Integrating leiomyoma genetics, epigenomics, and single-cell transcriptomics reveals causal genetic variants, genes, and cell types.

Uterine fibroids (UF), that can disrupt normal uterine function and cause significant physical and psychological health problems, are observed in nearly 70% of women of reproductive age. Although heritable genetics is a significant risk factor, specific genetic variations and gene targets causally associated with UF are poorly understood. Here, we performed a meta-analysis on existing fibroid genome-wide association studies (GWAS) and integrated the identified risk loci and potentially causal single nucleotide polymorphisms (SNPs) with epigenomics, transcriptomics, 3D chromatin organization from diverse cell types as well as primary UF patient's samples. This integrative analysis identifies 24 UF-associated risk loci that potentially target 394 genes, of which 168 are differentially expressed in UF tumors. Critically, integrating this data with single-cell gene expression data from UF patients reveales the causal cell types with aberrant expression of these target genes. Lastly, CRISPR-based epigenetic repression (dCas9-KRAB) or activation (dCas9-p300) in a UF disease-relevant cell type further refines and narrows down the potential gene targets. Our findings and the methodological approach indicate the effectiveness of integrating multi-omics data with locus-specific epigenetic editing approaches for identifying gene- and celt type-targets of disease-relevant risk loci.

Biology-driven therapy advances in high-grade serous ovarian cancer.

Following a period of slow progress, the completion of genome sequencing and the paradigm shift relative to the cell of origin for high grade serous ovarian cancer (HGSOC) led to a new perspective on the biology and therapeutic solutions for this deadly cancer. Experimental models were revisited to address old questions, and improved tools were generated. Additional pathways emerging as drivers of ovarian tumorigenesis and key dependencies for therapeutic targeting, in particular, VEGF-driven angiogenesis and homologous recombination deficiency, were discovered. Molecular profiling of histological subtypes of ovarian cancer defined distinct genetic events for each entity, enabling the first attempts toward personalized treatment. Armed with this knowledge, HGSOC treatment was revised to include new agents. Among them, PARP inhibitors (PARPis) were shown to induce unprecedented improvement in clinical benefit for selected subsets of patients. Research on mechanisms of resistance to PARPis is beginning to discover vulnerabilities and point to new treatment possibilities. This Review highlights these advances, the remaining challenges, and unsolved problems in the field.

Engineered MED12 mutations drive leiomyoma-like transcriptional and metabolic programs by altering the 3D genome compartmentalization.

Nearly 70% of Uterine fibroid (UF) tumors are driven by recurrent MED12 hotspot mutations. Unfortunately, no cellular models could be generated because the mutant cells have lower fitness in 2D culture conditions. To address this, we employ CRISPR to precisely engineer MED12 Gly44 mutations in UF-relevant myometrial smooth muscle cells. The engineered mutant cells recapitulate several UF-like cellular, transcriptional, and metabolic alterations, including altered Tryptophan/kynurenine metabolism. The aberrant gene expression program in the mutant cells is, in part, driven by a substantial 3D genome compartmentalization switch. At the cellular level, the mutant cells gain enhanced proliferation rates in 3D spheres and form larger lesions in vivo with elevated production of collagen and extracellular matrix deposition. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a platform for the broader scientific community to characterize genomics of recurrent MED12 mutations.

Engineered MED12 mutations drive uterine fibroid-like transcriptional and metabolic programs by altering the 3D genome compartmentalization.

Uterine fibroid (UF) tumors originate from a mutated smooth muscle cell (SMC). Nearly 70% of these tumors are driven by hotspot recurrent somatic mutations in the MED12 gene; however, there are no tractable genetic models to study the biology of UF tumors because, under culture conditions, the non-mutant fibroblasts outgrow the mutant SMC cells, resulting in the conversion of the population to WT phenotype. The lack of faithful cellular models hampered our ability to delineate the molecular pathways downstream of MED12 mutations and identify therapeutics that may selectively target the mutant cells. To overcome this challenge, we employed CRISPR knock-in with a sensitive PCR-based screening strategy to precisely engineer cells with mutant MED12 Gly44, which constitutes 50% of MED12 exon two mutations. Critically, the engineered myometrial SMC cells recapitulate several UF-like cellular, transcriptional and metabolic alterations, including enhanced proliferation rates in 3D spheres and altered Tryptophan/kynurenine metabolism. Our transcriptomic analysis supported by DNA synthesis tracking reveals that MED12 mutant cells, like UF tumors, have heightened expression of DNA repair genes but reduced DNA synthesis rates. Consequently, these cells accumulate significantly higher rates of DNA damage and are selectively more sensitive to common DNA-damaging chemotherapy, indicating mutation-specific and therapeutically relevant vulnerabilities. Our high-resolution 3D chromatin interaction analysis demonstrates that the engineered MED12 mutations drive aberrant genomic activity due to a genome-wide chromatin compartmentalization switch. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a novel platform for the broader scientific community to characterize genomics of recurrent MED12 mutations and discover potential therapeutic targets.

A Rapid Method for Direct Quantification of Antibody Binding-Site Concentration in Serum.

The quantitation of the available antibody binding-site concentration of polyclonal antibodies in serum is critical in defining the efficacy of vaccines against substances of abuse. We have conceptualized an equilibrium dialysis (ED)-based approach coupled with fluorimetry (ED-fluorimetry) to measure the antibody binding-site concentration to the ligand in an aqueous environment. The measured binding-site concentrations in monoclonal antibody (mAb) and sera samples from TT-6-AmHap-immunized rats by ED-fluorimetry are in agreement with those determined by a more established equilibrium dialysis coupled with ultraperformance liquid chromatography tandem mass spectrometry (ED-UPLC-MS/MS). Importantly, we have shown that the measured antibody binding-site concentrations to the ligand by ED-fluorimetry were not influenced by the sample serum matrix; thus, this method is valid for determining the binding-site concentration of polyclonal antibodies in sera samples. Further, we have demonstrated that under appropriate analytical conditions, this method resolved the total binding-site concentrations on a nanomolar scale with good accuracy and repeatability within the microliter sample volumes. This simple, rapid, and sample preparation-free approach has the potential to reliably perform quantitative antibody binding-site screening in serum and other more complex biological fluids.

Situación nutricional entre los percentiles ecuatorianos y la OMS en adolescentes del 12 años / Nutritional situation between ecuadorian and who percentile in 12-year-old adolescents

Se trata de la recopilación de información sobre estudios que reflejan la situación nutricional en Ecuador, y las comparaciones entre los percentiles ecuatorianos y los que sugiere la Organización Mundial de la Salud (OMS). Estudios refieren que este tipo de comparación difiere de las realidades de un país pues presenta diferente ubicación geográfica y por sus rasgos culturales, que difieren ancestralmente se presentan divergencias entre la alimentación, estilos de vida, bases socioeconómicas, rasgos antropométricos y otras variables que influyen y que han obligado a crear sus propios estándares de comparaciones. Objetivo: Describir la situación nutricional entre los percentiles ecuatorianos y la OMS en adolescentes de 12 años. Materiales y método: Es del tipo cualitativa, descriptiva, donde se recopila información relevante vinculada al descriptor en estudio, por eso la modalidad es tipo documental y descriptiva, transversal y observacional porque se le y observa información relevante de momento y se describe y señalan procedimientos como modelos para otras investigaciones. Resultados: Ecuador como país latinoamericano refleja una situación alimentaria y nutricional de amplio potencial productivo, de grandes oportunidades y capacidades socioeconómicas, necesarios para dar respuestas a las exigencias nutricionales de su población. Sin embargo, la información sobre el estado nutricional de la población infantil refleja el desarrollo del país, con una reducción significativa de la prevalencia de retardo de crecimiento, que prevalecen mucho más en grupos indígenas, por problemas sociales: mortalidad infantil, pobreza, indigencia y analfabetismo. Conclusiones: Estimar valores de desnutrición y sobrepeso a través de comparaciones por los percentiles internacionales (WHO) y los de Estados Unidos no reflejan la realidad de una población que difiere en todo con la de esos estándares comparativos. Por ello el esfuerzo de estudios por crear sus propios estándares nacionales es loable y muestra las realidades de una población(AU)

This is the collection of information on studies that reflect the nutritional situation in Ecuador, and comparisons between Ecuadorian percentiles and those suggested by the World Health Organization (WHO). Studies report that this type of comparison differs from the realities of a country because it has different geographical location and its cultural features, which differ ancestrally, there are divergences between food, lifestyles, socioeconomic bases, anthropometric traits and other variables that influence and that have forced to create their own standards of comparisons. Objective: To describe the nutritional situation between the Ecuadorian and WHO percentiles in 12-year-old adolescents Materials and methods: It is of the qualitative, descriptive type, where relevant information linked to the descriptor in study is collected, so the modality is documentary and descriptive, transversal and observational because relevant information is observed at the moment and procedures are described and indicated as models for another research. Results: Ecuador as a Latin American country reflects a food and nutritional situation with ample productive potential, great opportunities and socioeconomic capacities, necessary to respond to the nutritional demands of its population. However, information on the nutritional status of the child population reflects the development of the country, with a significant reduction in the prevalence of growth retardation, which is much more prevalent in indigenous groups, due to social problems: infant mortality, poverty, indigence and illiteracy. Conclusions: Estimating values of malnutrition and overweight through comparisons by the international percentiles (WHO) and those of the United States do not reflect the reality of a population that differs in everything with that of these comparative standards. That is why the effort of studies to create their own national standards is laudable and shows the realities of a population(AU)

Chemotherapy-Induced Distal Enhancers Drive Transcriptional Programs to Maintain the Chemoresistant State in Ovarian Cancer.

Chemoresistance is driven by unique regulatory networks in the genome that are distinct from those necessary for cancer development. Here, we investigate the contribution of enhancer elements to cisplatin resistance in ovarian cancers. Epigenome profiling of multiple cellular models of chemoresistance identified unique sets of distal enhancers, super-enhancers (SE), and their gene targets that coordinate and maintain the transcriptional program of the platinum-resistant state in ovarian cancer. Pharmacologic inhibition of distal enhancers through small-molecule epigenetic inhibitors suppressed the expression of their target genes and restored cisplatin sensitivity in vitro and in vivo. In addition to known drivers of chemoresistance, our findings identified SOX9 as a critical SE-regulated transcription factor that plays a critical role in acquiring and maintaining the chemoresistant state in ovarian cancer. The approach and findings presented here suggest that integrative analysis of epigenome and transcriptional programs could identify targetable key drivers of chemoresistance in cancers. SIGNIFICANCE: Integrative genome-wide epigenomic and transcriptomic analyses of platinum-sensitive and -resistant ovarian lines identify key distal regulatory regions and associated master regulator transcription factors that can be targeted by small-molecule epigenetic inhibitors.

A rapid solution-based method for determining the affinity of heroin hapten-induced antibodies to heroin, its metabolites, and other opioids.

We describe for the first time a method that utilizes microscale thermophoresis (MST) technology to determine polyclonal antibody affinities to small molecules. Using a novel type of heterologous MST, we have accurately measured a solution-based binding affinity of serum antibodies to heroin which was previously impossible with other currently available methods. Moreover, this mismatch approach (i.e., using a cross-reactive hapten tracer) has never been reported in the literature. When compared with equilibrium dialysis combined with ultra-performance liquid chromatography/tandem mass spectrometry (ED-UPLC/MS/MS), this novel MST method yields similar binding affinity values for polyclonal antibodies to the major heroin metabolites 6-AM and morphine. Additionally, we herein report the method of synthesis of this novel cross-reactive hapten, MorHap-acetamide-a useful analog for the study of heroin hapten-antibody interactions. Using heterologous MST, we were able to determine the affinities, down to nanomolar accuracies, of polyclonal antibodies to various abused opioids. While optimizing this method, we further discovered that heroin is protected from serum esterase degradation by the presence of these antibodies in a concentration-dependent manner. Lastly, using affinity data for a number of structurally different opioids, we were able to dissect the moieties that are crucial to antibody binding. The novel MST method that is presented herein can be extended to the analysis of any ligand that is prone to degradation and can be applied not only to the development of vaccines to substances of abuse but also to the analysis of small molecule/protein interactions in the presence of serum. Graphical abstract Strategy for the determination of hapten-induced antibody affinities using Microscale thermophoresis.