Objective To conduct a test of noninferiority for CardioCel (Admedus, Brisbane, Australia), a chemically engineered bovine pericardium over autologous pericardium treated intraoperatively with ...glutaraldehyde in a chronic juvenile sheep model of pulmonary valve (PV) and mitral valve (MV) reconstruction. Methods We replaced the posterior leaflet of the MV and of 1 PV cusp with patches in ewes aged 10 months. There were 2 groups: CardioCel (n = 6) and control (n = 4). All valves were competent. Echocardiography was performed before euthanasia. The collected data were function, macroscopy, histology, and calcium contents. The primary end points were thickening and calcium content. Results All animals survived until sacrifice after 7 months. The valves had normal echo. The macroscopic aspect of the valves was excellent. Examination of the slides for both groups revealed a continuous endothelium on both sides of the patch and a layer of new collagen developed on both sides between patch and endothelium and interstitial cells and smooth muscle cell in these layers. The patch had not thickened but the 2 layers of new collagen for the PV showed a median thickening of 37% in the CardioCel group and 111% in the control group ( P = .01), and for the MV a thickening of 108% and 251%, respectively, was seen ( P = .01). The median calcium content in the PV was 0.24 μg/mg (range, 0.19-0.30) in the CardioCel group versus 0.34 μg/mg (range, 0.24-0.62) in controls ( P = .20). In the MV it was 0.46 μg/mg (range, 0.30-1.0) in the CardioCel group and 0.47 μg/mg (range, 0.29-1.9) in controls ( P = 1.0). Conclusions In this growing lamb model the CardioCel patch allowed accurate valve repair at both systemic and pulmonary pressure. The mechanical properties of CardioCel after 7 months were preserved with a more controlled healing than the treated autologous pericardium and without calcification.
Genetically engineered mouse models only capture a small fraction of the genetic lesions that drive human cancer. Current CRISPR-Cas9 models can expand this fraction but are limited by their reliance ...on error-prone DNA repair. Here we develop a system for in vivo prime editing by encoding a Cre-inducible prime editor in the mouse germline. This model allows rapid, precise engineering of a wide range of mutations in cell lines and organoids derived from primary tissues, including a clinically relevant Kras mutation associated with drug resistance and Trp53 hotspot mutations commonly observed in pancreatic cancer. With this system, we demonstrate somatic prime editing in vivo using lipid nanoparticles, and we model lung and pancreatic cancer through viral delivery of prime editing guide RNAs or orthotopic transplantation of prime-edited organoids. We believe that this approach will accelerate functional studies of cancer-associated mutations and complex genetic combinations that are challenging to construct with traditional models.
A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, ...contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness. The contractile tail of Agrobacterium tumefaciens bacteriophage Milano is flexible and bent to varying degrees, which sets it apart from other contractile tail-like systems. Here, we report structures of the Milano tail including the sheath-tube complex, baseplate, and putative receptor-binding proteins. The flexible-to-rigid transformation of the Milano tail upon contraction can be explained by unique electrostatic properties of the tail tube and sheath. All components of the Milano tail, including sheath subunits, are crosslinked by disulfides, some of which must be reduced for contraction to occur. The putative receptor-binding complex of Milano contains a tailspike, a tail fiber, and at least two small proteins that form a garland around the distal ends of the tailspikes and tail fibers. Despite being flagellotropic, Milano lacks thread-like tail filaments that can wrap around the flagellum, and is thus likely to employ a different binding mechanism.
Women with biologically favorable early-stage breast cancer are increasingly treated with accelerated partial breast radiation (PBI). However, treatment-related morbidities have been linked to the ...large postoperative treatment volumes required for external beam PBI. Relative to external beam delivery, alternative PBI techniques require equipment that is not universally available. To address these issues, we designed a phase 1 trial utilizing widely available technology to 1) evaluate the safety of a single radiation treatment delivered preoperatively to the small-volume, intact breast tumor and 2) identify imaging and genomic markers of radiation response.
Women aged ≥55 years with clinically node-negative, estrogen receptor-positive, and/or progesterone receptor-positive HER2-, T1 invasive carcinomas, or low- to intermediate-grade in situ disease ≤2 cm were enrolled (n=32). Intensity modulated radiation therapy was used to deliver 15 Gy (n=8), 18 Gy (n=8), or 21 Gy (n=16) to the tumor with a 1.5-cm margin. Lumpectomy was performed within 10 days. Paired pre- and postradiation magnetic resonance images and patient tumor samples were analyzed.
No dose-limiting toxicity was observed. At a median follow-up of 23 months, there have been no recurrences. Physician-rated cosmetic outcomes were good/excellent, and chronic toxicities were grade 1 to 2 (fibrosis, hyperpigmentation) in patients receiving preoperative radiation only. Evidence of dose-dependent changes in vascular permeability, cell density, and expression of genes regulating immunity and cell death were seen in response to radiation.
Preoperative single-dose radiation therapy to intact breast tumors is well tolerated. Radiation response is marked by early indicators of cell death in this biologically favorable patient cohort. This study represents a first step toward a novel partial breast radiation approach. Preoperative radiation should be tested in future clinical trials because it has the potential to challenge the current treatment paradigm and provide a path forward to identify radiation response biomarkers.
Short tandem repeats (STRs) are enriched in eukaryotic
cis
-regulatory elements and alter gene expression, yet how they regulate transcription remains unknown. We found that STRs modulate ...transcription factor (TF)–DNA affinities and apparent on-rates by about 70-fold by directly binding TF DNA-binding domains, with energetic impacts exceeding many consensus motif mutations. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density, with impacts well predicted by statistical mechanics. Confirming that STRs also affect TF binding in cells, neural networks trained only on in vivo occupancies predicted effects identical to those observed in vitro. Approximately 90% of TFs preferentially bound STRs that need not resemble known motifs, providing a
cis
-regulatory mechanism to target TFs to genomic sites.
Editor’s summary
Short tandem repeats (STRs) are common within regulatory elements in eukaryotic genomes. Although changes in STR lengths often correlate with altered transcription, the mechanism by which they tune gene expression has remained mysterious. Horton
et al
. show that many transcription factor (TF) proteins directly bind STRs and that TF-preferred STRs need not resemble known binding sites (see the Perspective by Kuhlman). This binding can be explained and predicted by simple additive models in which repeated instances of low-affinity binding sites sum to have large effects. These findings suggest that STRs provide a regulatory mechanism to tune levels of TF binding and downstream gene expression. —Di Jiang
Transcription factors directly bind repetitive sequences that need not resemble known motifs.
INTRODUCTION
Gene expression is regulated by transcription factor (TF) proteins that bind DNA-regulatory elements in the genome. Despite decades of research cataloging TF “motifs,” these do not fully explain observed genomic binding in cells. Many TFs bind regions lacking motifs, whereas other regions with apparently strong motifs remain unoccupied, and emerging evidence suggests that the DNA sequence context surrounding motifs can strongly affect binding (see the figure, panel A). Short tandem repeats (STRs, consecutively repeated units of one to six nucleotides) provide a good example of these sequence contexts. STRs comprise ~5% of the human genome (compared with 1.5% for all protein-coding genes) and are enriched in enhancers. Variations in STR length have been associated with changes in gene expression and implicated in several complex phenotypes, such as schizophrenia, cancer, autism, and Crohn’s disease. However, the mechanism by which STRs affect transcription remains unknown.
RATIONALE
One mechanism by which STRs could affect gene expression is by altering the affinity and/or kinetics of TF binding to regulatory DNA (see the figure, panel A). To investigate this, we used various high-throughput microfluidic binding assays (i.e., MITOMI,
k
-MITOMI, and STAMMP) and bioinformatic analyses to systematically quantify the impacts of different sequence contexts on TF binding. We measured affinities (
K
d
s) and kinetics (
k
off
s) for two basic helix-loop-helix TFs that bind a CACGTG E-box motif (Pho4 from
Saccharomyces cerevisiae
and MAX from
Homo sapiens
) to DNA sequences with or without an E-box motif surrounded by random sequence or multiple different types of STRs (see the figure, panel B).
RESULTS
Measured binding constants (
K
d
s) for 609 distinct TF-DNA combinations revealed that different STRs can alter binding affinities by >70-fold (see the figure, panel C), approaching or exceeding effects from mutating the consensus motif. Preferred STRs differed for Pho4 and MAX TFs, demonstrating that motifs are not sufficient to predict preferred STRs. Gel-shift assays and additional experiments using TF truncation constructs established that TFs directly bind STRs (see the figure, panel C) through their DNA-binding domains in the presence or absence of motifs. Although not predicted by standard mononucleotide models, the observed STR binding is well explained by a simple partition function model from statistical mechanics in which multiple repeated weak binding sites contribute additively to binding affinity (see the figure, panel D). Measured apparent dissociation rates (
k
off
s) for 106 TF-DNA combinations and kinetic modeling suggested that STRs primarily alter macroscopic apparent association rates and increase the local density of DNA-bound TFs. Finally, neural networks trained only on in vivo genome-wide chromatin immunoprecipitation data predict effects identical to those measured in vitro, suggesting that STR preferences play a substantial role in properly localizing TFs in cells.
CONCLUSION
Analysis of previously published protein-binding microarray and SELEX data suggests that ~90% of eukaryotic TFs preferentially bind at least one type of STR (see the figure, panel E). Because STRs are highly mutable, we propose that they should be considered an easily evolvable class of
cis-
regulatory elements. Preferred STRs need not resemble known motifs, suggesting a mechanism by which TF paralogs can be recruited to different regulatory regions and regulate distinct target genes. Although STRs maximize the number of potential weak binding sites, we anticipate that nonrepetitive sequence contexts containing many low-affinity binding sites should similarly increase binding. Thus, we propose that STRs function as “rheostats” to tune local TF concentration and binding responses to regulate gene expression in disease, development, and homeostasis.
STRs directly bind TFs to alter gene expression.
(
A
) Schematic of enhancers, motifs, and STRs. (
B
) Schematic of TFs and DNA libraries tested in this study. (
C
) Favorable STRs alter energetic landscapes by directly binding TF DNA-binding domains. (
D
) Favorable STRs maximize potential preferred sites and contribute additively to binding energies. (
E
) STR binding by TFs is widespread.
Bromodomain-containing proteins frequently reside in multisubunit chromatin complexes with tissue or cell state-specific compositions. Recent studies have revealed tumor-specific dependencies on the ...BAF complex bromodomain subunit BRD9 that are a result of recurrent mutations afflicting the structure and composition of associated complex members. To enable the study of ligand engaged complex assemblies, we established a chemoproteomics approach using a functionalized derivative of the BRD9 ligand BI-9564 as an affinity matrix. Unexpectedly, in addition to known interactions with BRD9 and associated BAF complex proteins, we identify a previously unreported interaction with members of the NuA4 complex through the bromodomain-containing subunit BRD8. We apply this finding, alongside a homology-model-guided design, to develop chemical biology approaches for the study of BRD8 inhibition and to arrive at first-in-class selective and cellularly active probes for BRD8. These tools will empower further pharmacological studies of BRD9 and BRD8 within respective BAF and NuA4 complexes.
BackgroundPD-1/PD-L1 engagement and overexpression of galectin-3 (Gal-3) are critical mechanisms of tumor-induced immune suppression that contribute to immunotherapy resistance. We hypothesized that ...Gal-3 blockade with belapectin (GR-MD-02) plus anti-PD-1 (pembrolizumab) would enhance tumor response in patients with metastatic melanoma (MM) and head and neck squamous cell carcinoma (HNSCC).MethodsWe performed a phase I dose escalation study of belapectin+pembrolizumab in patients with advanced MM or HNSCC (NCT02575404). Belapectin was administered at 2, 4, or 8 mg/kg IV 60 min before pembrolizumab (200 mg IV every 3 weeks for five cycles). Responding patients continued pembrolizumab monotherapy for up to 17 cycles. Main eligibility requirements were a functional Eastern Cooperative Oncology Group status of 0–2, measurable or assessable disease, and no active autoimmune disease. Prior T-cell checkpoint antibody therapy was permitted.ResultsObjective response was observed in 50% of MM (7/14) and and 33% of HNSCC (2/6) patients. Belapectin+pembrolizumab was associated with fewer immune-mediated adverse events than anticipated with pembrolizumab monotherapy. There were no dose-limiting toxicities for belapectin within the dose range investigated. Significantly increased effector memory T-cell activation and reduced monocytic myeloid-derived suppressor cells (M-MDSCs) were observed in responders compared with non-responders. Increased baseline expression of Gal-3+ tumor cells and PD-1+CD8+ T cells in the periphery correlated with response as did higher serum trough levels of pembrolizumab.ConclusionsBelapectin+pembrolizumab therapy has activity in MM and HNSCC. Increased Gal-3 expression, expansion of effector memory T cells, and decreased M-MDSCs correlated with clinical response. Further investigation is planned.
BACKGROUNDThe Endocrine Self-Assessment Program In-Training Examination (ESAP-ITE) has the novel formative approach of allowing open access to all questions and answers after secure examination ...administration is complete, resulting in the creation of an entirely new in-training examination annually. OBJECTIVETo determine whether scores on the novel ESAP-ITE predict pass/fail outcomes on the American Board of Internal Medicine Endocrinology, Diabetes, and Metabolism Certification Examination (ABIM-ECE). METHODSAll endocrine fellows-in-training who took the ESAP-ITE between 2016 and 2019 and then subsequently attempted the ABIM-ECE within the same calendar year were included (n = 982). Primary analyses used the ESAP-ITE score from the final year of fellowship training. Covariates included sex, age on date of ABIM-ECE, medical school country, fellowship program region, pass/fail outcomes on the ABIM Internal Medicine Certification Examination, and ESAP-ITE score. All variables were analyzed using multivariable logistic regression. RESULTSESAP-ITE score (P < 0.001), ABIM Internal Medicine Certification Examination outcome (P < 0.001), and age (P = 0.005) were each significant predictors of passing the ABIM-ECE on the first attempt. ESAP-ITE score was the strongest predictor of passing the ABIM-ECE, and this relationship was such that a score of 75% correct yielded a 97% probability of passing the ABIM-ECE, whereas a score of 50% correct generated only a 70% probability of doing so. Sex, fellowship program region, and medical school country were not significant predictors of ABIM-ECE outcomes. CONCLUSIONSIn addition to serving as an important learning instrument for endocrine fellowship programs, ESAP-ITE is a robust predictive tool for pass/fail outcomes on the ABIM-ECE.
Effective prevention is needed to combat the worldwide epidemic of type 2 diabetes. We investigated the long-term extent of beneficial effects of lifestyle intervention and metformin on diabetes ...prevention, originally shown during the 3-year Diabetes Prevention Program (DPP), and assessed whether these interventions reduced diabetes-associated microvascular complications.
The DPP (1996-2001) was a randomised trial comparing an intensive lifestyle intervention or masked metformin with placebo in a cohort selected to be at very high risk of developing diabetes. All participants were offered lifestyle training at the end of the DPP. 2776 (88%) of the surviving DPP cohort were followed up in the DPP Outcomes Study (DPPOS, Sept 1, 2002, to Jan 2, 2014) and analysed by intention to treat on the basis of their original DPP assignment. During DPPOS, the original lifestyle intervention group was offered lifestyle reinforcement semi-annually and the metformin group received unmasked metformin. The primary outcomes were the development of diabetes and the prevalence of microvascular disease. For the assessment of microvascular disease, we used an aggregate microvascular outcome, composed of nephropathy, retinopathy, and neuropathy.
During a mean follow-up of 15 years, diabetes incidence was reduced by 27% in the lifestyle intervention group (hazard ratio 0·73, 95% CI 0·65-0·83; p<0·0001) and by 18% in the metformin group (0·82, 0·72-0·93; p=0·001), compared with the placebo group, with declining between-group differences over time. At year 15, the cumulative incidences of diabetes were 55% in the lifestyle group, 56% in the metformin group, and 62% in the placebo group. The prevalences at the end of the study of the aggregate microvascular outcome were not significantly different between the treatment groups in the total cohort (placebo 12·4%, 95% CI 11·1-13·8; metformin 13·0%, 11·7-14·5; lifestyle intervention 11·3%, 10·1-12·7). However, in women (n=1887) the lifestyle intervention was associated with a lower prevalence (8·7%, 95% CI 7·4-10·2) than in the placebo (11·0%, 9·6-12·6) and metformin (11·2%, 9·7-12·9) groups, with reductions in the lifestyle intervention group of 21% (p=0·03) compared with placebo and 22% (p=0·02) compared with metformin. Compared with participants who developed diabetes, those who did not develop diabetes had a 28% lower prevalence of microvascular complications (relative risk 0·72, 95% CI 0·63-0·83; p<0·0001).
Lifestyle intervention or metformin significantly reduced diabetes development over 15 years. There were no overall differences in the aggregate microvascular outcome between treatment groups; however, those who did not develop diabetes had a lower prevalence of microvascular complications than those who did develop diabetes. This result supports the importance of diabetes prevention.
National Institute of Diabetes and Digestive and Kidney Diseases.
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