In utero gene editing has the potential to prenatally treat genetic diseases that result in significant morbidity and mortality before or shortly after birth. We assessed the viral vector-mediated ...delivery of CRISPR-Cas9 or base editor 3 in utero, seeking therapeutic modification of Pcsk9 or Hpd in wild-type mice or the murine model of hereditary tyrosinemia type 1, respectively. We observed long-term postnatal persistence of edited cells in both models, with reduction of plasma PCSK9 and cholesterol levels following in utero Pcsk9 targeting and rescue of the lethal phenotype of hereditary tyrosinemia type 1 following in utero Hpd targeting. The results of this proof-of-concept work demonstrate the possibility of efficiently performing gene editing before birth, pointing to a potential new therapeutic approach for selected congenital genetic disorders.
Olivine-hosted melt inclusions are commonly used to determine pre-eruptive storage conditions. However, this approach relies on the assumption that co-erupted olivines have a simple association with ...their carrier melts. We show that primitive olivine crystal cargoes and their melt inclusions display a high degree of geochemical disequilibrium with their carrier melts at Kīlauea Volcano, Hawai'i. Within a given eruption, melt inclusions trapped in primitive olivine crystals exhibit compositional diversity exceeding that in erupted lava compositions since 1790 CE. This demonstrates that erupting liquids scavenge crystal cargoes from mush piles accumulating diverse melt inclusion populations over timescales of centuries or longer. Entrainment of hot primitive olivines into cooler, evolved carrier melts drives post-entrapment crystallization and sequestration of CO
into vapour bubbles, producing spurious barometric estimates. While scavenged melt inclusion records may not be suitable for the investigation of eruption-specific processes, they record timescales of crystal storage and remobilization within magmatic mush piles.
The distal appendages (DAPs) of centrioles have been proposed to anchor cilia to the plasma membrane, but their molecular composition, assembly, and exact function in ciliogenesis remain poorly ...understood. Using quantitative centrosome proteomics and superresolution microscopy, we identified five DAP components, including one previously described (CEP164), one partially characterized (CEP89 ccdc123), and three novel (CEP83 ccdc41, SCLT1, and FBF1) DAP proteins. Analyses of DAP assembly revealed a hierarchy. CEP83 recruits both SCLT1 and CEP89 to centrioles. Subsequent recruitment of FBF1 and CEP164 is independent of CEP89 but mediated by SCLT1. All five DAP components are essential for ciliogenesis; loss of CEP83 specifically blocks centriole-to-membrane docking. Undocked centrioles fail to recruit TTBK2 or release CP110, the two earliest modifications found on centrioles prior to cilia assembly, revealing centriole-to-membrane docking as a temporal and spatial cue promoting cilia initiation.
Two important challenges in the field of 19F magnetic resonance imaging (MRI) are the maintenance of high fluorine content without compromising imaging performance, and effective targeting of small ...particles to diseased tissue. To address these challenges, we have developed a series of perfluoropolyether (PFPE)-based hyperbranched (HBPFPE) nanoparticles with attached peptide aptamer as targeting ligands for specific in vivo detection of breast cancer with high 19F MRI sensitivity. A detailed comparison of the HBPFPE nanoparticles (NPs) with the previously reported trifluoroethyl acrylate (TFEA)-based polymers demonstrates that the mobility of fluorinated segments of the HBPFPE nanoparticles is significantly enhanced (19F T2 > 80 ms vs 31 ms), resulting in superior MR imaging sensitivity. Selective targeting was confirmed by auto- and pair correlation analysis of fluorescence microscopy data, in vitro immunofluorescence, in vivo 19F MRI, ex vivo fluorescence and 19F NMR. The results highlight the high efficiency of aptamers for targeting and the excellent sensitivity of the PFPE moieties for 19F MRI. Of relevance to in vivo applications, the PFPE-based polymers exhibit much faster clearance from the body than the previously introduced perfluorocarbon emulsions (t 1/2 ∼ 20 h vs up to months). Moreover, the aptamer-conjugated NPs show significantly higher tumor-penetration, demonstrating the potential of these imaging agents for therapeutic applications. This report of the synthesis of polymeric aptamer-conjugated PFPE-based 19F MRI CAs with high fluorine content (∼10 wt %) demonstrates that these NPs are exciting candidates for detecting diseases with high imaging sensitivity.
The only known function of human sebaceous glands is the provocation of acne. We assessed here whether sebum influences stratum corneum hydration or permeability barrier function in asebia J1 and 2 J ...mice, with profound sebaceous gland hypoplasia. Asebia J1 mice showed normal permeability barrier homeostasis and extracellular lamellar membrane structures, but they displayed epidermal hyperplasia, inflammation, and decreased (>50%) stratum corneum hydration, associated with a reduction in sebaceous gland lipids (wax diesters/monoesters, sterol esters). The triglyceride content of both asebia and control stratum corneum was low, consistent with high rates of triglyceride hydrolysis within the normal pilosebaceous apparatus, despite high rates of triglyceride synthesis. Although a mixture of synthetic, sebum-like lipids (sterol/wax esters, triglycerides) did not restore normal stratum corneum hydration to asebia skin, topical glycerol, the putative product of triglyceride hydrolysis in sebaceous glands, normalized stratum corneum hydration, and the glycerol content of asebia stratum corneum was 85% lower than in normal stratum corneum. In contrast, another potent endogenous humectant (urea) did not correct the abnormality. The importance of glycerol generation from triglyceride in sebaceous glands for stratum corneum hydration was demonstrated further by (i) the absence of sebaceous-gland-associated lipase activity in asebia mice, whereas abundant enzyme activity was present in the glands of control mice; and (ii) the inability of high concentrations of topical triglyceride to correct the hydration abnormality, despite the presence of abundant lipase activity in asebia stratum corneum. These results show that sebaceous-gland-derived glycerol is a major contributor to stratum corneum hydration.
This study examines differential effects of immersion, elevated oxygen partial pressure, and exercise on pulmonary function after series of five daily six-hour dives at 130 kPa (1.3 ATA), with 18 ...hours between dives. Five cohorts of 10 to 14 divers participated. The exposure phases were resting while breathing O2 or air in the water ("wetO2", "wetAir") or O2 in the hyperbaric chamber ("dryO2"), and exercise in the water while breathing O2 or air ("wetO2X", "wetAirX"). Respiratory symptoms were recorded during and after each dive, and pulmonary function (forced flow-volume) was measured twice at baseline before diving, after each dive both immediately and on the following morning, and three days post diving ("Day+3"). The incidences of symptoms and of flow volume changes from baseline greater than normal limits ("ΔFV") were assessed, as were mean ΔFV. The parameters examined were forced vital capacity (FVC), forced expired volume in 1 second (FEV1), and forced expired flow from 25% to 75% volume expired (FEF25-75). The phases ranked from greatest to least fraction of diver-days with symptoms were wetO2X (56%) > dryO2 (42%) > wetO2 (13%) > wetAir (2%) or wetAirX (1%) (p<0.05). FEV1 and FEF25-75 were depressed in the morning following wetO2 and wetO2X and on Day+3 after and wetO2X, but increased immediately following each wetAirX dive. O2 exposures caused symptoms and ΔFV suggestive of pulmonary oxygen toxicity,exacerbated by exercise. Indices of small airway function showed late (17-hour) post-O2 exposure deficits, but, particularly with exercise, improvement was evident early after exposure with or without O2. FEF25-75 and FEV1 remained depressed on Day+3 after wetO2 and wetO2X.
Daptomycin is a lipopeptide with bactericidal activity that acts on the cell membrane of enterococci and is often used off-label to treat patients infected with vancomycin-resistant enterococci. ...However, the emergence of resistance to daptomycin during therapy threatens its usefulness.
We performed whole-genome sequencing and characterization of the cell envelope of a clinical pair of vancomycin-resistant Enterococcus faecalis isolates from the blood of a patient with fatal bacteremia; one isolate (S613) was from blood drawn before treatment and the other isolate (R712) was from blood drawn after treatment with daptomycin. The minimal inhibitory concentrations (MICs) of these two isolates were 1 and 12 μg per milliliter, respectively. Gene replacements were made to exchange the alleles found in isolate S613 with those in isolate R712.
Isolate R712 had in-frame deletions in three genes. Two genes encoded putative enzymes involved in phospholipid metabolism, GdpD (which denotes glycerophosphoryl diester phosphodiesterase) and Cls (which denotes cardiolipin synthetase), and one gene encoded a putative membrane protein, LiaF (which denotes lipid II cycle-interfering antibiotics protein but whose exact function is not known). LiaF is predicted to be a member of a three-component regulatory system (LiaFSR) involved in the stress-sensing response of the cell envelope to antibiotics. Replacement of the liaF allele of isolate S613 with the liaF allele from isolate R712 quadrupled the MIC of daptomycin, whereas replacement of the gdpD allele had no effect on MIC. Replacement of both the liaF and gdpD alleles of isolate S613 with the liaF and gdpD alleles of isolate R712 raised the daptomycin MIC for isolate S613 to 12 μg per milliliter. As compared with isolate S613, isolate R712--the daptomycin-resistant isolate--had changes in the structure of the cell envelope and alterations in membrane permeability and membrane potential.
Mutations in genes encoding LiaF and a GdpD-family protein were necessary and sufficient for the development of resistance to daptomycin during the treatment of vancomycin-resistant enterococci. (Funded by the National Institute of Allergy and Infectious Diseases and the National Institutes of Health.).
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