CRISPR RNAs trigger innate immune responses in human cells

Here, we report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to similar to 80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting St-hydroxyl gRNAs in complex with Cas9 or Cpfl avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4(+) T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5'-hydroxyl group are much more efficient than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.

Prognostic Factors of Neurological Complications in Spinal Surgeries

Study Design Retrospective study. Purpose To determine prognostic factors of neurological complications (NCs) of posterior thoracolumbar surgeries. Overview of Literature There have been few reports on the prognosis of NCs according to the causes and treatment methods. Methods The subjects were 65 patients who had NCs for 19 years (1995–2013) after posterior thoracolumbar surgeries in Seoul Sacred Heart General Hospital. The degree of neurological injury was assessed using numeric scales as follows: G1, increased leg pain or sensory loss; G2, hemiparesis; G3, paraparesis; G4, cauda equine syndrome; and G5, complete paraplegia. The relative degree of neurological recovery was evaluated using four numeric scales as follows: Gr1, complete recovery; Gr2, almost complete recovery with residual sensory loss or numbness; Gr3, partial recovery with apparent neurological deficit; and Gr4, no recovery. The prognostic factors were investigated in terms of demographic and surgical variables that were available in a retrospective review. Results The causes were as follows: epidural hematoma (EH), 25 patients (38.5%); insufficient decompression and fusion, 14 patients (21.5%); mechanical injury, 11 patients (16.9%); insufficient discectomy, four patients (6.2%); and unknown, 11 patients (23.1%). The grade of neurological injury was as follows: G1, 11 patients (16.9%); G2, 34 patients (52.3%); G3, 15 patients (23.1%); G4, three patients (4.6%); and G5, two patients (3.1%). Thirteen patients received conservative treatment, and 52 underwent revision surgeries. Neurological recovery was as follows: Gr1, 21 patients (32.3%); Gr2, 17 patients (26.2%); Gr3, 20 patients (30.8%); and Gr4, seven patients (10.8%). The prognosis depended on the causes (p =0.041). The subgroup analysis of the revision group revealed a significant correlation between the degree of neurological recovery and the timing of revision, irrespective of causes (r =0.413, p =0.002). Conclusions The prognosis of NC depended on the causes. EH was the best and unknown was the worst prognostic factor. Revision should be performed as soon as possible for a better prognosis

Fasting Plasma Glucose Cutoff Value for the Prediction of Future Diabetes Development: A Study of Middle-Aged Koreans in a Health Promotion Center

We determined optimal fasting plasma glucose (FPG) cutoff values predictive of future diabetes development in a group of middle-aged Koreans who visited a health promotion center. The medical records of 2,964 subjects, who attended the Health Promotion Center in 1998 and 2003, were examined. Subjects were classified into four groups according to their baseline FPG values (Group 1:FPG <5.0 mM/L; Group 2: 5.0≤FPG <5.6 mM/L; Group 3: 5.6≤FPG <6.1 mM/L; Group 4: 6.1≤FPG <7.0 mM/L). No significant difference was observed between Group 1 and Group 2 in terms of diabetes incidence. However, incidence in Group 3 was significantly higher than that in Group 1 [hazards ratio 4.88 (1.65-14.41), p=0.004] and the hazards ratio in Group 4 for diabetes was 36.91 (13.11-103.61), p<0.001, versus Group 1. Receiver operator characteristics curve analysis showed that an FPG of 5.97 mM/L represents the lower limit and gives the best combination of sensitivity and specificity. Our data shows that the risk of future diabetes development started to increase below an FPG of 6.1 mM/L and suggests the importance of efforts to modify diabetes development risk factors at lower impaired fasting glucose levels

Anesthetic management of an adult patient with Rett syndrome and limited mouth opening -A case report-

Rett syndrome is a neurological disease that occurs only in females and it manifests with mental retardation, seizures, movement disorders, autistic behavior and abnormal breathing. A 19-year-old female with Rett syndrome underwent ophthalmologic surgery under general anesthesia at our institution. Airway control was difficult due to her limited mouth opening. We recommend that anesthesiologists should have proper knowledge about this disease and the patients to avoid the complications and problems that can be encountered during the perioperative period

Xenopus gpx3 Mediates Posterior Development by Regulating Cell Death during Embryogenesis

Glutathione peroxidase 3 (GPx3) belongs to the glutathione peroxidase family of selenoproteins and is a key antioxidant enzyme in multicellular organisms against oxidative damage. Downregulation of GPx3 affects tumor progression and metastasis and is associated with liver and heart disease. However, the physiological significance of GPx3 in vertebrate embryonic development remains poorly understood. The current study aimed to investigate the functional roles of gpx3 during embryogenesis. To this end, we determined gpx3&apos;s spatiotemporal expression using Xenopus laevis as a model organism. Using reverse transcription polymerase chain reaction (RT-PCR), we demonstrated the zygotic nature of this gene. Interestingly, the expression of gpx3 enhanced during the tailbud stage of development, and whole mount in situ hybridization (WISH) analysis revealed gpx3 localization in prospective tail region of developing embryo. gpx3 knockdown using antisense morpholino oligonucleotides (MOs) resulted in short post-anal tails, and these malformed tails were significantly rescued by glutathione peroxidase mimic ebselen. The gene expression analysis indicated that gpx3 knockdown significantly altered the expression of genes associated with Wnt, Notch, and bone morphogenetic protein (BMP) signaling pathways involved in tailbud development. Moreover, RNA sequencing identified that gpx3 plays a role in regulation of cell death in the developing embryo. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and phospho-histone 3 (PH3) staining confirmed the association of gpx3 knockdown with increased cell death and decreased cell proliferation in tail region of developing embryos, establishing the involvement of gpx3 in tailbud development by regulating the cell death. Furthermore, these findings are inter-related with increased reactive oxygen species (ROS) levels in gpx3 knockdown embryos, as measured by using a redox-sensitive fluorescent probe HyPer. Taken together, our results suggest that gpx3 plays a critical role in posterior embryonic development by regulating cell death and proliferation during vertebrate embryogenesis