The Cas9/guide RNA (Cas9/gRNA) system is commonly used for genome editing. mRNA expressing Cas9 can induce innate immune responses, reducing Cas9 expression. First-generation Cas9 mRNAs were modified ...with pseudouridine and 5-methylcytosine to reduce innate immune responses. We combined four approaches to produce more active, less immunogenic second-generation Cas9 mRNAs. First, we developed a novel co-transcriptional capping method yielding natural Cap 1. Second, we screened modified nucleotides in Cas9 mRNA to identify novel modifications that increase Cas9 activity. Third, we depleted the mRNA of uridines to improve mRNA activity. Lastly, we tested high-performance liquid chromatography (HPLC) purification to remove double-stranded RNAs. The activity of these mRNAs was tested in cell lines and primary human CD34+ cells. Cytokines were measured in whole blood and mice. These approaches yielded more active and less immunogenic mRNA. Uridine depletion (UD) most impacted insertion or deletion (indel) activity. Specifically, 5-methoxyuridine UD induced indel frequencies as high as 88% (average ± SD = 79% ± 11%) and elicited minimal immune responses without needing HPLC purification. Our work suggests that uridine-depleted Cas9 mRNA modified with 5-methoxyuridine (without HPLC purification) or pseudouridine may be optimal for the broad use of Cas9 both in vitro and in vivo.
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Glioblastomas have been proposed to be maintained by highly tumorigenic glioblastoma stem cells (GSCs) that are resistant to current therapy. Therefore, targeting GSCs is critical for developing ...effective therapies for glioblastoma. In this study, we identify the regulatory cascade of the nuclear receptor TLX and the DNA hydroxylase Ten eleven translocation 3 (TET3) as a target for human GSCs. We show that knockdown of TLX expression inhibits human GSC tumorigenicity in mice. Treatment of human GSC-grafted mice with viral vector-delivered TLX shRNA or nanovector-delivered TLX siRNA inhibits tumour development and prolongs survival. Moreover, we identify TET3 as a potent tumour suppressor downstream of TLX to regulate the growth and self-renewal in GSCs. This study identifies the TLX-TET3 axis as a potential therapeutic target for glioblastoma.
Systematic Review.
We sought to determine which method of the pedicle screw (PS) placement is most accurate and understand how the development of subsequent generations of robotic systems has changed ...placement accuracy over time.
Previous studies have demonstrated the success of robotic PS placement, but how this accuracy compares to other methods is unclear.
A systematic review following PRISMA Guidelines was performed on articles published between January 2000 and August 2021, comparing PS insertion methods with at least 10 screws per study arm. Single and multiple-arm trials were included. Data were extracted for patient outcomes, including optimal PS placement, misplacement, and accuracy. The logit-event rate of misplacement was calculated for each study. P values were adjusted for multiple comparisons using the Tukey post hoc correction.
Our search revealed 127 studies, and 156 comparative arms, with 77,360 pedicle screws placed using five different modalities. Meta-regression of pooled accuracy revealed no significant changes in PS accuracy over time for freehand, 2D fluoroscopic navigation, 3D fluoroscopic navigation, and computed tomography navigation. Robotic navigation had a significant increase in accuracy over time ( P =0.036). Pooled misplacement rates were also compared across all modalities. Robotics was found to have the lowest rates of misplacement for PS compared to freehand ( P =0.0015) and 2D fluoroscopic navigation ( P =0.026).
Our analysis is the largest study to date on pedicle screw placement. Pedicle screw placement through robotics was found to be superior due to its low misplacement rates compared with other modalities. Intraoperative 3D fluoroscopic navigation was found to have comparable misplacement rates. In addition, pedicle screw placement accuracy with robotics has continued to improve over time. This speaks to both the stability of the technology and the potential for continued improvement with new and more accurate robotic systems.
Purpsose
Cerebral radiation necrosis (RN) is often a delayed phenomenon occurring several months to years after the completion of radiation treatment. Differentiating RN from tumor recurrence ...presents a diagnostic challenge on standard MRI. To date, no evidence-based guidelines exist regarding imaging modalities best suited for this purpose. We aim to review the current literature and perform a diagnostic meta-analysis comparing various imaging modalities that have been studied to differentiate tumor recurrence and RN.
Methods
A systematic search adherent to PRISMA guidelines was performed using Scopus, PubMed/MEDLINE, and Embase. Pooled sensitivities and specificities were determined using a random-effects or fixed-effects proportional meta-analysis based on heterogeneity. Using diagnostic odds ratios, a diagnostic frequentist random-effects network meta-analysis was performed, and studies were ranked using P-score hierarchical ranking.
Results
The analysis included 127 studies with a total of 220 imaging datasets, including the following imaging modalities: MRI (n = 10), MR Spectroscopy (MRS) (n = 28), dynamic contrast-enhanced MRI (n = 7), dynamic susceptibility contrast MRI (n = 36), MR arterial spin labeling (n = 5), diffusion-weighted imaging (n = 13), diffusion tensor imaging (DTI) (n = 2), PET (n = 89), and single photon emission computed tomography (SPECT) (n = 30). MRS had the highest pooled sensitivity (90.7%). DTI had the highest pooled specificity (90.5%). Our hierarchical ranking ranked SPECT and MRS as most preferable, and MRI was ranked as least preferable.
Conclusion
These findings suggest SPECT and MRS carry greater utility than standard MRI in distinguishing RN from tumor recurrence.
Inactivation of the E6AP E3 ubiquitin ligase (UBE3A gene) causes Angelman syndrome, while aberrant degradation of p53 by E6AP is implicated in cervical cancers. Herein, we describe the development of ...photo-cross-linkers to discover catalytic residues of E6AP. Using these cross-linkers, we identified covalent modifications of the E6AP catalytic cysteine and two lysines: Lys847 and Lys799. Lys847 is required for the formation of Lys48-linked polyubiquitin chains, while the K799A E6AP mutant was more active at producing Lys48-linked polyubiquitin chains. Thus, opposing roles of Lys799 and Lys847 pave the path forward to pharmacological inhibitors or activators of E6AP for therapeutic purposes.
Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation
, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD8
. ...However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1
promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin-RING proteins.
E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates
. However, rather ...than functioning individually, many neddylated cullin-RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family-which together account for nearly half of all ubiquitin ligases in humans-form E3-E3 super-assemblies
. Here, by studying CRLs in the SKP1-CUL1-F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3-E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3-E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3-E3 super-assembly may therefore underlie widespread ubiquitylation.
Recent experiments have suggested that enzymes and other small molecules chemotax toward their substrates. However, the physical forces driving this chemotaxis are currently debated. In this work, we ...consider a simple thermodynamic theory for molecular chemotaxis that is based on the McMillan-Mayer theory of dilute solutions and Schellman's theory for macromolecular binding. Even in the absence of direct interactions, the chemical binding equilibrium introduces a coupling term into the relevant free energy, which then reduces the chemical potential of both enzymes and their substrates. Assuming a local thermodynamic equilibrium, this binding contribution to the chemical potential generates an effective thermodynamic force that promotes chemotaxis by driving each solute toward its binding partner. Our numerical simulations demonstrate that, although small, this thermodynamic force is qualitatively consistent with several experimental studies. Thus, our study may provide additional insight into the role of the thermodynamic binding free energy for molecular chemotaxis.
Optimization of a solid state polarizing bender for cold neutrons Shah, V.R.; Washington, A.L.; Stonaha, P. ...
Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment,
12/2014, Letnik:
768
Journal Article
Recenzirano
We have designed a solid state bender to polarize cold neutrons for the Spin Echo Scattering Angle Measurement (SESAME) instrument at the Low Energy Neutron Source (LENS) at Indiana University. The ...design attempts to achieve high neutron polarization across a wide range of neutron wavelengths and divergence angles by optimizing the supermirror coating materials. The transmission and polarizing efficiency of the bender were modeled using the VITESS software, then measured at both continuous-wave and pulsed neutron sources. While the measured peak neutron transmission and polarization agree reasonably well with simulations, neither quantity has been successfully modeled for long wavelength neutrons. These results imply an insufficient understanding of the magnetic microstructure of the supermirror coatings used.
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