CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic ...mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing.
Irreversible electroporation (IRE), a novel non-thermal ablation technique, is utilized to ablate unresectable solid tumors and demonstrates favorable safety and efficacy in the clinic. IRE applies ...electric pulses to alter the cell transmembrane voltage and causes nanometer-sized membrane defects or pores in the cells, which leads to loss of cell homeostasis and ultimately results in cell death. The major drawbacks of IRE are incomplete ablation and susceptibility to recurrence, which limit its clinical application. Recent studies have shown that IRE promotes the massive release of intracellular concealed tumor antigens that become an "
tumor vaccine," inducing a potential antitumor immune response to kill residual tumor cells after ablation and inhibiting local recurrence and distant metastasis. Therefore, IRE can be regarded as a potential immunomodulatory therapy, and combined with immunotherapy, it can exhibit synergistic treatment effects on malignant tumors, which provides broad application prospects for tumor treatment. This work reviewed the current status of the clinical efficacy of IRE in tumor treatment, summarized the characteristics of local and systemic immune responses induced by IRE in tumor-bearing organisms, and analyzed the specific mechanisms of the IRE-induced immune response. Moreover, we reviewed the current research progress of IRE combined with immunotherapy in the treatment of solid tumors. Based on the findings, we present deficiencies of current preclinical studies of animal models and analyze possible reasons and solutions. We also propose possible demands for clinical research. This review aimed to provide theoretical and practical guidance for the combination of IRE with immunotherapy in the treatment of malignant tumors.
Abstract
Monogenic mutations in the SHANK3 gene, which encodes a postsynaptic scaffold protein, play a causative role in autism spectrum disorder (ASD). Although a number of mouse models with Shank3 ...mutations have been valuable for investigating the pathogenesis of ASD, species-dependent differences in behaviors and brain structures post considerable challenges to use small animals to model ASD and to translate experimental therapeutics to the clinic. We have used clustered regularly interspersed short palindromic repeat/CRISPR-associated nuclease 9 to generate a cynomolgus monkey model by disrupting SHANK3 at exons 6 and 12. Analysis of the live mutant monkey revealed the core behavioral abnormalities of ASD, including impaired social interaction and repetitive behaviors, and reduced brain network activities detected by positron-emission computed tomography (PET). Importantly, these abnormal behaviors and brain activities were alleviated by the antidepressant fluoxetine treatment. Our findings provide the first demonstration that the genetically modified non-human primate can be used for translational research of therapeutics for ASD.
Dear Editor, Despite substantial progress made toward under- standing the molecular changes contributing to autism spectrum disorders （ASD）, the neuropathophysiology underlying ASD remains poorly ...understood 1, 2. Struc- tural brain imaging in humans is valuable, but lacks res- olution at the cellular level. Studies of neuropathology in humans have been hampered by the lack of high quality postmortem brains from individuals with ASD 2. For more than decades, mutant mice have served as major tools to dissect the pathophysiology of ASD because of the wealth of molecular and neurobiological techniques developed for studies with rodents.
The radiative heating increase due to increased CO
2
concentration is the primary source for the rapid adjustment of atmospheric circulation and clouds. In this study, we investigate the rapid ...adjustment resulting from an instantaneous doubling of CO
2
and its physical mechanism using a multiscale modeling framework (MMF). The cloud-resolving model component of this MMF includes a sophisticated third-order turbulence closure and the MMF simulates realistic shallow and deep cloud climatology and boundary layer turbulence. Although the simulated cloud adjustment and its mechanism generally agree with earlier studies with conventional global climate models and another MMF with a lower-order turbulence closure, this MMF simulates an increase in the global-mean shortwave and net cloud radiative cooling and a negative cloud radiative effect change due to cloud adjustment. This result is related to the large increase in low-level clouds over the extratropical and subtropical oceans, resulting from reduced cloud-top entrainment implied from strengthened inversion. The downshift of planetary boundary layer and low-level clouds is generally weaker than that simulated by other models, which is due to reduction of shallow cumulus in the ascending and weak subsidence circulation regimes but to increase of stratocumulus in the strongest subsidence regime. Optically thicker stratocumulus compensates for reduced cooling by shallow cumulus. The reduced strength of all oceanic circulation regimes, which may be contributed by weakened energy transport resulting from water vapor and cloud CO
2
masking effects, not only reduces optical depth of convective clouds but also shifts cloud coverage to lands where deep convection is enhanced.
Background
This article presents a new method using a dermis–outer orbicularis fascia–orbicularis–levator (DOOL) fixation technique for double-eyelid blepharoplasty.
Methods
Our surgical technique ...preserves the preorbicular venous network (POVN) and uses mattress sutures to fix the dermis, outer fascia of the orbicularis oculi muscle, and orbicularis oculi muscle with pretarsal levator aponeurosis (DOOL). Between January 2016 and July 2018, 335 patients were treated with this POVN-preserving DOOL technique (321 women and 14 men; mean age, 29.6 y). The patients were followed up for 6–30 months. The complications were documented, and the overall outcomes of the upper eyelid folds were evaluated by both surgeons and patients as good, fair, or poor.
Results
Among 335 patients, 307 (91.6%) had good results, 17 (5.1%) had fair results, and 11 (3.3%) had poor results. Postoperative complications included partial (
n
=4) or complete (
n
=3) loss of the double-eyelid line and asymmetric folds (
n
=4). Hypertrophic/depressed scars did not occur.
Conclusions
With less invasiveness and secure internal fixation, the DOOL fixation technique with POVN preservation can achieve a stable and natural double-eyelid appearance.
Level of evidence IV
This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors
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.
The sensitivity of nested WRF simulations of precipitating shallow marine cumuli and cold pools to microphysical parameterization is examined. The simulations differ only in their use of two widely ...used double-moment rain microphysical schemes: the Thompson and Morrison schemes. Both simulations produce similar mesoscale variability, with the Thompson scheme producing more weak cold pools and the Morrison scheme producing more strong cold pools, which are associated with more intense shallow convection. The most robust difference is that the cloud cover and LWP are significantly larger in the Morrison simulation than in the Thompson simulation. One-dimensional kinematic simulations confirm that dynamical feedbacks do not mask the impact of microphysics. These also help elucidate that a slower autoconversion process along with a stronger accretion process explains the Morrison scheme's higher cloud fraction for a similar rain mixing ratio. Differences in the raindrop terminal fall speed parameters explain the higher evaporation rate of the Thompson scheme at moderate surface rain rates. Given the implications of the cloud-cover differences for the radiative forcing of the expansive trade wind regime, the microphysical scheme should be considered carefully when simulating precipitating shallow marine cumulus.
The benefits of hypoxia for maintaining the stemness of cultured human bone marrow-derived endothelial progenitor cells (BM EPCs) have previously been demonstrated but the mechanisms responsible ...remain unclear. Growing evidences suggest that cellular metabolism plays an important role in regulating stem cell fate and self-renewal. Here we aimed to detect the changes of glucose metabolism and to explore its role on maintaining the stemness of BM EPCs under hypoxia. We identified the metabolic status of BM EPCs by using extracellular flux analysis, LC-MS/MS, and
C tracing HPLC-QE-MS, and found that hypoxia induced glucose metabolic reprogramming, which manifested as increased glycolysis and pentose phosphate pathway (PPP), decreased tricarboxylic acid (TCA) and mitochondrial respiration. We further pharmacologically altered the metabolic status of cells by employing various of inhibitors of key enzymes of glycolysis, PPP, TCA cycle and mitochondria electron transport chain (ETC). We found that inhibiting glycolysis or PPP impaired cell proliferation either under normoxia or hypoxia. On the contrary, inhibiting pyruvate oxidation, TCA or ETC promoted cell proliferation under normoxia mimicking hypoxic conditions. Moreover, promoting pyruvate oxidation reverses the maintenance effect of hypoxia on cell stemness. Taken together, our data suggest that hypoxia induced glucose metabolic reprogramming maintains the stemness of BM EPCs, and artificial manipulation of cell metabolism can be an effective way for regulating the stemness of BM EPCs, thereby improving the efficiency of cell expansion in vitro.
Train stopping patterns and schedules play critical roles in the service design of metro lines with express/local mode. Previous studies on express/local mode generally handle the design of train ...stopping patterns and schedules independently, which cannot ensure the overall optimality of service provision. In this paper, a mixed integer nonlinear programming model is developed to collaboratively adjust train stopping patterns and schedules in order to minimize passenger travel time in express/local mode. With two types of train services provided, overtaking is allowed and a diverse set of passenger route choices is also incorporated into the proposed model. Linearization techniques are applied to transform this model from nonlinear to linear, which enables the proposed model to be handled by commercial linear solvers. To enhance computing efficiency in large-scale problems, a guided branch-and-cut algorithm is designed. The numerical examples on a test line and a real-world metro line are implemented to demonstrate the effectiveness of the proposed model and approach. The proposed approach is compared to existing approaches to identify the benefits of integrating train stop planning and scheduling decisions. Based on the computational time and the objective value, the guided branch-and-cut algorithm outperforms the direct use of the CPLEX solver.
Glutamate is excitotoxic to neurons. The entry of glutamine or glutamate from the blood into the brain is limited. To overcome this, branched-chain amino acids (BCAAs) catabolism replenishes the ...glutamate in brain cells. Branched-chain amino acid transaminase 1 (BCAT1) activity is silenced by epigenetic methylation in
mutant gliomas. However, glioblastomas (GBMs) express wild type
. Here, we investigated how oxidative stress promotes BCAAs' metabolism to maintain intracellular redox balance and, consequently, the rapid progression of GBMs. We found that reactive oxygen species (ROS) accumulation promoted the nuclear translocation of lactate dehydrogenase A (LDHA), which triggered DOT1L (disruptor of telomeric silencing 1-like)-mediated histone H3K79 hypermethylation and enhanced BCAA catabolism in GBM cells. Glutamate derived from BCAAs catabolism participates in antioxidant thioredoxin (TxN) production. The inhibition of BCAT1 decreased the tumorigenicity of GBM cells in orthotopically transplanted nude mice, and prolonged their survival time. In GBM samples, BCAT1 expression was negatively correlated with the overall survival time (OS) of patients. These findings highlight the role of the non-canonical enzyme activity of LDHA on BCAT1 expression, which links the two major metabolic pathways in GBMs. Glutamate produced by the catabolism of BCAAs was involved in complementary antioxidant TxN synthesis to balance the redox state in tumor cells and promote the progression of GBMs.
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