was prevented either by separating the cells using Transwell culture or by impairing mitochondrial transfer with nocodazole or colcemid treatment. In conclusion, as a novel means of intercellular communication, MNTs rescue distressed CMs from apoptosis by transporting mitochondria along microtubules via KIF5B.

Clinical features and microsurgical treatment of pediatric patients with cerebral cavernous malformation.

The aim of the present study was to describe the clinical features and to evaluate the surgical treatment outcomes of pediatric patients with cerebral cavernous malformations (CCM). We investigated 85 children (53 boys and 32 girls), aged from 6 months to 17.9 years with CCM. Seizures and symptomatic hemorrhages, which were the most frequent symptoms, occurred in 81 patients. Nine patients had a positive family history of CCM. Eighty patients underwent microsurgical treatment after strict operative indications were met. Neuronavigation, combined with intraoperative ultrasonography or functional MRI, was used for precise localization of the lesions. The principles of minimally invasive techniques were followed during surgery. A total of 89 lesions were removed in 80 patients, and there were no deaths. During their hospital stay, only nine patients suffered from postoperative seizures, which were controlled with medication. Postoperative neurological deficits improved in 27 patients, were unchanged in nine, and worsened in two. With the help of advanced neuroimaging, a satisfactory surgical outcome was achieved for 10 lesions located in eloquent brain areas and four lesions in the brain stem. A follow-up study of 66 patients showed that all of these patients remained seizure-free, and nine patients with postoperative neurological deficits gradually recovered. Microsurgical treatment should be performed early for pediatric patients with CCM. Accurate localization of the lesions and the use of minimally invasive techniques and functional MRI monitoring were the key features of the surgical procedures.