Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing ...mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.
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•Cas9-mediated DSB induction and repair by end joining occurs within hours•End joining provides an efficient way to restore reading frames without mosaicism•Unrepaired DSBs persist through mitosis and result in frequent chromosome loss•Off-target effects of Cas9 cause indels as well as chromosome loss
CRISPR-Cas9 gene editing in early human embryos leads to frequent loss of the targeted chromosome, indicating that human germline gene editing would pose a substantial risk for aneuploidy and other adverse genetic consequences
Intraventricular hemorrhage (IVH) occurs in 15-45% of all very low birth weight (VLBW) preterm infants. Despite improvements in the perinatal care, the incidence of IVH remains high. As more preterm ...infants survive, there will be a larger burden of neurodevelopmental abnormalities borne by former preterm infants.
The objective of this study was to develop a predictive clinical model of IVH risk within the first few hours of life in an effort to augment perinatal counseling and guide the timing of future targeted therapies aimed at preventing or slowing the progression of disease.
This is a prospective observational cohort study of VLBW infants born in the NICU at John's Hopkins Children's Center from 2011 to 2019. The presence and severity of IVH was defined on standard head ultrasound screening (HUS) using the modified Papile classification. Clinical variables were identified as significant using absolute risk regression from a general linear model. The model predictors included clinically meaningful variables that were not collinear.
This study took place at the Johns Hopkins Children's Center Level IV NICU.
The study sample included VLBW infants treated in the neonatal intensive care unit (NICU) at John's Hopkins Children's Center from 2011 to 2019. A total of 683 infants included in the study had no or grade I IVH, and 115 infants had grades II through IV IVH. Exclusion criteria included admission to the JHH NICU after 24 h of age, BW > 1500 g, and failure to consent.
The main outcome of this study was the presence of grades II-IV IVH on standard head ultrasound screening using the modified Papile classification 1.
A total of 798 VLBW infants were studied in this cohort and 14.4% had moderate to severe IVH. Fifty four percent of the cohort was black, 33% white, and half of the cohort was male. A higher gestational age, 5-min Apgar score, hematocrit, and platelet count were significantly associated with decreased incidence of IVH in a multi-predictor model (ROC 0.826).
In the face of continued lack of treatments for IVH, prevention is still a primary goal to avoid long-term developmental sequela. This model can be used for perinatal counseling and may provide important information during the narrow therapeutic window for targeted prevention therapies.
Pendrin (Slc26a4) localizes to type B and non-A, non-B intercalated cells in the distal convoluted tubule, the connecting tubule, and the cortical collecting duct (CCD), where it mediates apical ...Cl(-)/HCO(3)(-) exchange. The purpose of this study was to determine whether angiotensin II increases transepithelial net chloride transport, J(Cl), in mouse CCD through a pendrin-dependent mechanism. J(Cl) and transepithelial voltage, V(T), were measured in CCDs perfused in vitro from wild-type and Slc26a4 null mice ingesting a NaCl-replete diet or a NaCl-replete diet and furosemide. In CCDs from wild-type mice ingesting a NaCl-replete diet, V(T) and J(Cl) were not different from zero either in the presence or absence of angiotensin II (10(-8) M) in the bath. Thus further experiments employed mice given the high-NaCl diet and furosemide to upregulate renal pendrin expression. CCDs from furosemide-treated wild-type mice had a lumen-negative V(T) and absorbed Cl(-). With angiotensin II in the bath, Cl(-) absorption doubled although V(T) did not become more lumen negative. In contrast, in CCDs from furosemide-treated Slc26a4 null mice, Cl(-) secretion and a V(T) of approximately 0 were observed, neither of which changed with angiotensin II application. Inhibiting ENaC with benzamil abolished V(T) although J(Cl) fell only approximately 50%. Thus substantial Cl(-) absorption is observed in the absence of an electromotive force. Attenuating apical anion exchange with the peritubular application of the H(+)-ATPase inhibitor bafilomycin abolished benzamil-insensitive Cl(-) absorption. In conclusion, angiotensin II increases transcellular Cl(-) absorption in the CCD through a pendrin- and H(+)-ATPase-dependent process.
Objectives:
Effective health communication can increase intent to vaccinate. We compared 8 messages that may influence parents’ intent to vaccinate their children against COVID-19.
Methods:
In a ...cross-sectional survey of adults in the United States administered online in August 2021, 1837 parents and legal guardians were exposed to 8 messages (individual choice, gain/practical benefits, nonexpert, health care provider recommendation, altruism/community good, safety/effectiveness, safety, and effectiveness) to determine message reception and influence on intent to vaccinate their children. Parents responded to 10 questions using a Likert scale. We computed odds ratios (ORs) for each message, with an OR >1.0 indicating greater observed odds of participant agreement with the follow-up statement as compared with a reference message. We compared outcomes individually across messages with ordinal logistic regression fit using generalized estimating equations.
Results:
The individual choice message had the highest odds of agreement for understanding intent (OR = 2.10; 95% CI, 1.94-2.27), followed by the health care provider recommendation message (OR = 1.58; 95% CI, 1.46-1.71). The individual choice message had the highest odds of memorability, relatability, and trustworthiness. The altruism/community good message was at or near second best. The altruism/community good message had the highest or near-highest odds of increasing parents’ intent to vaccinate their children, asking friends and family for their thoughts, and searching for additional information. The message that most motivated parents to vaccinate their children depended on parental intent to vaccinate prior to being exposed to the tested messages.
Conclusions:
Messages with themes of individual choice, health care provider recommendation, and altruism/community good may be used in future message campaigns. Further research is needed to refine message concepts related to altruism/community good.
Pravastatin sodium (PV) is a potent cholesterol-lowering agent that acts by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Biotransformation profiles of PV in pooled human urine, plasma, ...and feces from healthy male volunteers given single 19.2-mg oral or 9.9-mg iv doses of 14CPV were determined by HPLC. The predominant drug-related component in urine, plasma, and feces corresponded to intact PV; in the pooled urine samples, PV constituted 29 and 69% of the radioactivity after the po and iv doses, respectively. The delta 4.5-3 alpha-hydroxy isomer of PV constituted 10% (po) and 2% (iv), and 6-epi-PV constituted 3% (po) and 1% (iv) of the urinary radioactivity. Negligible amounts of the lactones of PV or its isomers were detected in urine, plasma, or feces. At least 15 other metabolites were also present; none of these accounted for more than 6% of the total urinary radioactivity. For metabolite isolation, an aliquot of pooled urine samples, obtained after administration of the radioactive dose, was added as a tracer to urine samples obtained from healthy subjects after administration of single nonradiolabeled 40-mg oral doses of PV. Urinary metabolites were concentrated on an XAD-2 column, extracted with ethyl acetate, and purified by extensive preparative HPLC. In addition to isolation and identification of unchanged drug and the two isomeric metabolites described above, eight other metabolites were isolated and structural assignments were made based on HPLC, UV spectra, mass spectral analysis, and proton NMR.
We describe a stream-based analysis pipeline to detect gravitational waves from the merger of binary neutron stars, binary black holes, and neutron-star-black-hole binaries within ~ 1 minute of the ...arrival of the merger signal at Earth. Such low-latency detection is crucial for the prompt response by electromagnetic facilities in order to observe any fading electromagnetic counterparts that might be produced by mergers involving at least one neutron star. Even for systems expected not to produce counterparts, low-latency analysis of the data is useful for deciding when not to point telescopes, and as feedback to observatory operations. Analysts using this pipeline were the first to identify GW151226, the second gravitational-wave event ever detected. The pipeline also operates in an offline mode, in which it incorporates more refined information about data quality and employs acausal methods that are inapplicable to the online mode. The pipeline's offline mode was used in the detection of the first two gravitational-wave events, GW150914 and GW151226, as well as the identification of a third candidate, LVT151012.
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