As the most common subtype of Leber congenital amaurosis (LCA), LCA10 is a severe retinal dystrophy caused by mutations in the CEP290 gene. The most frequent mutation found in patients with LCA10 is ...a deep intronic mutation in CEP290 that generates a cryptic splice donor site. The large size of the CEP290 gene prevents its use in adeno-associated virus (AAV)-mediated gene augmentation therapy. Here, we show that targeted genomic deletion using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system represents a promising therapeutic approach for the treatment of patients with LCA10 bearing the CEP290 splice mutation. We generated a cellular model of LCA10 by introducing the CEP290 splice mutation into 293FT cells and we showed that guide RNA pairs coupled with SpCas9 were highly efficient at removing the intronic splice mutation and restoring the expression of wild-type CEP290. In addition, we demonstrated that a dual AAV system could effectively delete an intronic fragment of the Cep290 gene in the mouse retina. To minimize the immune response to prolonged expression of SpCas9, we developed a self-limiting CRISPR/Cas9 system that minimizes the duration of SpCas9 expression. These results support further studies to determine the therapeutic potential of CRISPR/Cas9-based strategies for the treatment of patients with LCA10.
LCA10 is caused by mutations in CEP290, which is too large for the gene therapy vector AAV. Here, Ruan et al. show that gene editing using CRISPR/Cas9 represents a promising therapeutic approach to treat LCA10. They also develop a self-limiting CRISPR/Cas9 system to limit duration of Cas9 expression.
Herein, we report that vascular endothelial growth factor A (VEGF‐A) engages the PI3K/Akt pathway by a previously unknown mechanism that involves three tyrosine kinases. Upon VEGF‐A‐dependent ...activation of VEGF receptor‐2 (VEGFR‐2), and subsequent TSAd‐mediated activation of Src family kinases (SFKs), SFKs engage the receptor tyrosine kinase Axl via its juxtamembrane domain to trigger ligand‐independent autophosphorylation at a pair of YXXM motifs that promotes association with PI3K and activation of Akt. Other VEGF‐A‐mediated signalling pathways are independent of Axl. Interfering with Axl expression or function impairs VEGF‐A‐ but not bFGF‐dependent migration of endothelial cells. Similarly, Axl null mice respond poorly to VEGF‐A‐induced vascular permeability or angiogenesis, whereas other agonists induce a normal response. These results elucidate the mechanism by which VEGF‐A activates PI3K/Akt, and identify previously unappreciated potential therapeutic targets of VEGF‐A‐driven processes.
VEGF‐A, but not other angiogenic growth factors, regulate endothelial cell migration by activating PI3K/AKT signalling through a novel pathway involving autophosphorylation of the receptor tyrosine kinase Axl.
Although a high level of lactate is quintessential to both tumors and wound healing, the manner by which lactate impacts endothelial cells to promote angiogenesis and thereby create or restore ...vascular perfusion to growing tissues has not been fully elucidated. Here we report that lactate activated the PI3K/Akt pathway in primary human endothelial cells. Furthermore, activating this signaling pathway was required for lactate-stimulated organization of endothelial cells into tubes and for sprouting of vessels from mouse aortic explants. Lactate engaged the PI3K/Akt pathway via ligand-mediated activation of the three receptor tyrosine kinases Axl, Tie2, and VEGF receptor 2. Neutralizing the ligands for these receptor tyrosine kinases, pharmacologically inhibiting their kinase activity or suppressing their expression largely eliminated the ability of cells and explants to respond to lactate. Elucidating the mechanism by which lactate communicates with endothelial cells presents a previously unappreciated opportunity to improve our understanding of the angiogenic program and to govern it.
Background: The signaling events by which lactate instructs endothelial cells to undergo angiogenesis are incompletely resolved.
Results: Lactate activated Akt in endothelial cells by promoting ligand-dependent activation of multiple RTKs.
Conclusion: Metabolic products such as lactate engage established regulators of angiogenesis.
Significance: Our results advance our appreciation of the interface between metabolism and angiogenesis.
Objective
The study objective was to examine the association between phentermine/topiramate therapy and weight loss and adverse events in adults with overweight or obesity by meta‐analysis and ...systematic review.
Methods
Medical Subject Headings and free‐text terms were selected to search for eligible trials in PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), and Embase up to April 18, 2020. The quality of randomized controlled trials was evaluated by the Cochrane risk‐of‐bias tool. Meta‐analysis was performed using random‐effect models.
Results
Phentermine/topiramate therapy resulted in an average weight loss of 7.73 kg (95% CI: 6.60‐8.85) in general compared with placebo. The weight loss was related to the dose of phentermine/topiramate. Compared with placebo, the average weight loss was 3.55 kg (95% CI: 2.22‐4.88) for 3.75/23 mg, 7.27 kg (95% CI: 6.40‐8.13) for 7.5/46 mg, and 8.25 kg (95% CI: 6.92‐9.79) for 15/92 mg. For phentermine/topiramate participants in different weight‐loss subgroups, the weight loss of participants with ≥5%, ≥10%, and ≥15% baseline weight loss was 3.18 (95% CI: 2.75‐3.67), 5.32 (95% CI: 4.53‐6.25), and 5.65 (95% CI: 3.55‐9.01), respectively. Compared with placebo, the adverse events associated with the treatment mainly included dysgeusia (odds ratio OR = 8.86, 95% CI: 5.65‐13.89), paresthesia (OR = 8.51, 95% CI: 6.20‐11.67), dry mouth (OR = 6.71, 95% CI: 5.03‐8.94), disturbance in attention (OR = 4.48, 95% CI: 2.39‐8.41), irritability (OR = 4.10, 95% CI: 2.29‐7.33), hypoesthesia (OR = 3.81, 95% CI: 1.32‐11.00), constipation (OR = 2.43, 95% CI: 2.02‐2.93), and dizziness (OR = 2.26, 95% CI: 1.72‐2.98). Phentermine/topiramate also reduced waist circumference, blood pressure, blood sugar levels, and lipid levels.
Conclusions
Phentermine/topiramate has considerable benefit in reducing body weight, and the efficacy was closely related to the dosage. However, it increased the risk of nervous system‐related adverse events.
Austenite stainless steels with gradient nanostructure exhibit exceptional combination of high yield strength and high ductility. In order to describe their structure-property relation, a theoretical ...model is proposed in this work, in which the depth-dependent bimodal grain size distribution and nanotwin-nanograin composite structure are taken into account. The micromechanical model and the Voigt rule of mixture are adopted in deriving the constitutive relations. Furthermore, the evolution and influence of the nano/micro cracks/voids are considered for predicting the failure strain. The numerical results based on the theoretical model agree well with experimental results in terms of the yield strength, ductility, and the strain hardening rate, demonstrating that the proposed model can well describe the mechanical properties of gradient-nanostructured austenite stainless steels. We further study the variations of the yield strength and ductility of gradient-nanograined and gradient-nanotwinned 304 stainless steels with different distribution of grain size and twin spacing along the depth, which shows that the present model can be applied to optimize the combination of strength and ductility of the gradient-nanostructured metals by tuning depth-dependent distributions of microstructures.
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Microbial dysbiosis in the upper digestive tract is linked to an increased risk of esophageal squamous cell carcinoma (ESCC). Overabundance of Porphyromonas gingivalis is associated with shorter ...survival of ESCC patients. We investigated the molecular mechanisms driving aggressive progression of ESCC by P. gingivalis. Intracellular invasion of P. gingivalis potentiated proliferation, migration, invasion, and metastasis abilities of ESCC cells via transforming growth factor-β (TGFβ)-dependent Drosophila mothers against decapentaplegic homologs (Smads)/Yes-associated protein (YAP)/Transcriptional coactivator with PDZ-binding motif (TAZ) activation. Smads/YAP/TAZ/TEA domain transcription factor1 (TEAD1) complex formation was essential to initiate downstream target gene expression, inducing an epithelial-mesenchymal transition (EMT) and stemness features. Furthermore, P. gingivalis augmented secretion and bioactivity of TGFβ through glycoprotein A repetitions predominant (GARP) up-regulation. Accordingly, disruption of either the GARP/TGFβ axis or its activated Smads/YAP/TAZ complex abrogated the tumor-promoting role of P. gingivalis. P. gingivalis signature genes based on its activated effector molecules can efficiently distinguish ESCC patients into low- and high-risk groups. Targeting P. gingivalis or its activated effectors may provide novel insights into clinical management of ESCC.
The retina is both a sensory organ and a self-sustained circadian clock. Gene targeting studies have revealed that mammalian circadian clocks generate molecular circadian rhythms through coupled ...transcription/translation feedback loops which involve 6 core clock genes, namely Period (Per) 1 and 2, Cryptochrome (Cry) 1 and 2, Clock, and Bmal1 and that the roles of individual clock genes in rhythms generation are tissue-specific. However, the mechanisms of molecular circadian rhythms in the mammalian retina are incompletely understood and the extent to which retinal neural clocks share mechanisms with the suprachiasmatic nucleus (SCN), the central neural clock, is unclear. In the present study, we examined the rhythmic amplitude and period of real-time bioluminescence rhythms in explants of retina from Per1-, Per2-, Per3-, Cry1-, Cry2-, and Clock-deficient mice that carried transgenic PERIOD2::LUCIFERASE (PER2::LUC) or Period1::luciferase (Per1::luc) circadian reporters. Per1-, Cry1- and Clock-deficient retinal and SCN explants showed weakened or disrupted rhythms, with stronger effects in retina compared to SCN. Per2, Per3, and Cry2 were individually dispensable for sustained rhythms in both tissues. Retinal and SCN explants from double knockouts of Cry1 and Cry2 were arrhythmic. Gene effects on period were divergent with reduction in the number of Per1 alleles shortening circadian period in retina, but lengthening it in SCN, and knockout of Per3 substantially shortening retinal clock period, but leaving SCN unaffected. Thus, the retinal neural clock has a unique pattern of clock gene dependence at the tissue level that it is similar in pattern, but more severe in degree, than the SCN neural clock, with divergent clock gene regulation of rhythmic period.
Stereoselectively-fluorinated analogs of pipecolic acid have been investigated through a combined theoretical and experimental approach. Three of the four possible diastereoisomers of ...4,5-difluoropipecolic acid were successfully synthesized via deoxyfluorination chemistry, navigating a complex reaction network that included neighboring group participation, rearrangement, and elimination pathways. A DFT-based conformational study, supported by NMR J-based analysis, revealed that the different diastereoisomers of 4,5-difluoropipecolic acid preferentially adopt different puckers of the six-membered ring. These findings could have future relevance for the conformational control of biologically active peptides.
The influence of the mammalian retinal circadian clock on retinal physiology and function is widely recognized, yet the cellular elements and neural regulation of retinal circadian pacemaking remain ...unclear due to the challenge of long-term culture of adult mammalian retina and the lack of an ideal experimental measure of the retinal circadian clock. In the current study, we developed a protocol for long-term culture of intact mouse retinas, which allows retinal circadian rhythms to be monitored in real time as luminescence rhythms from a PERIOD2::LUCIFERASE (PER2::LUC) clock gene reporter. With this in vitro assay, we studied the characteristics and location within the retina of circadian PER2::LUC rhythms, the influence of major retinal neurotransmitters, and the resetting of the retinal circadian clock by light. Retinal PER2::LUC rhythms were routinely measured from whole-mount retinal explants for 10 d and for up to 30 d. Imaging of vertical retinal slices demonstrated that the rhythmic luminescence signals were concentrated in the inner nuclear layer. Interruption of cell communication via the major neurotransmitter systems of photoreceptors and ganglion cells (melatonin and glutamate) and the inner nuclear layer (dopamine, acetylcholine, GABA, glycine, and glutamate) did not disrupt generation of retinal circadian PER2::LUC rhythms, nor did interruption of intercellular communication through sodium-dependent action potentials or connexin 36 (cx36)-containing gap junctions, indicating that PER2::LUC rhythms generation in the inner nuclear layer is likely cell autonomous. However, dopamine, acting through D1 receptors, and GABA, acting through membrane hyperpolarization and casein kinase, set the phase and amplitude of retinal PER2::LUC rhythms, respectively. Light pulses reset the phase of the in vitro retinal oscillator and dopamine D1 receptor antagonists attenuated these phase shifts. Thus, dopamine and GABA act at the molecular level of PER proteins to play key roles in the organization of the retinal circadian clock.
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