Enhanced membrane protein production in HEK293T cells via ATF4 gene knockout: A CRISPR-Cas9 mediated approach

HEK293T cells are extensively utilized for therapeutic protein production due to their human origin, which enables accurate post-translational modifications. This study aimed to enhance membrane protein production in HEK293T cells by knocking out the ATF4 gene using CRISPR-Cas9 technology. The ATF4 gene was edited by infecting HEK293T cells with a lentivirus carrying optimized single-guide RNA (ATF4-KO-3) and Cas9 genes. Comparative evaluations were conducted using all-in-one and two-vector systems. Genome sequencing and membrane protein productivity of ATF4-knockout (KO) cells were compared to wild-type (WT) cells using next-generation sequencing (NGS) and a membrane protein isolation kit, respectively. Single-cell analysis confirmed gene editing patterns, with NGS verifying the intended deletions. Membrane protein production was also assessed indirectly via flow cytometry, analyzing cells expressing Membrane-GFP. Compared to WT cells, ATF4-KO cells exhibited a significant increase in membrane protein production, with a 52.2 ± 19.0% improvement. Gene editing efficiency was compared between the two delivery systems, with the two-vector system demonstrating higher efficiency based on T7 endonuclease I assays. Western blot analysis confirmed ATF4 suppression and increased expression of membrane proteins, including E-cadherin and CD63. Quantitative analysis via PAGE revealed a 77.2 ± 30.6% increase in purified membrane protein yields, consistent with the observed enhancements. Flow cytometry using Membrane-GFP further demonstrated a 22.9 ± 9.7% increase in productivity. In summary, ATF4 knockout significantly enhances membrane protein production in HEK293T cells, offering potential improvements in biopharmaceutical manufacturing by enabling more efficient protein synthesis

Recessive C10orf2 mutations in a family with infantile-onset spinocerebellar ataxia, sensorimotor polyneuropathy, and myopathy

Recessive mutations in chromosome 10 open reading frame 2 (C10orf2) are relevant in infantile-onset spinocerebellar ataxia (IOSCA). In this study, we investigated the causative mutation in a Korean family with combined phenotypes of IOSCA, sensorimotor polyneuropathy, and myopathy. We investigated recessive mutations in a Korean family with two individuals affected by IOSCA. Causative mutations were investigated using whole exome sequencing. Electrophysiological analyses and muscle and nerve biopsies were performed, along with magnetic resonance imaging (MRI) of the brain and lower extremities. Compound heterozygous mutations c.1460C>T and c.1485-1G>A in C10orf2 were identified as causative of IOSCA. Skeletal muscle showed mitochondrial DNA (mtDNA) deletions. Both patients showed a period of normal development until 12–15 months, followed by ataxia, athetosis, hearing loss, and intellectual disability. Electrophysiological findings indicated motor and sensory polyneuropathies. Muscle biopsy revealed variations in the size and shape of myofibers with scattered, small, and angulated degenerating myofibers containing abnormal mitochondria; these observations are consistent with myopathy and may be the result of mtDNA deletions. Sural nerve biopsy revealed an axonal neuropathy. High-signal-intensity lesions in the middle cerebellar peduncles were correlated with clinical severity, and MRI of the lower legs was compatible with the hypothesis of length-dependent axonal degeneration. We identified novel compound heterozygous mutations of the C10orf2 gene as the cause of IOSCA with sensorimotor polyneuropathy and myopathy. Signs of motor neuropathy and myopathy were discovered for the first time in IOSCA patients with C10orf2 mutations. These results suggest that the clinical spectrum of IOSCA caused by C10orf2 mutations may be more variable than previously reported. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10048-014-0405-1) contains supplementary material, which is available to authorized users

Hashimoto thyroiditis with an unusual presentation of cardiac tamponade in Noonan syndrome

Noonan syndrome is an autosomal dominant, multisystem disorder. Autoimmune thyroiditis with hypothyroidism is an infrequent feature in patients with Noonan syndrome. A 16-year-old boy was admitted because of chest discomfort and dyspnea; an echocardiogram revealed pericardial effusion. Additional investigations led to a diagnosis of severe hypothyroidism due to Hashimoto thyroiditis. The patient was treated with L-thyroxine at 0.15 mg daily. However, during admission, he developed symptoms of cardiac tamponade. Closed pericardiostomy was performed, after which the patient's chest discomfort improved, and his vital signs stabilized. Herein, we report a case of an adolescent with Noonan syndrome, who was diagnosed with Hashimoto thyroiditis with an unusual presentation of cardiac tamponade

A giant retroperitoneal lymphangioma in a patient with neurofibromatosis type 1

Neurofibromatosis type 1 (NF-1) is a genetically inherited disorder that may cause skin abnormalities and tumors that form on nerve tissues. These tumors can be small or large and can occur anywhere in the body, including the brain, spinal cord, or other peripheral nerves. Retroperitoneal lymphangiomas are very rare benign malformations of the lymphatic system. About 95% lymphangiomas occur in the skin and the subcutaneous tissues of the head, neck and axillary region and the remaining 5% appear in other parts of the body such as lungs, pleura, pericardium, liver, gallbladder, kidney, and the mesentery. Herein, we report the case of a giant retroperitoneal lymphangioma in a patient with NF-1 with a review of the literature

A Family Harboring CMT1A Duplication and HNPP Deletion

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with duplication of chromosome 17p11.2-p12, whereas hereditary neuropathy with liability to pressure palsies (HNPP), which is an autosomal dominant neuropathy showing characteristics of recurrent pressure palsies, is associated with 17p11.2-p12 deletion. An altered gene dosage of PMP22 is believed to the main cause underlying the CMT1A and HNPP phenotypes. Although CMT1A and HNPP are associated with the same locus, there has been no report of these two mutations within a single family. We report a rare family harboring CMT1A duplication and HNPP deletion

The Impact of Intima-media Thickness of Radial Artery on Early Failure of Radiocephalic Arteriovenous Fistula in Hemodialysis Patients

This study was performed to investigate the impact of intima-media thickness (IMT) of radial artery on early failure of radiocephalic arteriovenous fistula (AVF) in hemodialysis (HD) patients. Ninety uremic patients undergoing radiocephalic AVF operation were included in this study. During the operation, 10-mm long partial arterial walls were removed with elliptical form for microscopic analysis. Specimens were stained with trichrome and examined by a pathologist blinded to the clinical data. And then AVF patency was followed up for 1 yr after the operation. Of the total 90 patients, 31 patients (34%) had AVF failure within 1 yr after the operation. Mean IMT was thicker in failed group (n=31) than in patent group (n=59) (486±130 µm vs. 398±130 µm, p=0.004). The AVF patency rate within 1 yr after the operation was lower in patients with IMT ≥500 µm (n=26) than in patients with IMT <500 µm (n=64) (p=0.017). Age was an independent risk factor of IMT. Diabetes mellitus tended to be independent risk factor but not statistically significant. Our data suggest that increased radial artery IMT is closely associated with early failure of radiocephalic AVF in HD patients