We report that epigenetic silencing causes the loss of function of multi-transcript unit constructs that are integrated using CRISPR-Cas9. Using a modular two color reporter system flanked by ...selection markers, we demonstrate that expression heterogeneity does not correlate with sequence alteration but instead correlates with chromosomal accessibility. We partially reverse this epigenetic silencing via small-molecule inhibitors of methylation and histone deacetylation. We then correlate each heterogeneously-expressing phenotype with its expected epigenetic state by employing ATAC-seq. The stability of each expression phenotype is reinforced by selective pressure, which indicates that ongoing epigenetic remodeling can occur for over one month after integration. Collectively, our data suggests that epigenetic silencing limits the utility of multi-transcript unit constructs that are integrated via double-strand repair pathways. Our research implies that mammalian synthetic biologists should consider localized epigenetic outcomes when designing complex genetic circuits.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Highly effective vaccines elicit specific, robust, and durable adaptive immune responses. To advance informed vaccine design, it is critical that we understand the cellular dynamics underlying ...responses to different antigen formats. Here, we sought to understand how antigen-specific B and T cells were activated and participated in adaptive immune responses within the mucosal site. Using a human tonsil organoid model, we tracked the differentiation and kinetics of the adaptive immune response to influenza vaccine and virus modalities. Each antigen format elicited distinct B and T cell responses, including differences in their magnitude, diversity, phenotype, function, and breadth. These differences culminated in substantial changes in the corresponding antibody response. A major source of antigen format-related variability was the ability to recruit naive vs. memory B and T cells to the response. These findings have important implications for vaccine design and the generation of protective immune responses in the upper respiratory tract.
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•Used tonsil organoids to compare influenza vaccine formats within an individual•Transcriptional fate of Ag-specific B cells is affected by vaccine format•Type I IFNs mediate some but not all of the vaccine-format-specific differences•Inactivated flu vaccine depends on pre-existing memory cells
Understanding the cellular dynamics underlying responses to different antigen formats is critical for rational vaccine design. Using a human immune organoid model, Kastenschmidt et al. evaluate the effect of influenza vaccine modalities on adaptive immunity and reveal key differences in the magnitude, breadth, and quality of B cell, T cell, and antibody responses.
Sex-based differences in immune cell composition and function can contribute to distinct adaptive immune responses. Prior work has quantified these differences in peripheral blood, but little is ...known about sex differences within human lymphoid tissues. Here, we characterized the composition and phenotypes of adaptive immune cells from male and female ex vivo tonsils and evaluated their responses to influenza antigens using an immune organoid approach. In a pediatric cohort, female tonsils had more memory B cells compared to male tonsils direct ex vivo and after stimulation with live-attenuated but not inactivated vaccine, produced higher influenza-specific antibody responses. Sex biases were also observed in adult tonsils but were different from those measured in children. Analysis of peripheral blood immune cells from
vaccinated adults also showed higher frequencies of tissue homing CD4 T cells in female participants. Together, our data demonstrate that distinct memory B and T cell profiles are present in male vs. female lymphoid tissues and peripheral blood respectively and suggest that these differences may in part explain sex biases in response to vaccines and viruses.
•A method to generate bone metastases in over 95% of mice.•Tumors can be detected within one to two weeks.•Low rates of vital organ metastases, relative to other methods.•Consistent tumor ...localization in lower body.•Growth rate and consistency of tumors can be controlled by quantity of cancer cells injected.
Many cancers metastasize to the bones, particularly in cases of breast and prostate cancers. Due to the “vicious cycle” of cancer cells inducing bone resorption, which promotes further tumor growth, they are difficult to treat and may lead to extreme pain. These factors increase the urgency for emerging therapeutics that target bone metastases more specifically and effectively. Animal studies are essential to the development of any therapeutics, but also require robust animal models of human diseases. Robust animal models are often challenging to develop in the case of bone metastasis studies. Previous methods to induce bone metastasis include intracardiac, intravenous, subcutaneous via mammary fat pad, and intraosseous cancer cell injections, but these methods all have limitations. By contrast, the caudal artery route of injection offers more robust bone metastasis, while also resulting in a lower rate of vital organ metastases than that of other routes of tumor implantation. A syngeneic animal model of bone metastasis is necessary in many cancer studies, because it allows the use of immunocompetent animals, which more accurately mimic cancer development observed in immunocompetent humans. Here we present a detailed method to generate robust and easily monitored 4T1-CLL1 syngeneic bone metastases with over 95% occurrence in BALB/c mice, within two weeks. This method can potentially increase consistency between animals in bone cancer metastasis studies and reduce the number of animals needed for studying bone metastases in mice.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background
Exercise has been shown to reduce fatigue in early breast cancer survivors (EBCS), though it is unclear if these results translate to community-based exercise settings. Mechanisms that ...influence changes in fatigue seen after exercise are also poorly understood. This study sought to evaluate the impact of community-based exercise and identify associations of fatigue in EBCS.
Methods
Twenty-nine EBCS and 13 non-cancer controls (CON) enrolled. Pre/post-intervention measurements included measures of fitness/function, balance, and adherence/compliance as well as self-reported measures of fatigue, health-related quality of life (HRQOL), well-being, self-efficacy, and physical activity. Both groups participated in a supervised 16-week aerobic + resistance exercise intervention. A mixed model ANOVA and Cohen’s D effect size assessed fatigue changes, and univariable linear regressions identified fatigue associations.
Results
Fatigue improved for EBCS (– 2.6,
Cohen’s D
= 0.51) but not CON (0.0,
Cohen’s D
= 0.02); no interaction effect was observed. Post-intervention fatigue in EBCS was associated with better QOL (
R
2
= 0.387;
p
< 0.01), depression (
R
2
= 0.251;
p
< 0.01), self-efficacy, (
R
2
= 0.453;
p
< 0.01), outcome expectations from exercise (
R
2
= 0.254;
p
< 0.01), balance (
R
2
= 0.167;
p
< 0.05), and the 6-minute walk test
(R
2
= 0.193;
p
< 0.05). EBCS improvements in fatigue were associated with improvements in self-reported physical health (
R
2
= 0.425;
p
< 0.01), depression (
R
2
= 0.233;
p
< 0.01), pain (
R
2
= 0.157;
p
< 0.05), outcome expectations from exercise (
R
2
= 0.420;
p
< 0.01), and the 6-minute walk test (
R
2
= 0.172;
p
< 0.05). Less fatigue in the CON group was shown be associated with better sleep quality (
R
2
= 0.309;
p
< 0.05) and pain (
R
2
= 0.259;
p
< 0.05).
Conclusion
Community-based exercise appears beneficial for alleviating fatigue in EBCS. These improvements may be driven by parallel improvements in psychosocial outcomes and objectively measured functional outcomes.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The "2023 ACC/AHA/ACCP/HRS Guideline for the Diagnosis and Management of Patients With Atrial Fibrillation" provides recommendations to guide clinicians in the treatment of patients with atrial ...fibrillation.
A comprehensive literature search was conducted from May 12, 2022, to November 3, 2022, encompassing studies, reviews, and other evidence conducted on human subjects that were published in English from PubMed, EMBASE, the Cochrane Library, the Agency for Healthcare Research and Quality, and other selected databases relevant to this guideline. Additional relevant studies, published through November 2022, during the guideline writing process, were also considered by the writing committee and added to the evidence tables, where appropriate.
Atrial fibrillation is the most sustained common arrhythmia, and its incidence and prevalence are increasing in the United States and globally. Recommendations from the "2014 AHA/ACC/HRS Guideline for the Management of Patients With Atrial Fibrillation" and the "2019 AHA/ACC/HRS Focused Update of the 2014 AHA/ACC/HRS Guideline for the Management of Patients With Atrial Fibrillation" have been updated with new evidence to guide clinicians. In addition, new recommendations addressing atrial fibrillation and thromboembolic risk assessment, anticoagulation, left atrial appendage occlusion, atrial fibrillation catheter or surgical ablation, and risk factor modification and atrial fibrillation prevention have been developed.
Purpose
Examine baseline fatigue levels in early-breast cancer survivors (EBCS) compared to inactive controls (CON) and identify associated physical and psychosocial factors with fatigue prior to ...community-based exercise.
Methods
A total of 33 EBCS (53.9 ± 11.4 years) and 21 CON (54.0 ± 8.0 years) were recruited. Participants completed questionnaires for demographics and patient-reported outcome measures pertaining to fatigue, quality of life, mental health, and physical activity, and completed a 6-min walk test, balance assessment, cardiopulmonary exercise test (VO
2peak
), and muscular strength test. A Mann–Whitney
U
test compared fatigue between groups and unadjusted univariable linear regressions were used to explore relationships with fatigue.
Results
Fatigue in EBCS was not statistically different from CON (EBCS: 16.9 ± 5.75; CON: 14.2 ± 3.4,
p
= 0.121). Univariable analyses showed lower fatigue in EBCS was associated with better Physical and Mental Health (both
R
2
= 0.435;
p
< 0.01), better outcome expectations for exercise (
R
2
= 0.237;
p
< 0.01), better self-efficacy (
R
2
= 0.407;
p
< 0.01), lower depression (
R
2
= 0.383;
p
< 0.001), lower anxiety (
R
2
= 0.104;
p
< 0.05), and better balance (
R
2
= 0.265;
p
< 0.01). Lower fatigue in the CON group was associated with better sleep quality (
R
2
= 0.263;
p
< 0.05) and self-efficacy (
R
2
= 0.417;
p
< 0.05).
Conclusions
Mild fatigue was prevalent in EBCS, whereas moderate/severe fatigue was not. This discrepancy should be explored provided the benefits of exercise for fatigue management. Further, fatigue in EBCS was associated with multiple psychosocial and functional outcomes, which emphasized both its multi-factorial nature and uniqueness to the EBCS population.
Trial registration.
ClinicalTrials.gov Number: NCT03760536.
Abstract
Drug-induced gastrointestinal toxicities (GITs) rank among the most common clinical side effects. Preclinical efforts to reduce incidence are limited by inadequate predictivity of in vitro ...assays. Recent breakthroughs in in vitro culture methods support intestinal stem cell maintenance and continual differentiation into the epithelial cell types resident in the intestine. These diverse cells self-assemble into microtissues with in vivo-like architecture. Here, we evaluate human GI microtissues grown in transwell plates that allow apical and/or basolateral drug treatment and 96-well throughput. Evaluation of assay utility focused on predictivity for diarrhea because this adverse effect correlates with intestinal barrier dysfunction which can be measured in GI microtissues using transepithelial electrical resistance (TEER). A validation set of widely prescribed drugs was assembled and tested for effects on TEER. When the resulting TEER inhibition potencies were adjusted for clinical exposure, a threshold was identified that distinguished drugs that induced clinical diarrhea from those that lack this liability. Microtissue TEER assay predictivity was further challenged with a smaller set of drugs whose clinical development was limited by diarrhea that was unexpected based on 1-month animal studies. Microtissue TEER accurately predicted diarrhea for each of these drugs. The label-free nature of TEER enabled repeated quantitation with sufficient precision to develop a mathematical model describing the temporal dynamics of barrier damage and recovery. This human 3D GI microtissue is the first in vitro assay with validated predictivity for diarrhea-inducing drugs. It should provide a platform for lead optimization and offers potential for dose schedule exploration.
IMPORTANCE: Atrial fibrillation (AF) is the most common arrhythmia affecting 1% of the population. Young individuals with AF have a strong genetic association with the disease, but the mechanisms ...remain incompletely understood. OBJECTIVE: To perform large-scale whole-genome sequencing to identify genetic variants related to AF. DESIGN, SETTING, AND PARTICIPANTS: The National Heart, Lung, and Blood Institute’s Trans-Omics for Precision Medicine Program includes longitudinal and cohort studies that underwent high-depth whole-genome sequencing between 2014 and 2017 in 18 526 individuals from the United States, Mexico, Puerto Rico, Costa Rica, Barbados, and Samoa. This case-control study included 2781 patients with early-onset AF from 9 studies and identified 4959 controls of European ancestry from the remaining participants. Results were replicated in the UK Biobank (346 546 participants) and the MyCode Study (42 782 participants). EXPOSURES: Loss-of-function (LOF) variants in genes at AF loci and common genetic variation across the whole genome. MAIN OUTCOMES AND MEASURES: Early-onset AF (defined as AF onset in persons <66 years of age). Due to multiple testing, the significance threshold for the rare variant analysis was P = 4.55 × 10−3. RESULTS: Among 2781 participants with early-onset AF (the case group), 72.1% were men, and the mean (SD) age of AF onset was 48.7 (10.2) years. Participants underwent whole-genome sequencing at a mean depth of 37.8 fold and mean genome coverage of 99.1%. At least 1 LOF variant in TTN, the gene encoding the sarcomeric protein titin, was present in 2.1% of case participants compared with 1.1% in control participants (odds ratio OR, 1.76 95% CI, 1.04-2.97). The proportion of individuals with early-onset AF who carried a LOF variant in TTN increased with an earlier age of AF onset (P value for trend, 4.92 × 10−4), and 6.5% of individuals with AF onset prior to age 30 carried a TTN LOF variant (OR, 5.94 95% CI, 2.64-13.35; P = 1.65 × 10−5). The association between TTN LOF variants and AF was replicated in an independent study of 1582 patients with early-onset AF (cases) and 41 200 control participants (OR, 2.16 95% CI, 1.19-3.92; P = .01). CONCLUSIONS AND RELEVANCE: In a case-control study, there was a statistically significant association between an LOF variant in the TTN gene and early-onset AF, with the variant present in a small percentage of participants with early-onset AF (the case group). Further research is necessary to understand whether this is a causal relationship.
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