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
Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable ...integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering.
Racial disparities in breast cancer survival between Black and White women persist across all stages of breast cancer. The metabolic syndrome (MetS) of insulin resistance disproportionately affects ...more Black than White women. It has not been discerned if insulin resistance mediates the link between race and poor prognosis in breast cancer. We aimed to determine whether insulin resistance mediates in part the association between race and breast cancer prognosis, and if insulin receptor (IR) and insulin-like growth factor receptor (IGF-1R) expression differs between tumors from Black and White women.
We conducted a cross-sectional, multi-center study across ten hospitals. Self-identified Black women and White women with newly diagnosed invasive breast cancer were recruited. The primary outcome was to determine if insulin resistance, which was calculated using the homeostatic model assessment of insulin resistance (HOMA-IR), mediated the effect of race on prognosis using the multivariate linear mediation model. Demographic data, anthropometric measurements, and fasting blood were collected. Poor prognosis was defined as a Nottingham Prognostic Index (NPI) > 4.4. Breast cancer pathology specimens were evaluated for IR and IGF-1R expression by immunohistochemistry (IHC).
Five hundred fifteen women were recruited (83% White, 17% Black). The MetS was more prevalent in Black women than in White women (40% vs 20%, p < 0.0001). HOMA-IR was higher in Black women than in White women (1.9 ± 1.2 vs 1.3 ± 1.4, p = 0.0005). Poor breast cancer prognosis was more prevalent in Black women than in White women (28% vs 15%. p = 0.004). HOMA-IR was positively associated with NPI score (r = 0.1, p = 0.02). The mediation model, adjusted for age, revealed that HOMA-IR significantly mediated the association between Black race and poor prognosis (β = 0.04, 95% CI 0.005-0.009, p = 0.002). IR expression was higher in tumors from Black women than in those from White women (79% vs 52%, p = 0.004), and greater IR/IGF-1R ratio was also associated with higher NPI score (IR/IGF-1R > 1: 4.2 ± 0.8 vs IR/IGF-1R = 1: 3.9 ± 0.8 vs IR/IGF-1R < 1: 3.5 ± 1.0, p < 0.0001).
In this multi-center, cross-sectional study of US women with newly diagnosed invasive breast cancer, insulin resistance is one factor mediating part of the association between race and poor prognosis in breast cancer.
Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate ...the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic.
•Study focused on a sample of buprenorphine-exposed infants.•Maternal gestational buprenorphine dose is a positive predictor of NAS severity.•Polysubstance exposure most potently predicted NAS ...severity.•Findings underscore the need for comprehensive addictions treatment during pregnancy.
Maternal buprenorphine maintenance predisposes the infant to exhibit neonatal abstinence syndrome (NAS), but there is insufficient published information regarding the nature of NAS and factors that contribute to its severity in buprenorphine-exposed infants.
The present study evaluated forty-one infants of buprenorphine-maintained women in comprehensive substance use disorder treatment who participated in an open-label study examining the effects of maternal buprenorphine maintenance on infant outcomes. Modifiers of the infant outcomes, including maternal treatment and substance use disorder parameters, were also evaluated.
Fifty-nine percent of offspring exhibited NAS that required pharmacologic management. Both maternal buprenorphine dose as well as prenatal polysubstance exposure to illicit substance use/licit substance misuse were independently associated with NAS expression. Polysubstance exposure was associated with more severe NAS expression after controlling for the effects of buprenorphine dose. Other exposures, including cigarette smoking and SRI use, were not related to outcomes. Maternal buprenorphine dose was positively associated with lower birth weight and length.
Polysubstance exposure was the most potent predictor of NAS severity in this sample of buprenorphine-exposed neonates. This finding suggests the need for interventions that reduce maternal polysubstance use during medication assisted treatment for opioid use disorder, and highlights the necessity of a comprehensive approach, beyond buprenorphine treatment alone, for the optimal care for pregnant women with opioid use disorders.
Background
Despite the survival benefit associated with adjuvant chemotherapy in early-stage breast cancer, many do not complete treatment. This study identified factors associated with noncompletion ...of adjuvant chemotherapy among a select population of women with early-stage breast cancer.
Methods
The study sample was obtained from a multicenter study designed to evaluate patient-assistance program usage among early-stage breast cancer patients requiring adjuvant therapy. In this study, 333 patients with stages I and II breast cancer undergoing surgery from October 2006 to September 2009 completed 6-month follow-up surveys assessing their experiences with care, health status, social support, self-efficacy, and treatment beliefs. In- and outpatient medical records were abstracted to assess treatment completion. Of the 333 patients, 198 initiated adjuvant chemotherapy and formed our study cohort. The study compared patients who did and did not complete adjuvant chemotherapy.
Results
The median patient age was 53 years (range 28–86 years). According to self-identification, 41 % of the patients were non-Hispanic white and 21 % were black. A total of 13 patients (7 %) did not complete adjuvant chemotherapy. In the bivariate analysis, the patients not completing chemotherapy were more likely to be black and unmarried women with low emotional social support and a poor body image after treatment. In the multivariate analysis, black race odds ratio (OR) 5.62; 95 % confidence interval (CI) 1.63–20.36 and poor body image (OR 9.75; 95 % CI 2.12–95.95) were independently associated with noncompletion of chemotherapy.
Conclusions
Overall chemotherapy noncompletion rates were low among women exposed to patient-assistance programs. However, poor body image and black race were independent predictors of uncompleted chemotherapy. The true impact of race in this group may result from social factors that occur more often among black women, including poor social support.
Although breast cancer incidence is higher among white women, black women are more likely to have aggressive tumors with less favorable histology, and to have a worse prognosis. Obesity and alcohol ...consumption have been identified as two modifiable risk factors for breast cancer, while physical activity may offer protection. Little however is known about the association of these factors with race on the severity of breast cancer.
Data collected as part of a large prospective study looking at insulin resistance and race among women with breast cancer was queried for patient characteristics, lifestyle factors and tumor characteristics. The association with Nottingham Prognostic Index (NPI) was assessed with different models using univariate and multivariate linear regression.
Among 746 women in our cohort, 82% (n = 615) were white and 18% (n = 131) were black, mean age 58 years. Black patients were more likely to have high BMI (31.0 vs. 26.7, p < 0.0001), comorbidities (69% vs 55%, p = 0.01), self-reported poor diet (70% vs 42%, p < 0.001), be sedentary (56% vs 46%, p = 0.03) and were less likely to consume alcohol (8% vs 32%, p < 0.0001) compared to white patients. Overall, 137 (18%) of the patients had poorer prognosis (NPI > 4.4), which was significantly associated with younger age (55.6 vs 58.5 years, p = 0.02), black race (27% vs 15%, p = 0.001), triple negative cancer (15% vs 6%, p = 0.003), and poor diet (54% vs 45%, p = 0.046) compared to patients with better prognosis (NPI ≤ 4.4).
On multivariate analysis, (model R2 = 0.12; p < 0.001), age (β = −0.011 per year, p = 0.002), healthy diet (β = −0.195, p = 0.02), and exercise (β = −0.004, p = 0.02) were associated with better prognosis, while black race (β = 0.247, p = 0.02) and triple negative cancer (β = 0.908, p < 0.0001) were associated with poor prognosis. Neither alcohol use nor BMI was significantly associated with NPI.
Among modifiable risk factors, diet and exercise are associated with NPI. Unmodifiable factors including race and biologic subtype remain the most important determinants of prognosis.
•Black breast cancer patients more likely to have high BMI, and be sedentary.•Poorer breast cancer prognosis associated with poor diet.•Neither alcohol use nor BMI associated with prognosis.•Among modifiable risk factors, diet and exercise are associated with NPI.
Background
Currently electronic cigarettes are becoming popular, especially among youth (middle and high school students). Their usage is being debated concerning their role in smoking cessation. The ...electronic cigarette, also known as e-cigarette, is an electronic nicotine delivery system. It is a mechanical device designed to mimic regular cigarettes, looks like conventional cigarette, delivers nicotine through inhaling vapors without burning tobacco. Globally, as well as in the US, tobacco smoking has been the leading cause of preventable death. In 2015, an estimated 5 million middle and high school students were users of all kinds of tobacco products in the USA. Out of which, 3 million students have experimented with e-cigarettes.
Objective
To assess the extent of e-cigarette experimenting among youth from 2011 to 2015 grade-wise and gender-wise. To analyze the 2015 data more extensively.
Methods
National Youth Tobacco Survey (NYTS) data is used from CDC, USA. Peer-reviewed articles from 2000 to 2016 were identified through electronic database Medline and Pub-Med on the electronic cigarettes, were reviewed and analyzed.
Results
The trend of experimenting with e-cigarettes at different ages and grades has been increasing steeply among youth. In the year 2015, about 45% of 12th graders experimented with e-cigarettes. Also, about 24% of females and 29% of male students have reported experimenting with e-cigarettes. Significantly, more Mexicans and Native Hawaiians, and fewer Asians tried e-cigarettes compared to the rest. There was no significant difference in experimenting with e-cigarettes among whites versus non-whites and African Americans versus non-African Americans.
Conclusions
Our findings emphasize the need to make policy makers and school administrators aware of the extent of usage of e-cigarettes, especially, among juniors and seniors. Since the prevalence of e-cigarette use is steeply increasing over the past years, proper intervention programs should be implemented in schools for the benefit of targeted youth.
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