Morphogenesis of mammalian facial processes requires coordination of cellular proliferation, migration, and apoptosis to develop intricate features. Cleft lip and/or palate (CL/P), the most frequent ...human craniofacial birth defect, can be caused by perturbation of any of these programs. Mutations of WNT, P63, and IRF6 yield CL/P in humans and mice; however, how these genes are regulated remains elusive. We generated mouse lines lacking Pbx genes in cephalic ectoderm and demonstrated that they exhibit fully penetrant CL/P and perturbed Wnt signaling. We also characterized a midfacial regulatory element that Pbx proteins bind to control the expression of Wnt9b-Wnt3, which in turn regulates p63. Altogether, we establish a Pbx-dependent Wnt-p63-Irf6 regulatory module in midfacial ectoderm that is conserved within mammals. Dysregulation of this network leads to localized suppression of midfacial apoptosis and CL/P. Ectopic Wnt ectodermal expression in Pbx mutants rescues the clefting, opening avenues for tissue repair.
► Pbx genes promote midfacial morphogenesis via direct activation of Wnt9b-Wnt3 ► A Pbx-Wnt-p63-Irf6 module induces apoptosis at the embryonic lambdoidal junction (λ) ► Disruption of the Pbx-Wnt-p63-Irf6 module, conserved in mammals, causes cleft lip ► Ectopic Wnt1 expression can rescue genetically cleft lip in Pbx mutants
CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin ...mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated KrasG12D mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.
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•Generation of mouse lines with Cre-dependent and constitutive Cas9 expression•Viral/nonviral delivery of sgRNA to the brain, vasculature, immune cells, and lung•Modeling of competition between gain- and loss-of-function mutations in lung cancer•A convenient platform for achieving efficient genome editing in vivo
Viral and nonviral delivery of sgRNAs in CRISPR-Cas9 knockin mice enables diverse genome engineering applications in biology and disease modeling.
People routinely hear and understand speech at rates of 120–200 words per minute 1, 2. Thus, speech comprehension must involve rapid, online neural mechanisms that process words’ meanings in an ...approximately time-locked fashion. However, electrophysiological evidence for such time-locked processing has been lacking for continuous speech. Although valuable insights into semantic processing have been provided by the “N400 component” of the event-related potential 3–6, this literature has been dominated by paradigms using incongruous words within specially constructed sentences, with less emphasis on natural, narrative speech comprehension. Building on the discovery that cortical activity “tracks” the dynamics of running speech 7–9 and psycholinguistic work demonstrating 10–12 and modeling 13–15 how context impacts on word processing, we describe a new approach for deriving an electrophysiological correlate of natural speech comprehension. We used a computational model 16 to quantify the meaning carried by words based on how semantically dissimilar they were to their preceding context and then regressed this measure against electroencephalographic (EEG) data recorded from subjects as they listened to narrative speech. This produced a prominent negativity at a time lag of 200–600 ms on centro-parietal EEG channels, characteristics common to the N400. Applying this approach to EEG datasets involving time-reversed speech, cocktail party attention, and audiovisual speech-in-noise demonstrated that this response was very sensitive to whether or not subjects understood the speech they heard. These findings demonstrate that, when successfully comprehending natural speech, the human brain responds to the contextual semantic content of each word in a relatively time-locked fashion.
•EEG reflects semantic processing of continuous natural speech•Mapping function of semantic features to neural response shares traits with the N400•Computational language models capture neural-related measures of semantic dissimilarity•Index of semantic processing is sensitive to attention and speech intelligibility
Electrophysiological studies eliciting semantic-related neural activity typically involve paradigms using incongruous words within specially constructed sentences. Broderick et al. use computational language modeling and linear regression to successfully index the semantic processing of continuous, natural speech using EEG.
Summary Background Based on the early results of the Placement of Aortic Transcatheter Valves (PARTNER) trial, transcatheter aortic valve replacement (TAVR) is an accepted treatment for patients with ...severe aortic stenosis who are not suitable for surgery. However, little information is available about the late clinical outcomes in such patients. Methods We did this randomised controlled trial at 21 experienced valve centres in Canada, Germany, and the USA. We enrolled patients with severe symptomatic inoperable aortic stenosis and randomly assigned (1:1) them to transfemoral TAVR or to standard treatment, which often included balloon aortic valvuloplasty. Patients and their treating physicians were not masked to treatment allocation. The randomisation was done centrally, and sites learned of the assignment only after a patient had been screened, consented, and entered into the database. The primary outcome of the trial was all-cause mortality at 1 year in the intention-to-treat population, here we present the prespecified findings after 5 years. This study is registered with ClinicalTrials.gov , number NCT00530894. Findings We screened 3015 patients, of whom 358 were enrolled (mean age 83 years, Society of Thoracic Surgeons Predicted Risk of Mortality 11·7%, 54% female). 179 were assigned to TAVR treatment and 179 were assigned to standard treatment. 20 patients crossed over from the standard treatment group and ten withdrew from study, leaving only six patients at 5 years, of whom five had aortic valve replacement treatment outside of the study. The risk of all-cause mortality at 5 years was 71·8% in the TAVR group versus 93·6% in the standard treatment group (hazard ratio 0·50, 95% CI 0·39–0·65; p<0·0001). At 5 years, 42 (86%) of 49 survivors in the TAVR group had New York Heart Association class 1 or 2 symptoms compared with three (60%) of five in the standard treatment group. Echocardiography after TAVR showed durable haemodynamic benefit (aortic valve area 1·52 cm2 at 5 years, mean gradient 10·6 mm Hg at 5 years), with no evidence of structural valve deterioration. Interpretation TAVR is more beneficial than standard treatment for treatment of inoperable aortic stenosis. TAVR should be strongly considered for patients who are not surgical candidates for aortic valve replacement to improve their survival and functional status. Appropriate selection of patients will help to maximise the benefit of TAVR and reduce mortality from severe comorbidities. Funding Edwards Lifesciences.
Summary Background The Placement of Aortic Transcatheter Valves (PARTNER) trial showed that mortality at 1 year, 2 years, and 3 years is much the same with transcatheter aortic valve replacement ...(TAVR) or surgical aortic valve replacement (SAVR) for high-risk patients with aortic stenosis. We report here the 5-year outcomes. Methods We did this randomised controlled trial at 25 hospitals, in Canada (two), Germany (one), and the USA (23). We used a computer-generated randomisation sequence to randomly assign high-risk patients with severe aortic stenosis to either SAVR or TAVR with a balloon-expandable bovine pericardial tissue valve by either a transfemoral or transapical approach. Patients and their treating physicians were not masked to treatment allocation. The primary outcome of the trial was all-cause mortality in the intention-to-treat population at 1 year, we present here predefined outcomes at 5 years. The study is registered with ClinicalTrials.gov , number NCT00530894. Findings We screened 3105 patients, of whom 699 were enrolled (348 assigned to TAVR, 351 assigned to SAVR). Overall mean Society of Thoracic Surgeons Predicted Risk of Mortality score was 11·7%. At 5 years, risk of death was 67·8% in the TAVR group compared with 62·4% in the SAVR group (hazard ratio 1·04, 95% CI 0·86–1·24; p=0·76). We recorded no structural valve deterioration requiring surgical valve replacement in either group. Moderate or severe aortic regurgitation occurred in 40 (14%) of 280 patients in the TAVR group and two (1%) of 228 in the SAVR group (p<0·0001), and was associated with increased 5-year risk of mortality in the TAVR group (72·4% for moderate or severe aortic regurgitation vs 56·6% for those with mild aortic regurgitation or less; p=0·003). Interpretation Our findings show that TAVR as an alternative to surgery for patients with high surgical risk results in similar clinical outcomes. Funding Edwards Lifesciences.
Sex determination can be robustly genetic, strongly environmental, or genetic subject to environmental perturbation. The genetic basis of sex determination is unknown for zebrafish (Danio rerio), a ...model for development and human health. We used RAD-tag population genomics to identify sex-linked polymorphisms. After verifying this "RAD-sex" method on medaka (Oryzias latipes), we studied two domesticated zebrafish strains (AB and TU), two natural laboratory strains (WIK and EKW), and two recent isolates from nature (NA and CB). All four natural strains had a single sex-linked region at the right tip of chromosome 4, enabling sex genotyping by PCR. Genotypes for the single nucleotide polymorphism (SNP) with the strongest statistical association to sex suggested that wild zebrafish have WZ/ZZ sex chromosomes. In natural strains, "male genotypes" became males and some "female genotypes" also became males, suggesting that the environment or genetic background can cause female-to-male sex reversal. Surprisingly, TU and AB lacked detectable sex-linked loci. Phylogenomics rooted on D. nigrofasciatus verified that all strains are monophyletic. Because AB and TU branched as a monophyletic clade, we could not rule out shared loss of the wild sex locus in a common ancestor despite their independent domestication. Mitochondrial DNA sequences showed that investigated strains represent only one of the three identified zebrafish haplogroups. Results suggest that zebrafish in nature possess a WZ/ZZ sex-determination mechanism with a major determinant lying near the right telomere of chromosome 4 that was modified during domestication. Strains providing the zebrafish reference genome lack key components of the natural sex-determination system but may have evolved variant sex-determining mechanisms during two decades in laboratory culture.
The induction of a strong cytotoxic T cell response is an important prerequisite for successful immunotherapy against many viral diseases and tumors. Nucleotide vaccines, including mRNA vaccines with ...their intracellular antigen synthesis, have been shown to be potent activators of a cytotoxic immune response. The intracellular delivery of mRNA vaccines to the cytosol of antigen presenting immune cells is still not sufficiently well understood. Here, we report on the development of a lipid nanoparticle formulation for the delivery of mRNA vaccines to induce a cytotoxic CD 8 T cell response. We show transfection of dendritic cells, macrophages, and neutrophils. The efficacy of the vaccine was tested in an aggressive B16F10 melanoma model. We found a strong CD 8 T cell activation after a single immunization. Treatment of B16F10 melanoma tumors with lipid nanoparticles containing mRNA coding for the tumor-associated antigens gp100 and TRP2 resulted in tumor shrinkage and extended the overall survival of the treated mice. The immune response can be further increased by the incorporation of the adjuvant LPS. In conclusion, the lipid nanoparticle formulation presented here is a promising vector for mRNA vaccine delivery, one that is capable of inducing a strong cytotoxic T cell response. Further optimization, including the incorporation of different adjuvants, will likely enhance the potency of the vaccine.
Insulated Molecular Wires Frampton, Michael J.; Anderson, Harry L.
Angewandte Chemie (International ed.),
01/2007, Letnik:
46, Številka:
7
Journal Article
Recenzirano
An astonishing assortment of structures have been described as “insulated molecular wires” (IMWs), thus illustrating the diversity of approaches to molecular‐scale insulation. These systems ...demonstrate the scope of encapsulation in the molecular engineering of optoelectronic materials and organic semiconductors. This Review surveys the synthesis and structural characterization of IMWs, and highlights emerging structure–property relationships to determine how insulation can enhance the behavior of a molecular wire. We focus mainly on three IMW architectures: polyrotaxanes, polymer‐wrapped π systems, and dendronized polymers, and compare the properties of these systems with those of conjugated polymers threaded through mesoporous frameworks and zeolites. Encapsulation of molecular wires can enhance properties as diverse as luminescence, electrical transport, and chemical stability, which points to applications in electroluminescent displays, sensors, and the photochemical generation of hydrogen.
Plastic nanoelectronics! Threading a molecular organic semiconductor inside an insulating sheath (see example) provides control of properties such as luminescence, electrical transport, and chemical stability. Applications in electroluminescent displays, sensors, and hydrogen generators are now within reach.
We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by ...immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed that Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric acidemia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.
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•Lysine glutarylation is a protein posttranslational modification•SIRT5 can catalyze the enzymatic removal of lysine glutarylation•Proteomic analyses identify a link between lysine glutarylation and metabolism•Glutarylation suppresses CPS1 activity, which is targeted by SIRT5 for removal
Tan et al. report a new type of evolutionarily conserved posttranslational modification, lysine glutarylation, targeted by SIRT5 which impacts metabolic processes. CPS1, the rate-limiting enzyme in urea cycle, is suppressed by glutarylation in glutaric academia type I disease and is targeted by SIRT5 for deglutarylation.
The scientific community has responded to the coronavirus disease 2019 (COVID-19) pandemic by rapidly undertaking research to find effective strategies to reduce the burden of this disease. ...Encouragingly, researchers from a diverse array of fields are collectively working towards this goal. Research with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undertaken in high-containment laboratories; however, it is often desirable to work with samples at lower-containment levels. To facilitate the transfer of infectious samples from high-containment laboratories, we have tested methods commonly used to inactivate virus and prepare the sample for additional experiments. Incubation at 80°C, a range of detergents, Trizol reagents, and UV energies were successful at inactivating a high titer of SARS-CoV-2. Methanol and paraformaldehyde incubation of infected cells also inactivated the virus. These protocols can provide a framework for in-house inactivation of SARS-CoV-2 in other laboratories, ensuring the safe use of samples in lower-containment levels.
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