Transposons contribute to the functional diversification of the head, gut, and ovary transcriptomes across Drosophila natural strains.

Transcriptomes are dynamic, with cells, tissues, and body parts expressing particular sets of transcripts. Transposable elements (TEs) are a known source of transcriptome diversity; however, studies often focus on a particular type of chimeric transcript, analyze single body parts or cell types, or are based on incomplete TE annotations from a single reference genome. In this work, we have implemented a method based on de novo transcriptome assembly that minimizes the potential sources of errors while identifying a comprehensive set of gene-TE chimeras. We applied this method to the head, gut, and ovary dissected from five Drosophila melanogaster natural strains, with individual reference genomes available. We found that ∼19% of body part-specific transcripts are gene-TE chimeras. Overall, chimeric transcripts contribute a mean of 43% to the total gene expression, and they provide protein domains for DNA binding, catalytic activity, and DNA polymerase activity. Our comprehensive data set is a rich resource for follow-up analysis. Moreover, because TEs are present in virtually all species sequenced to date, their role in spatially restricted transcript expression is likely not exclusive to the species analyzed in this work.

DrosOmics: A Browser to Explore -omics Variation Across High-Quality Reference Genomes From Natural Populations of Drosophila melanogaster.

The advent of long-read sequencing technologies has allowed the generation of multiple high-quality de novo genome assemblies for multiple species, including well-known model species such as Drosophila melanogaster. Genome assemblies for multiple individuals of the same species are key to discover the genetic diversity present in natural populations, especially the one generated by transposable elements, the most common type of structural variant. Despite the availability of multiple genomic data sets for D. melanogaster populations, we lack an efficient visual tool to display different genome assemblies simultaneously. In this work, we present DrosOmics, a population genomic-oriented browser currently containing 52 high-quality reference genomes of D. melanogaster, including annotations from a highly reliable set of transposable elements, and functional transcriptomics and epigenomics data for 26 genomes. DrosOmics is based on JBrowse 2, a highly scalable platform, which allows the visualization of multiple assemblies at once, key to unraveling structural and functional features of D. melanogaster natural populations. DrosOmics is an open access browser and is freely available at http://gonzalezlab.eu/drosomics.

From whole bodies to single cells: A guide to transcriptomic approaches for ecology and evolutionary biology.

RNA sequencing (RNAseq) methodology has experienced a burst of technological developments in the last decade, which has opened up opportunities for studying the mechanisms of adaptation to environmental factors at both the organismal and cellular level. Selecting the most suitable experimental approach for specific research questions and model systems can, however, be a challenge and researchers in ecology and evolution are commonly faced with the choice of whether to study gene expression variation in whole bodies, specific tissues, and/or single cells. A wide range of sometimes polarised opinions exists over which approach is best. Here, we highlight the advantages and disadvantages of each of these approaches to provide a guide to help researchers make informed decisions and maximise the power of their study. Using illustrative examples of various ecological and evolutionary research questions, we guide the readers through the different RNAseq approaches and help them identify the most suitable design for their own projects.

PopHumanVar: an interactive application for the functional characterization and prioritization of adaptive genomic variants in humans.

Adaptive challenges that humans faced as they expanded across the globe left specific molecular footprints that can be decoded in our today's genomes. Different sets of metrics are used to identify genomic regions that have undergone selection. However, there are fewer methods capable of pinpointing the allele ultimately responsible for this selection. Here, we present PopHumanVar, an interactive online application that is designed to facilitate the exploration and thorough analysis of candidate genomic regions by integrating both functional and population genomics data currently available. PopHumanVar generates useful summary reports of prioritized variants that are putatively causal of recent selective sweeps. It compiles data and graphically represents different layers of information, including natural selection statistics, as well as functional annotations and genealogical estimations of variant age, for biallelic single nucleotide variants (SNVs) of the 1000 Genomes Project phase 3. Specifically, PopHumanVar amasses SNV-based information from GEVA, SnpEFF, GWAS Catalog, ClinVar, RegulomeDB and DisGeNET databases, as well as accurate estimations of iHS, nSL and iSAFE statistics. Notably, PopHumanVar can successfully identify known causal variants of frequently reported candidate selection regions, including EDAR in East-Asians, ACKR1 (DARC) in Africans and LCT/MCM6 in Europeans. PopHumanVar is open and freely available at https://pophumanvar.uab.cat.

The genomic basis of copper tolerance in Drosophila is shaped by a complex interplay of regulatory and environmental factors.

BACKGROUND: Escalation in industrialization and anthropogenic activity have resulted in an increase of pollutants released into the environment. Of these pollutants, heavy metals such as copper are particularly concerning due to their bio-accumulative nature. Due to its highly heterogeneous distribution and its dual nature as an essential micronutrient and toxic element, the genetic basis of copper tolerance is likely shaped by a complex interplay of genetic and environmental factors. RESULTS: In this study, we utilized the natural variation present in multiple populations of Drosophila melanogaster collected across Europe to screen for variation in copper tolerance. We found that latitude and the degree of urbanization at the collection sites, rather than any other combination of environmental factors, were linked to copper tolerance. While previously identified copper-related genes were not differentially expressed in tolerant vs. sensitive strains, genes involved in metabolism, reproduction, and protease induction contributed to the differential stress response. Additionally, the greatest transcriptomic and physiological responses to copper toxicity were seen in the midgut, where we found that preservation of gut acidity is strongly linked to greater tolerance. Finally, we identified transposable element insertions likely to play a role in copper stress response. CONCLUSIONS: Overall, by combining genome-wide approaches with environmental association analysis, and functional analysis of candidate genes, our study provides a unique perspective on the genetic and environmental factors that shape copper tolerance in natural D. melanogaster populations and identifies new genes, transposable elements, and physiological traits involved in this complex phenotype.

Drosophila Evolution over Space and Time (DEST): A New Population Genomics Resource.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome data sets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate data sets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in >20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This data set, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental metadata. A web-based genome browser and web portal provide easy access to the SNP data set. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan data set. Our resource will enable population geneticists to analyze spatiotemporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

The optimal 18F-fluoromisonidazole PET threshold to define tumor hypoxia in preclinical squamous cell carcinomas using pO2 electron paramagnetic resonance imaging as reference truth.

PURPOSE: To identify the optimal threshold in 18F-fluoromisonidazole (FMISO) PET images to accurately locate tumor hypoxia by using electron paramagnetic resonance imaging (pO2 EPRI) as ground truth for hypoxia, defined by pO2 [Formula: see text] 10 mmHg. METHODS: Tumor hypoxia images in mouse models of SCCVII squamous cell carcinoma (n = 16) were acquired in a hybrid PET/EPRI imaging system 2 h post-injection of FMISO. T2-weighted MRI was used to delineate tumor and muscle tissue. Dynamic contrast enhanced (DCE) MRI parametric images of Ktrans and ve were generated to model tumor vascular properties. Images from PET/EPR/MRI were co-registered and resampled to isotropic 0.5 mm voxel resolution for analysis. PET images were converted to standardized uptake value (SUV) and tumor-to-muscle ratio (TMR) units. FMISO uptake thresholds were evaluated using receiver operating characteristic (ROC) curve analysis to find the optimal FMISO threshold and unit with maximum overall hypoxia similarity (OHS) with pO2 EPRI, where OHS = 1 shows perfect overlap and OHS = 0 shows no overlap. The means of dice similarity coefficient, normalized Hausdorff distance, and accuracy were used to define the OHS. Monotonic relationships between EPRI/PET/DCE-MRI were evaluated with the Spearman correlation coefficient ([Formula: see text]) to quantify association of vasculature on hypoxia imaged with both FMISO PET and pO2 EPRI. RESULTS: FMISO PET thresholds to define hypoxia with maximum OHS (both OHS = 0.728 [Formula: see text] 0.2) were SUV [Formula: see text] 1.4 [Formula: see text] SUVmean and SUV [Formula: see text] 0.6 [Formula: see text] SUVmax. Weak-to-moderate correlations (|[Formula: see text]|< 0.70) were observed between PET/EPRI hypoxia images with vascular permeability (Ktrans) or fractional extracellular-extravascular space (ve) from DCE-MRI. CONCLUSION: This is the first in vivo comparison of FMISO uptake with pO2 EPRI to identify the optimal FMISO threshold to define tumor hypoxia, which may successfully direct hypoxic tumor boosts in patients, thereby enhancing tumor control.

iMKT: the integrative McDonald and Kreitman test.

The McDonald and Kreitman test (MKT) is one of the most powerful and widely used methods to detect and quantify recurrent natural selection using DNA sequence data. Here we present iMKT (acronym for integrative McDonald and Kreitman test), a novel web-based service performing four distinct MKT types. It allows the detection and estimation of four different selection regimes -adaptive, neutral, strongly deleterious and weakly deleterious- acting on any genomic sequence. iMKT can analyze both user's own population genomic data and pre-loaded Drosophila melanogaster and human sequences of protein-coding genes obtained from the largest population genomic datasets to date. Advanced options in the website allow testing complex hypotheses such as the application example showed here: do genes located in high recombination regions undergo higher rates of adaptation? We aim that iMKT will become a reference site tool for the study of evolutionary adaptation in massive population genomics datasets, especially in Drosophila and humans. iMKT is a free resource online at https://imkt.uab.cat.

PopHumanScan: the online catalog of human genome adaptation.

Since the migrations that led humans to colonize Earth, our species has faced frequent adaptive challenges that have left signatures in the landscape of genetic variation and that we can identify in our today's genomes. Here, we (i) perform an outlier approach on eight different population genetic statistics for 22 non-admixed human populations of the Phase III of the 1000 Genomes Project to detect selective sweeps at different historical ages, as well as events of recurrent positive selection in the human lineage; and (ii) create PopHumanScan, an online catalog that compiles and annotates all candidate regions under selection to facilitate their validation and thoroughly analysis. Well-known examples of human genetic adaptation published elsewhere are included in the catalog, as well as hundreds of other attractive candidates that will require further investigation. Designed as a collaborative database, PopHumanScan aims to become a central repository to share information, guide future studies and help advance our understanding of how selection has modeled our genomes as a response to changes in the environment or lifestyle of human populations. PopHumanScan is open and freely available at https://pophumanscan.uab.cat.

Magnetic resonance angiography reveals increased arterial blood supply and tumorigenesis following high fat feeding in a mouse model of triple-negative breast cancer.

Breast cancer is the second most commonly diagnosed malignancy among women globally. Past MRI studies have linked a high animal fat diet (HAFD) to increased mammary cancer risk in the SV40Tag mouse model of triple-negative breast cancer. Here, serial MRI examines tumor progression and measures the arterial blood volume feeding mammary glands in low fat diet (LFD) or HAFD fed mice. Virgin female C3(1)SV40Tag mice (n = 8), weaned at 3 weeks old, were assigned to an LFD (n = 4, 3.7 kcal/g, 17.2% kcal from vegetable oil) or an HAFD (n = 4, 5.3 kcal/g, 60% kcal from lard) group. From ages 8 to 12 weeks, weekly fast spin echo MR images and time-of-flight (TOF) MR angiography of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed. Inguinal mammary glands were excised and fixed for ex vivo MRI and histology. Tumor, blood, and mammary gland volumes for each time point were measured from manually traced regions of interest; tumors were classified as invasive by histopathology-blinded observers. Our analysis confirmed a strong correlation between total tumor volume and blood volume in the mammary gland. Tumor growth rates from weeks 8-12 were twice as high in HAFD-fed mice (0.42 ± 0.14/week) as in LFD-fed mice (0.21 ± 0.03/week), p < 0.004. Mammary gland blood volume growth rate was 2.2 times higher in HAFD mice (0.29 ± 0.11/week) compared with LFD mice (0.13 ± 0.06/week), p < 0.02. The mammary gland growth rate of HAFD-fed mice (0.071 ± 0.011/week) was 2.7 times larger than that of LFD-fed mice (0.026 ± 0.009/week), p < 0.01. This is the first non-invasive, in vivo MRI study to demonstrate a strong correlation between an HAFD and increased cancer burden and blood volume in mammary cancer without using contrast agents, strengthening the evidence supporting the adverse effects of an HAFD on mammary cancer. These results support the potential future use of TOF angiography to evaluate vasculature of suspicious lesions.

Mapping Selection within Drosophila melanogaster Embryo's Anatomy.

We present a survey of selection across Drosophila melanogaster embryonic anatomy. Our approach integrates genomic variation, spatial gene expression patterns, and development with the aim of mapping adaptation over the entire embryo's anatomy. Our adaptation map is based on analyzing spatial gene expression information for 5,969 genes (from text-based annotations of in situ hybridization data directly from the BDGP database, Tomancak et al. 2007) and the polymorphism and divergence in these genes (from the project DGRP, Mackay et al. 2012).The proportion of nonsynonymous substitutions that are adaptive, neutral, or slightly deleterious are estimated for the set of genes expressed in each embryonic anatomical structure using the distribution of fitness effects-alpha method (Eyre-Walker and Keightley 2009). This method is a robust derivative of the McDonald and Kreitman test (McDonald and Kreitman 1991). We also explore whether different anatomical structures differ in the phylogenetic age, codon usage, or expression bias of the genes they express and whether genes expressed in many anatomical structures show more adaptive substitutions than other genes.We found that: 1) most of the digestive system and ectoderm-derived structures are under selective constraint, 2) the germ line and some specific mesoderm-derived structures show high rates of adaptive substitution, and 3) the genes that are expressed in a small number of anatomical structures show higher expression bias, lower phylogenetic ages, and less constraint.

Magnetic Resonance Imaging and Molecular Characterization of a Hormone-Mediated Murine Model of Prostate Enlargement and Bladder Outlet Obstruction.

Urinary complications resulting from benign prostatic hyperplasia and bladder outlet obstruction continue to be a serious health problem. Novel animal model systems and imaging approaches are needed to understand the mechanisms of disease initiation, and to develop novel therapies for benign prostatic hyperplasia. Long-term administration of both estradiol and testosterone in mice can result in prostatic enlargement and recapitulate several clinical components of lower urinary tract symptoms. Herein, we use longitudinal magnetic resonance imaging and histological analyses to quantify changes in prostatic volume, urethral volume, and genitourinary vascularization over time in response to estradiol-induced prostatic enlargement. Our data demonstrate significant prostatic enlargement by 12 weeks after treatment, with no detectable immune infiltration by macrophages or T- or B-cell populations. Importantly, the percentage of cell death, as measured by terminal deoxynucleotidyl transferase dUTP nick-end labeling, was significantly decreased in the prostatic epithelium of treated animals as compared to controls. We found no significant change in prostate cell proliferation in treated mice when compared to controls. These studies highlight the utility of magnetic resonance imaging to quantify changes in prostatic and urethral volumes over time. In conjunction with histological analyses, this approach has the high potential to enable mechanistic studies of initiation and progression of clinically relevant lower urinary tract symptoms. In addition, this model is tractable for investigation and testing of therapeutic interventions to ameliorate or potentially reverse prostatic enlargement.

Adaptive Evolution Is Substantially Impeded by Hill-Robertson Interference in Drosophila.

Hill-Robertson interference (HRi) is expected to reduce the efficiency of natural selection when two or more linked selected sites do not segregate freely, but no attempt has been done so far to quantify the overall impact of HRi on the rate of adaptive evolution for any given genome. In this work, we estimate how much HRi impedes the rate of adaptive evolution in the coding genome of Drosophila melanogaster. We compiled a data set of 6,141 autosomal protein-coding genes from Drosophila, from which polymorphism levels in D. melanogaster and divergence out to D. yakuba were estimated. The rate of adaptive evolution was calculated using a derivative of the McDonald-Kreitman test that controls for slightly deleterious mutations. We find that the rate of adaptive amino acid substitution at a given position of the genome is positively correlated to both the rate of recombination and the mutation rate, and negatively correlated to the gene density of the region. These correlations are robust to controlling for each other, for synonymous codon bias and for gene functions related to immune response and testes. We show that HRi diminishes the rate of adaptive evolution by approximately 27%. Interestingly, genes with low mutation rates embedded in gene poor regions lose approximately 17% of their adaptive substitutions whereas genes with high mutation rates embedded in gene rich regions lose approximately 60%. We conclude that HRi hampers the rate of adaptive evolution in Drosophila and that the variation in recombination, mutation, and gene density along the genome affects the HRi effect.

MRI reveals increased tumorigenesis following high fat feeding in a mouse model of triple-negative breast cancer.

High animal fat consumption is associated with an increase in triple-negative breast cancer (TNBC) risk. Based on previous MRI studies demonstrating the feasibility of detecting very early non-palpable mammary cancers in simian virus 40 large T antigen (SV40TAg) mice, we examined the effect of dietary fat fed from weaning to young adulthood in this model of TNBC. Virgin female C3(1)SV40TAg mice (n = 16) were weaned at 3-4 weeks of age and then fed either a low fat diet (LFD) (n = 8, 3.7 kcal/g; 17.2% kcal from vegetable oil) or a high animal fat diet (HAFD) (n = 8, 5.3 kcal/g; 60% kcal from lard). After 8 weeks on the diet (12 weeks of age), fast spin echo MR images of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed and inguinal mammary glands were excised and formalin fixed for ex vivo MRI. 3D volume-rendered MR images were then correlated with mammary gland histology to assess the glandular parenchyma and tumor burden. Using in vivo MRI, an average of 3.88 ± 1.03 tumors were detected per HAFD-fed mouse compared with an average of 1.25 ± 1.16 per LFD-fed mouse (p < 0.007). Additionally, the average tumor volume was significantly higher following HAFD feeding (0.53 ± 0.45 mm3 ) compared with LFD feeding (0.20 ± 0.08 mm3 , p < 0.02). Analysis of ex vivo MR and histology images demonstrated that HAFD mouse mammary glands had denser parenchyma, irregular and enlarged ducts, dilated blood vessels, increased white adipose tissue, and increased tumor invasion. MRI and histological studies of the SV40TAg mice demonstrated that HAFD feeding also resulted in higher cancer incidence and larger mammary tumors. Unlike other imaging methods for assessing environmental effects on mammary cancer growth, MRI allows routine serial measurements and reliable detection of small cancers as well as accurate tumor volume measurements and assessment of the three-dimensional distribution of tumors over time.

MRI accurately identifies early murine mammary cancers and reliably differentiates between in situ and invasive cancer: correlation of MRI with histology.

MRI methods that accurately identify various stages of mouse mammary cancer could provide new knowledge that may have a direct impact on the management of breast cancer in patients. This research investigates whether we can accurately follow the progression from in situ to invasive cancer by the evaluation of in vivo and ex vivo MRI, and in comparison with histology as the gold standard for the diagnosis and staging of cancer. Six C3(1)SV40Tag virgin female mice, aged 12-16 weeks, were studied. At this age, these mice develop in situ cancer that resembles human ductal carcinoma in situ (DCIS). Fast spin-echo images of inguinal mammary glands were acquired at 9.4 T. After in vivo MRI, mice were sacrificed; inguinal mammary glands were excised and fixed in formalin for ex vivo MRI. Three-dimensional, volume-rendered, in vivo and ex vivo MR images were then correlated with histology. High-resolution ex vivo scans facilitated the comparison of in vivo scans with histology. The sizes of mammary cancers classified as in situ on the basis of histology ranged from 150 to 400 µm in largest diameter, and the average signal intensity relative to muscle was 1.40 ± 0.18 on T2 -weighted images. Cancers classified as invasive on the basis of histology were >400 µm in largest diameter, and the average intensity relative to muscle on T2 -weighted images was 2.34 ± 0.26. Using a cut-off of 400 µm in largest diameter to distinguish between in situ and invasive cancers, a T2 -weighted signal intensity of at least 1.4 times that of muscle for in situ cancer, and at least 2.3 times that of muscle for invasive cancer, 96% of in situ and 100% of invasive cancers were correctly identified on in vivo MRI, using histology as the gold standard. Precise MRI-histology correlation demonstrates that MRI reliably detects early in situ cancer and differentiates in situ from invasive cancers in the SV40Tag mouse model of human breast cancer.

IV Administered Gadodiamide Enters the Lumen of the Prostatic Glands: X-Ray Fluorescence Microscopy Examination of a Mouse Model.

OBJECTIVE: Dynamic contrast-enhanced MRI (DCE-MRI) has become a standard component of multiparametric protocols for MRI examination of the prostate, and its use is incorporated into current guidelines for prostate MRI examination. Analysis of DCE-MRI data for the prostate is usually based on the distribution of gadolinium-based agents, such as gadodiamide, into two well-mixed compartments, and it assumes that gadodiamide does not enter into the glandular lumen. However, this assumption has not been directly tested. The purpose of this study was to use x-ray fluorescence microscopy (XFM) imaging in situ to measure the concentration of gadodiamide in the epithelia and lumens of the prostate of healthy mice after IV injection of the contrast agent. MATERIALS AND METHODS: Six C57Bl6 male mice (age, 28 weeks) were sacrificed 10 minutes after IV injection of gadodiamide (0.13 mmol/kg), and three mice were sacrificed after saline injection. Prostate tissue samples obtained from each mouse were harvested and frozen; 7-µm-thick slices were sectioned for XFM imaging, and adjacent 5-µm-thick slices were sectioned for H and E staining. Elemental concentrations were determined from XFM images. RESULTS: A mean (± SD) baseline concentration of gadolinium of 0.01 ± 0.01 mM was determined from XFM measurements of prostatic tissue samples when no gadodiamide was administered, and it was used to determine the measurement error. When gadodiamide was added, the mean concentrations of gadolinium in the epithelia and lumens in 32 prostatic glands from six mice were 1.00 ± 0.13 and 0.36 ± 0.09 mM, respectively. CONCLUSION: Our data suggest that IV administration of gadodiamide results in uptake of contrast agent by the glandular lumens of the mouse prostate. We were able to quantitatively determine gadodiamide distributions in mouse prostatic epithelia and lumens.

Mammary cancer initiation and progression studied with magnetic resonance imaging.

INTRODUCTION: Previous work from this laboratory demonstrated that magnetic resonance imaging (MRI) detects early murine mammary cancers and reliably differentiates between in situ and invasive cancer. Based on this previous work, we used MRI to study initiation and progression of murine mammary cancer, and monitor the transition from the in situ to the invasive phase. METHODS: In total, seven female C3(1) SV40 Tag mice were imaged every two weeks between the ages of 8 to 23 weeks. Lesions were identified on T2-weighted images acquired at 9.4 Tesla based on their morphology and growth rates. Lesions were traced manually on MR images of each slice. Volume of each lesion was calculated by adding measurements from individual slices. Plots of lesion volume versus time were analyzed to obtain the specific growth rate (SGR). The time at which in situ cancers (referred to as 'mammary intraepithelial neoplasia (MIN)') and invasive cancers were first detected; and the time at which in situ cancers became invasive were recorded. RESULTS: A total of 121 cancers (14 to 25 per mouse) were identified in seven mice. On average the MIN lesions and invasive cancers were first detected when mice were 13 and 18 weeks old, respectively. The average SGR was 0.47 ± 0.18 week(-1) and there were no differences (P >0.05) between mice. 74 lesions had significantly different tumor growth rates before and after ~17 weeks of age; with average doubling times (DT) of 1.88 and 1.27 weeks, respectively. The average DT was significantly shorter (P <0.0001) after 17 weeks of age. However, the DT for some cancers was longer after 17 weeks of age, and about 10% of the cancers detected did not progress to the invasive stage. CONCLUSIONS: A wide range of growth rates were observed in SV40 mammary cancers. Most cancers transitioned to a more aggressive phenotype at approximately 17 weeks of age, but some cancers became less aggressive. The results suggest that the biology of mammary cancers is extremely heterogeneous. This work is a first step towards use of MRI to improve understanding of factors that control and/or signal the development of aggressive breast cancer.

Experimental Validation of Transposable Element Insertions Using the Polymerase Chain Reaction (PCR).

Transposable elements (TEs), also known as transposons, are repetitive DNA sequences, present in virtually all organisms, that can move from one genomic position to another. TEs can be a source of mutations with important consequences for organisms. Despite their interest, its repetitive nature has made their study very challenging. However, the emergence of new sequencing technologies that allow obtaining long-read sequences, has improved the in silico de novo detection and annotation of TEs. The de novo annotation of TEs has already been performed in several organisms including the fruit fly Drosophila melanogaster. Yet, experimental validation can be used to confirm the presence of TEs in specific D. melanogaster natural populations. Here, we present a step-by-step protocol to experimentally validate by polymerase chain reaction (PCR) the presence and/or absence of TEs in natural populations of D. melanogaster. This detailed protocol has been implemented in the participant high schools of the Citizen Fly Lab activity that is part of the international citizen science project Melanogaster: Catch the Fly! ( https://melanogaster.eu ). Specifically, the students collaborate with the scientists of the European Drosophila Population Genomics Consortium (DrosEU) in the experimental validation of new genetic variants, previously identified using bioinformatic techniques.

Reversal of Lactate and PD-1-mediated Macrophage Immunosuppression Controls Growth of PTEN/p53-deficient Prostate Cancer.

PURPOSE: Phosphatase and tensin homolog (PTEN) loss of function occurs in approximately 50% of patients with metastatic castrate-resistant prostate cancer (mCRPC), and is associated with poor prognosis and responsiveness to standard-of-care therapies and immune checkpoint inhibitors. While PTEN loss of function hyperactivates PI3K signaling, combinatorial PI3K/AKT pathway and androgen deprivation therapy (ADT) has demonstrated limited anticancer efficacy in clinical trials. Here, we aimed to elucidate mechanism(s) of resistance to ADT/PI3K-AKT axis blockade, and to develop rational combinatorial strategies to effectively treat this molecular subset of mCRPC. EXPERIMENTAL DESIGN: Prostate-specific PTEN/p53-deficient genetically engineered mice (GEM) with established 150-200 mm3 tumors, as assessed by ultrasound, were treated with either ADT (degarelix), PI3K inhibitor (copanlisib), or anti-PD-1 antibody (aPD-1), as single agents or their combinations, and tumors were monitored by MRI and harvested for immune, transcriptomic, and proteomic profiling, or ex vivo co-culture studies. Single-cell RNA sequencing on human mCRPC samples was performed using 10X Genomics platform. RESULTS: Coclinical trials in PTEN/p53-deficient GEM revealed that recruitment of PD-1-expressing tumor-associated macrophages (TAM) thwarts ADT/PI3Ki combination-induced tumor control. The addition of aPD-1 to ADT/PI3Ki combination led to TAM-dependent approximately 3-fold increase in anticancer responses. Mechanistically, decreased lactate production from PI3Ki-treated tumor cells suppressed histone lactylation within TAM, resulting in their anticancer phagocytic activation, which was augmented by ADT/aPD-1 treatment and abrogated by feedback activation of Wnt/ß-catenin pathway. Single-cell RNA-sequencing analysis in mCRPC patient biopsy samples revealed a direct correlation between high glycolytic activity and TAM phagocytosis suppression. CONCLUSIONS: Immunometabolic strategies that reverse lactate and PD-1-mediated TAM immunosuppression, in combination with ADT, warrant further investigation in patients with PTEN-deficient mCRPC.

Complete mitochondrial DNA profile in stroke: A geographical matched case-control study in Spanish population.

INTRODUCTION: Stroke, the second leading cause of death worldwide, is a complex disease influenced by many risk factors among which we can find reactive oxygen species (ROS). Since mitochondria are the main producers of cellular ROS, nowadays studies are trying to elucidate the role of these organelles and its DNA (mtDNA) variation in stroke risk. The aim of the present study was to perform a comprehensive evaluation of the association between mtDNA mutations and mtDNA content and stroke risk. MATERIAL AND METHODS: Homoplasmic and heteroplasmic mutations of the mtDNA were analysed in a case-controls study using 110 S cases and their corresponding control individuals. Mitochondrial DNA copy number (mtDNA-CN) was analysed in 73 of those case-control pairs. RESULTS: Our results suggest that haplogroup V, specifically variants m.72C > T, m.4580G > A, m.15904C > T and m.16298 T > C have a protective role in relation to stroke risk. On the contrary, variants m.73A > G, m.11719G > A and m.14766C > T appear to be genetic risk factors for stroke. In this study, we found no statistically significant association between stroke risk and mitochondrial DNA copy number. CONCLUSIONS: These results demonstrate the possible role of mtDNA genetics on the pathogenesis of stroke, probably through alterations in mitochondrial ROS production.