N 1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells.

Synthetic messenger RNA (mRNA) tools often use pseudouridine and 5-methyl cytidine as substitutions for uridine and cytidine to avoid the immune response and cytotoxicity induced by introducing mRNA into cells. However, the influence of base modifications on the functionality of the RNA tools is poorly understood. Here we show that synthetic mRNA switches containing N1-methylpseudouridine (m1Ψ) as a substitution of uridine substantially out-performed all other modified bases studied, exhibiting enhanced microRNA and protein sensitivity, better cell-type separation ability, and comparably low immune stimulation. We found that the observed phenomena stem from the high protein expression from m1Ψ containing mRNA and efficient translational repression in the presence of target microRNAs or proteins. In addition, synthetic gene circuits with m1Ψ significantly improve performance in cells. These findings indicate that synthetic mRNAs with m1Ψ modification have enormous potentials in the research and application of biofunctional RNA tools.

Relationship between Vertebral Instability and the Cross-Sectional Area of Lumbar Muscles in Postmenopausal Acute Osteoporotic Vertebral Fractures.

INTRODUCTION: Vertebral instability (VI) in osteoporotic vertebral fractures (OVFs) varies from mild to severe. The relationship between the VI of OVFs and independent factors, such as bone mineral density (BMD) and lumbar muscle volume, is unclear. This study aimed to investigate whether BMD and the cross-sectional area (CSA) of lumbar muscles are related to VI in OVFs. METHODS: On the basis of the thoracolumbar lateral radiographs of 95 acute OVFs in postmenopausal women (mean age 80.6 years; range: 64-103 years), supine and standing vertebral collapse rates (CRsp and CRst, respectively) were determined. Subsequently, VI was defined as follows: VI=CRst-CRsp. Using axial T2-weighted magnetic resonance imaging (MRI), CSA of the psoas major, erector spinae, and multifidus muscles at the L3/4 intervertebral disc level were measured. The BMD of the lumbar spine and proximal femur (total hip) was measured for all participants using dual-energy X-ray absorptiometry. The patients were classified into group 1 (VI <20%) and group 2 (VI ≥20%). RESULTS: We observed a negative correlation between VI and CSA of the erector spinae muscle (r=-0.3962, P<0.0001). No significant correlations were observed between VI and BMD. The CSA of the erector spinae muscle in group 2 was significantly lower than that in group 1 (P=0.0002). No significant difference in the BMD or the CSA of the psoas major or multifidus muscles was observed between the two groups. A multivariable analysis of factors of VI was performed. Both age (odds ratio [OR], 1.099; 95% confidence interval [CI], 1.015-1.189; P=0.020) and the CSA of the erector spinae (OR, 0.996; 95% CI, 0.993-0.999; P=0.020) were significant predictors of high VI. CONCLUSIONS: Although the severity of OVFs was related to the CSA of the erector spinae muscle, it was not associated with BMD.

Effects of conservative treatment of 2-week rigorous bed rest on muscle disuse atrophy in osteoporotic vertebral fracture patients.

Objective: Osteoporotic vertebral fracture (OVF) is conventionally treated with conservative management such as bed rest, but a relatively prolonged bed rest has the potential risk of muscle disuse atrophy. This study aimed to examine whether the 2-week of rigorous bed rest affects muscle disuse atrophy in OVF patients. Patients and Methods: A total of 54 OVF patients (16 males; 38 females; mean age, 80.2 ± 9.2 years) were treated with an initial 2-week rigorous bed rest by hospitalization with persistent rehabilitation. Cognitive function, swallowing function, grip strength, and lower extremity circumference were evaluated at three-time points (admission, end of bed rest, and discharge). Results: Of the 51 patients who were able to walk independently before the injury, one patient (2.0%) had to use a wheelchair after the injury. During hospitalization, cognitive function decline was observed in 33.3% of patients, but not in patients with Revised Hasegawa's Dementia Scale score ≥25 at admission. Swallowing function decline was observed in one patient, and none of the patients developed aspiration pneumonia during hospitalization. The grip strength significantly improved both at the end of bed rest (P=0.04) and discharge (P=0.02). Although the lower extremity circumference significantly decreased at the end of bed rest (P<0.01), it was recovered afterward. The lower extremity circumference did not significantly differ between the admission and discharge (P=0.17). Conclusion: Our results suggested that conservative treatment of OVF through an initial 2-week rigorous bed rest with persistent hospital rehabilitation poses a low risk of muscle disuse atrophy. If cognitive dysfunction is observed on admission, close monitoring for exacerbation should be performed during the hospital stay.

Author Correction: Synthetic RNA-based logic computation in mammalian cells.

The original version of this Article contained an error in the fourth sentence of the second paragraph of the 'Improving the performance of miRNA-responsive circuits' section of the Results, which incorrectly read 'We confirmed a significant fold-change between ON and OFF states (from 3.5- to 9.0-fold) in 293FT cells (Supplementary Figure 3).' The correct version states '4.6' in place of '3.5'. This has been corrected in both the PDF and HTML versions of the Article.The original version of the Supplementary Information contained a corresponding error in Supplementary Figure 3. The HTML has been updated to include a corrected version of the Supplementary Information.

MicroRNA-Based Separation of Cortico-Fugal Projection Neuron-Like Cells Derived From Embryonic Stem Cells.

The purification of pluripotent stem cell-derived cortico-fugal projection neurons (PSC-CFuPNs) is useful for disease modeling and cell therapies related to the dysfunction of cortical motor neurons, such as amyotrophic lateral sclerosis (ALS) or stroke. However, no CFuPN-specific surface markers for the purification are known. Recently, microRNAs (miRNAs) have been reported as alternatives to surface markers. Here, we investigated this possibility by applying the miRNA switch, an mRNA technology, to enrich PSC-CFuPNs. An array study of miRNAs in mouse fetal brain tissue revealed that CFuPNs highly express miRNA-124-3p at E14.5 and E16.5. In response, we designed a miRNA switched that responds to miRNA-124-3p and applied it to mouse embryonic stem cell (ESC)-derived cortical neurons. Flow cytometry and quantitative polymerase chain reaction (qPCR) analyses showed the miRNA-124-3p switch enriched CFuPN-like cells from this population. Immunocytechemical analysis confirmed vGlut1/Emx1/Bcl11b triple positive CFuPN-like cells were increased from 6.5 to 42%. Thus, our miRNA-124-3p switch can uniquely enrich live CFuPN-like cells from mouse ESC-derived cortical neurons.

Synthetic RNA-based logic computation in mammalian cells.

Synthetic biological circuits are designed to regulate gene expressions to control cell function. To date, these circuits often use DNA-delivery methods, which may lead to random genomic integration. To lower this risk, an all RNA system, in which the circuit and delivery method are constituted of RNA components, is preferred. However, the construction of complexed circuits using RNA-delivered devices in living cells has remained a challenge. Here we show synthetic mRNA-delivered circuits with RNA-binding proteins for logic computation in mammalian cells. We create a set of logic circuits (AND, OR, NAND, NOR, and XOR gates) using microRNA (miRNA)- and protein-responsive mRNAs as decision-making controllers that are used to express transgenes in response to intracellular inputs. Importantly, we demonstrate that an apoptosis-regulatory AND gate that senses two miRNAs can selectively eliminate target cells. Thus, our synthetic RNA circuits with logic operation could provide a powerful tool for future therapeutic applications.

Simple quantification of Cu,Zn-superoxide dismutase activity after separation by non-denaturing isoelectric focusing.

The Cu,Zn-superoxide dismutase (SOD) activity in bovine retina cytosol was separated from retinal pigment using short-length non-denaturing isoelectric focusing (IEF) (15-mm long x 1.3-mm i.d. column) and detected using non-denaturing two-dimensional electrophoresis (2-DE). After the SOD and pigment in the retina cytosol are separated, SOD activity can be quantified by water-soluble tetrazolium salt. We also found that SOD separated by this IEF retained its native function.

Intracellular expression of the Salmonella plasmid virulence protein, SpvB, causes apoptotic cell death in eukaryotic cells.

The spv genes carried on the Salmonella virulence plasmid are commonly associated with severe systemic infection in experimental animals. The SpvB virulence-associated protein has been shown to ADP-ribosylate actin, and this enzymatic activity is essential for virulence in mice. Here, we present evidence that intracellular expression of SpvB protein induces not only disruption of actin filaments but also apoptotic cell death in eukaryotic cells.

Relationship between cerebral interstitial levels of amino acids and phosphorylation potential during secondary energy failure in hypoxic-ischemic newborn piglets.

The aim of this study was to determine the validity of the hypothesis that excitatory amino acids are related to phosphorylation potential during primary and secondary cerebral energy failure observed in asphyxiated infants. We report here the results of experiments using newborn piglets subjected to severe transient cerebral hypoxia-ischemia followed by resuscitation. We examined cerebral energy metabolism by phosphorus nuclear magnetic resonance spectroscopy and changes in levels of amino acid neurotransmitters in the cortex by microdialysis before, during, and up to 24 h after the hypoxic-ischemic insult. The concentrations of aspartate, glutamate, taurine, and gamma-aminobutyric acid were significantly elevated during the hypoxic-ischemic insult compared with prebaseline values. Shortly after resuscitation, glutamate, taurine, and gamma-aminobutyric acid concentrations decreased but then began to increase again. These secondary elevations were greater than the primary elevations. A negative linear correlation was found between primary interstitial levels of glutamate and taurine and minimum values of phosphocreatine/inorganic phosphate during the secondary energy failure. The cerebral energy state depended on the time course of changes in excitatory amino acids, suggesting that amino acids play distinct roles during the early and delayed phases of injury.