RESUMEN
Deliberate and effortful attempts to gaze at the bodies of women is emerging as a valuable marker of sexual objectification in men. Some preliminary evidence suggests that pervasive body gaze behavior may also accompany insidious attitudes which can facilitate sexual assault. The present study aimed to further explore this potential by examining pervasive body gaze associations with explicit, implicit, and physiological sexual assault propensity measures. We presented 110 heterosexual male participants with images of fully and partially dressed women with and without injuries while measuring their skin conductance responses. We also captured implicit and explicit sexual assault measures in addition to self-reported pervasive body gaze behavior. Pervasive body gaze behavior was significantly correlated with rape myth acceptance attitudes, prior perpetration of sexual assault, a stronger implicit association between erotica and aggression, and lower physiological reactivity during exposure to partially dressed injured women. These findings suggest that body gaze towards women could be a behavioral marker for inclinations to victim blame, preferences for rough sexual conduct, and a physiological desensitization towards female victims. This study further validates a five item self-reported body gaze measure as a valuable tool for detecting deviant sexual objectification attitudes and affective states. As such, measurement and observation of body gaze behavior could be useful for developing risk assessments, estimating intervention efficacy, and enhancing public awareness.
RESUMEN
The pathogenesis of duodenal tumors in the inherited tumor syndromes familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) is poorly understood. This study aimed to identify genes that are significantly mutated in these tumors and to explore the effects of these mutations. Whole exome and whole transcriptome sequencing identified recurrent somatic coding variants of phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PIGA) in 19/70 (27%) FAP and MAP duodenal adenomas, and further confirmed the established driver roles for APC and KRAS. PIGA catalyzes the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Flow cytometry of PIGA-mutant adenoma-derived and CRISPR-edited duodenal organoids confirmed loss of GPI anchors in duodenal epithelial cells and transcriptional profiling of duodenal adenomas revealed transcriptional signatures associated with loss of PIGA. IMPLICATIONS: PIGA somatic mutation in duodenal tumors from patients with FAP and MAP and loss of membrane GPI-anchors may present new opportunities for understanding and intervention in duodenal tumorigenesis.
Asunto(s)
Poliposis Adenomatosa del Colon , Neoplasias Duodenales , Glicosilfosfatidilinositoles , Mutación , Humanos , Glicosilfosfatidilinositoles/metabolismo , Glicosilfosfatidilinositoles/genética , Neoplasias Duodenales/genética , Neoplasias Duodenales/metabolismo , Neoplasias Duodenales/patología , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/metabolismo , Poliposis Adenomatosa del Colon/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Carcinogénesis/genética , Masculino , FemeninoRESUMEN
Structural changes to microvascular networks are increasingly highlighted as markers of pathogenesis in a wide range of disease, e.g. Alzheimer's disease, vascular dementia and tumour growth. This has motivated the development of dedicated 3D imaging techniques, alongside the creation of computational modelling frameworks capable of using 3D reconstructed networks to simulate functional behaviours such as blood flow or transport processes. Extraction of 3D networks from imaging data broadly consists of two image processing steps: segmentation followed by skeletonisation. Much research effort has been devoted to segmentation field, and there are standard and widely-applied methodologies for creating and assessing gold standards or ground truths produced by manual annotation or automated algorithms. The Skeletonisation field, however, lacks widely applied, simple to compute metrics for the validation or optimisation of the numerous algorithms that exist to extract skeletons from binary images. This is particularly problematic as 3D imaging datasets increase in size and visual inspection becomes an insufficient validation approach. In this work, we first demonstrate the extent of the problem by applying 4 widely-used skeletonisation algorithms to 3 different imaging datasets. In doing so we show significant variability between reconstructed skeletons of the same segmented imaging dataset. Moreover, we show that such a structural variability propagates to simulated metrics such as blood flow. To mitigate this variability we introduce a new, fast and easy to compute super metric that compares the volume, connectivity, medialness, bifurcation point identification and homology of the reconstructed skeletons to the original segmented data. We then show that such a metric can be used to select the best performing skeletonisation algorithm for a given dataset, as well as to optimise its parameters. Finally, we demonstrate that the super metric can also be used to quickly identify how a particular skeletonisation algorithm could be improved, becoming a powerful tool in understanding the complex implication of small structural changes in a network.
Asunto(s)
Imagenología Tridimensional , Neoplasias , Humanos , Imagenología Tridimensional/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Algoritmos , Simulación por ComputadorRESUMEN
Chronic hypoxia (CH) from birth attenuates the acute hypoxic ventilatory response (HVR) in rats and other mammals, but CH is often reported to augment the HVR in adult mammals. To test the hypothesis that this transition - from blunting to augmenting the HVR - occurs in the third or fourth postnatal week in rats, juvenile and adult rats were exposed to normobaric CH (12% O2) for 7 days and the HVR was assessed by whole-body plethysmography. No transition was observed, however, and the acute HVR was reduced by 61 - 85% across all ages studied. The failure to observe an augmented HVR in adult rats could not be explained by the substrain of Sprague Dawley rats used, the duration of the CH exposure, the order in which test gases were presented, the level of hypoxia used for CH and to assess the HVR, or the effects of CH on the metabolic response to hypoxia and the hypercapnic ventilatory response. A literature survey revealed several distinct patterns of ventilatory acclimatization to hypoxia (VAH) in adult rats, with most studies (77%) revealing a decrease or no change in the acute HVR after CH. In conclusion, the effects of CH on respiratory control are qualitatively similar across age groups, at least within the populations of Sprague Dawley rats used in the present study, and there does not appear to be one "typical" pattern for VAH in adult rats.
Asunto(s)
Hipoxia , Ventilación Pulmonar , Animales , Ratas , Ventilación Pulmonar/fisiología , Ratas Sprague-Dawley , Hipoxia/metabolismo , Pletismografía Total , MamíferosRESUMEN
Whilst the use of various blended learning models preceded the COVID-19 pandemic, the abrupt shift to remote delivery served as catalyst within the sector in enhancing digital solutions to meet immediate student needs. As we emerge from the pandemic, a return to purely didactic and impersonal in-person teaching seems anticlimactic, with the return to the lecture theatre seeing many lecturers trialling various digital tools in creating more interactive in-person, synchronous, and asynchronous sessions. In evaluating students' experiences of the various tools and approaches applied by academic staff, a survey was developed by a multidisciplinary team of educators at Cardiff University's School of Medicine exploring student perceptions of e-learning resources (ELRs), as well as student experiences of various blended learning approaches. The primary aim of this study was to evaluate student experience, satisfaction, and engagement with ELRs and blended learning. A total of 179 students (undergraduate and postgraduate) completed the survey. 97% confirmed that e-learning resources were blended within the teaching they received, with 77% rating the quality of e-learning as good-to-excellent and 66% reporting a preference for asynchronous resources that enable them to learn at their own pace. A variety of platforms, tools, and approaches were identified by students as meeting their diverse learning needs. We therefore propose a personalised, evidence-based and inclusive learning (PEBIL) model enabling the application of digital technologies both on and offline.
RESUMEN
Background: Genome, transcriptome and methylome-wide association studies have identified single-nucleotide polymorphisms (SNPs) or genes at 258 loci associated with colorectal cancer (CRC) risk. We studied the relationship between these and patient outcome. Methods: We studied 1926 unrelated patients with advanced CRC from COIN and COIN-B. Of 205 CRC-risk SNPs, 19 were directly genotyped and 162 were imputed, and of 53 risk genes, 52 were tested. An additive Cox model for overall survival was adjusted for known prognostic factors. For nominally significant SNPs or genes, we considered a recessive model with a Bonferroni corrected threshold of P = 2.1 × 10-4. We examined SNPs as expression quantitative trait loci (eQTL) and the relationship between gene expression in colorectal tumours and survival in 597 unrelated patients. Results: Eleven SNPs or genes were nominally associated with survival under an additive model. Only rs117079142 mapping to UTP23 and EIF3H (Hazard Ratio [HR] = 2.79, 95% Confidence Intervals [CI] = 1.70-4.58, P = 4.7 × 10-5) and rs9924886 mapping to CDH1 and CDH3 (HR = 1.24, 95% CI = 1.12-1.38, P = 5.2 × 10-5) passed the multiple testing threshold under a recessive model. rs117079142 was an eQTL for UTP23 and rs9924886 for CDH1, CDH3 and ZFP90. Decreased CDH1 expression in CRCs was associated with worse survival (HR = 2.18, 95% CI = 1.3-3.5, P = 1.8 × 10-3). Conclusion: rs117079142 and rs9924886 may represent potential prognostic biomarkers for CRC.
RESUMEN
Cellular DNA damage caused by reactive oxygen species is repaired by the base excision repair (BER) pathway which includes the DNA glycosylase MUTYH. Inherited biallelic MUTYH mutations cause predisposition to colorectal adenomas and carcinoma. However, the mechanistic progression from germline MUTYH mutations to MUTYH-Associated Polyposis (MAP) is incompletely understood. Here, we sequence normal tissue DNAs from 10 individuals with MAP. Somatic base substitution mutation rates in intestinal epithelial cells were elevated 2 to 4-fold in all individuals, except for one showing a 31-fold increase, and were also increased in other tissues. The increased mutation burdens were of multiple mutational signatures characterised by C > A changes. Different mutation rates and signatures between individuals are likely due to different MUTYH mutations or additional inherited mutations in other BER pathway genes. The elevated base substitution rate in normal cells likely accounts for the predisposition to neoplasia in MAP. Despite ubiquitously elevated mutation rates, individuals with MAP do not display overt evidence of premature ageing. Thus, accumulation of somatic mutations may not be sufficient to cause the global organismal functional decline of ageing.
Asunto(s)
Poliposis Adenomatosa del Colon , Neoplasias Colorrectales , ADN Glicosilasas/genética , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , ADN Glicosilasas/metabolismo , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Humanos , Mutación , Tasa de MutaciónRESUMEN
Pulsatility is important to islet function. As islets mature into fully developed insulin-secreting micro-organs, their ability to produce oscillatory intracellular calcium ([Ca2+]i) patterns in response to glucose also matures. In this study, we measured [Ca2+]i using fluorescence imaging to characterize oscillations from neonatal mice on postnatal (PN) days 0, 4, and 12 in comparison to adult islets. Under substimulatory (3-mM) glucose levels, [Ca2+]i was low and quiescent for adult islets as expected, as well as for PN day 12 islets. In contrast, one-third of islets on PN day 0 and 4 displayed robust [Ca2+]i oscillations in low glucose. In stimulatory glucose (11 mM) conditions, oscillations were present on all neonatal days but differed from patterns in adults. By PN day 12, [Ca2+]i oscillations were approaching characteristics of fully developed islets. The immature response pattern of neonatal islets was due, at least in part, to differences in adenosine 5'-triphosphate (ATP)-sensitive K+-channel activity estimated by [Ca2+]i responses to KATP channel agents diazoxide and tolbutamide. Neonatal [Ca2+]i patterns were also strikingly similar to patterns observed in mature islets exposed to hyperglycemic conditions (20 mM glucose for 48 hours): elevated [Ca2+]i and oscillations in low glucose along with reduced pulse mass in high glucose. Since a hallmark of diabetic islets is dedifferentiation, we propose that diabetic islets display features of "reverse maturation," demonstrating similar [Ca2+]i dynamics as neonatal islets. Pulsatility is thus an important emergent feature of neonatal islets. Our findings may provide insight into reversing ß-cell dedifferentiation and to producing better functioning ß cells from pluripotent stem cells.
Asunto(s)
Hiperglucemia , Islotes Pancreáticos , Animales , Animales Recién Nacidos , Calcio/metabolismo , Señalización del Calcio , Glucosa/metabolismo , Glucosa/farmacología , Hiperglucemia/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , RatonesRESUMEN
We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5'-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management.
Asunto(s)
Poliposis Adenomatosa del Colon , Neoplasias Colorrectales , Neoplasias de la Úvea , Poliposis Adenomatosa del Colon/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Endodesoxirribonucleasas/genética , Predisposición Genética a la Enfermedad , Células Germinativas/patología , Mutación de Línea Germinal/genética , Humanos , Neoplasias de la Úvea/genéticaRESUMEN
Advances in biological imaging have accelerated our understanding of human physiology in both health and disease. As these advances have developed, the opportunities gained by integrating with cutting-edge mathematical models have become apparent yet remain challenging. Combined imaging-modelling approaches provide unprecedented opportunity to correlate data on tissue architecture and function, across length and time scales, to better understand the mechanisms that underpin fundamental biology and also to inform clinical decisions. Here we discuss the opportunities and challenges of such approaches, providing literature examples across a range of organ systems. Given the breadth of the field we focus on the intersection of continuum modelling and in vivo imaging applied to the vasculature and blood flow, though our rationale and conclusions extend widely. We propose three key research pillars (image acquisition, image processing, mathematical modelling) and present their respective advances as well as future opportunity via better integration. Multidisciplinary efforts that develop imaging and modelling tools concurrently, and share them open-source with the research community, provide exciting opportunity for advancing these fields.
Asunto(s)
Fenómenos Biológicos , Modelos Teóricos , Diagnóstico por Imagen , Humanos , Procesamiento de Imagen Asistido por Computador , Modelos BiológicosRESUMEN
BACKGROUND: While genome-wide association studies (GWAS) have identified germline variants influencing the risk of developing colorectal cancer (CRC), there has been limited examination of the possible role of inherited variation as a determinant of patient outcome. PATIENTS AND METHODS: We performed a GWAS for overall survival (OS) in 1926 patients with advanced CRC from the COIN and COIN-B clinical trials. For single nucleotide polymorphisms (SNPs) showing an association with OS (P < 1.0 × 10-5), we conducted sensitivity analyses based on the time from diagnosis to death and sought independent replications in 5675 patients from the Study of Colorectal Cancer in Scotland (SOCCS) and 16,964 patients from the International Survival Analysis in Colorectal cancer Consortium (ISACC). We analysed the Human Protein Atlas to determine if ERBB4 expression was associated with survival in 438 patients with colon adenocarcinomas. RESULTS: The most significant SNP associated with OS was rs79612564 in ERBB4 (hazard ratio [HR] = 1.24, 95% confidence interval [CI] = 1.16-1.32, P = 1.9 × 10-7). SNPs at 17 loci had suggestive associations for OS and all had similar effects on the time from diagnosis to death. No lead SNPs were independently replicated in the meta-analysis of all patients from SOCCS and ISACC. However, rs79612564 was significant in stage-IV patients from SOCCS (P = 2.1 × 10-2) but not ISACC (P = 0.89) and SOCCS combined with COIN and COIN-B attained genome-wide significance (P = 1.7 × 10-8). Patients with high ERBB4 expression in their colon adenocarcinomas had worse survival (HR = 1.50, 95% CI = 1.1-1.9, P = 4.6 × 10-2). CONCLUSIONS: Genetic and expression data support a potential role for rs79612564 in the receptor tyrosine kinase ERBB4 as a predictive biomarker of survival.
Asunto(s)
Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Receptor ErbB-4/genética , Adenocarcinoma/mortalidad , Neoplasias Colorrectales/mortalidad , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
Chemotherapies administered at normal therapeutic dosages can cause significant side-effects and may result in early treatment discontinuation. Inter-individual variation in toxicity highlights the need for biomarkers to personalise treatment. We sought to identify such biomarkers by conducting 40 genome-wide association studies, together with gene and gene set analyses, for any toxicity and 10 individual toxicities in 1800 patients with advanced colorectal cancer treated with oxaliplatin and fluoropyrimidine chemotherapy ± cetuximab from the MRC COIN and COIN-B trials (385 patients received FOLFOX, 360 FOLFOX + cetuximab, 707 XELOX and 348 XELOX + cetuximab). Single nucleotide polymorphisms (SNPs), genes and gene sets that reached genome-wide or suggestive significance were validated in independent patient groups. We found that MROH5 was significantly associated with neutropenia in MAGMA gene analyses in patients treated with XELOX (P = 6.6 × 10-7 ) and was independently validated in those receiving XELOX + cetuximab; pooled P = 3.7 × 10-7 . rs13260246 at 8q21.13 was significantly associated with vomiting in patients treated with XELOX (odds ratio = 5.0, 95% confidence interval = 3.0-8.3, P = 9.8 × 10-10 ) but was not independently replicated. SNPs at 139 loci had suggestive associations for toxicities and lead SNPs at five of these were independently validated (rs6030266 with diarrhoea, rs1546161 with hand-foot syndrome, rs9601722 with neutropenia, rs13413764 with lethargy and rs4600090 with nausea; all with pooled P's < 5.0 × 10-6 ). In conclusion, the association of MROH5 with neutropenia and five other putative biomarkers warrant further investigation for their potential clinical utility. Despite our comprehensive genome-wide analyses of large, well-characterised, clinical trials, we found a lack of common variants with modest effect sizes associated with toxicities.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/genética , Estudio de Asociación del Genoma Completo/métodos , Polimorfismo de Nucleótido Simple , Adenocarcinoma/genética , Adenocarcinoma/patología , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Capecitabina/administración & dosificación , Capecitabina/efectos adversos , Cetuximab/administración & dosificación , Cetuximab/efectos adversos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Diarrea/inducido químicamente , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/efectos adversos , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Oxaliplatino/administración & dosificación , Oxaliplatino/efectos adversos , Ensayos Clínicos Controlados Aleatorios como Asunto , Proteínas Ribosómicas/genética , Factores de Empalme Serina-Arginina/genética , Vómitos/inducido químicamenteRESUMEN
Nanoparticles have the potential to increase the efficacy of anticancer drugs whilst reducing off-target side effects. However, there remain uncertainties regarding the cellular uptake kinetics of nanoparticles which could have implications for nanoparticle design and delivery. Polymersomes are nanoparticle candidates for cancer therapy which encapsulate chemotherapy drugs. Here we develop a mathematical model to simulate the uptake of polymersomes via endocytosis, a process by which polymersomes bind to the cell surface before becoming internalised by the cell where they then break down, releasing their contents which could include chemotherapy drugs. We focus on two in vitro configurations relevant to the testing and development of cancer therapies: a well-mixed culture model and a tumour spheroid setup. Our mathematical model of the well-mixed culture model comprises a set of coupled ordinary differential equations for the unbound and bound polymersomes and associated binding dynamics. Using a singular perturbation analysis we identify an optimal number of ligands on the polymersome surface which maximises internalised polymersomes and thus intracellular chemotherapy drug concentration. In our mathematical model of the spheroid, a multiphase system of partial differential equations is developed to describe the spatial and temporal distribution of bound and unbound polymersomes via advection and diffusion, alongside oxygen, tumour growth, cell proliferation and viability. Consistent with experimental observations, the model predicts the evolution of oxygen gradients leading to a necrotic core. We investigate the impact of two different internalisation functions on spheroid growth, a constant and a bond dependent function. It was found that the constant function yields faster uptake and therefore chemotherapy delivery. We also show how various parameters, such as spheroid permeability, lead to travelling wave or steady-state solutions.
Asunto(s)
Antineoplásicos , Portadores de Fármacos , Endocitosis , Modelos Biológicos , Nanopartículas/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Transporte Biológico , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacología , Humanos , Cinética , Nanopartículas/químicaRESUMEN
In the endocrine pancreas, growth hormone (GH) is known to promote pancreatic islet growth and insulin secretion. In this study, we show that GH receptor (GHR) loss in the germline and in adulthood impacts islet mass in general but more profoundly in male mice. GHR knockout (GHRKO) mice have enhanced insulin sensitivity and low circulating insulin. We show that the total cross-sectional area of isolated islets (estimated islet mass) was reduced by 72% in male but by only 29% in female GHRKO mice compared with wild-type controls. Also, islets from GHRKO mice secreted â¼50% less glucose-stimulated insulin compared with size-matched islets from wild-type mice. We next used mice with a floxed Ghr gene to knock down the GHR in adult mice at 6 mo of age (6mGHRKO) and examined the impact on glucose and islet metabolism. By 12 mo of age, female 6mGHRKO mice had increased body fat and reduced islet mass but had no change in glucose tolerance or insulin sensitivity. However, male 6mGHRKO mice had nearly twice as much body fat, substantially reduced islet mass, and enhanced insulin sensitivity, but no change in glucose tolerance. Despite large losses in islet mass, glucose-stimulated insulin secretion from isolated islets was not significantly different between male 6mGHRKO and controls, whereas isolated islets from female 6mGHRKO mice showed increased glucose-stimulated insulin release. Our findings demonstrate the importance of GH to islet mass throughout life and that unique sex-specific adaptations to the loss of GH signaling allow mice to maintain normal glucose metabolism.NEW & NOTEWORTHY Growth hormone (GH) is important for more than just growth. GH helps to maintain pancreatic islet mass and insulin secretion throughout life. Sex-specific adaptations to the loss of GH signaling allow mice to maintain normal glucose regulation despite losing islet mass.
Asunto(s)
Células Germinativas/metabolismo , Hormona del Crecimiento/deficiencia , Islotes Pancreáticos/crecimiento & desarrollo , Islotes Pancreáticos/fisiología , Receptores de Somatotropina/genética , Factores de Edad , Animales , Proliferación Celular/genética , Femenino , Células Germinativas/fisiología , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos/genética , Receptores de Somatotropina/deficiencia , Receptores de Somatotropina/metabolismo , Caracteres Sexuales , Transducción de Señal/genéticaRESUMEN
Insufficient insulin secretion is a key component of both type 1 and type 2 diabetes. Since insulin is released by the islets of Langerhans, obtaining viable and functional islets is critical for research and transplantation. The effective and efficient isolation of these small islands of endocrine cells from the sea of exocrine tissue that is the rest of the pancreas is not necessarily simple or quick. Choosing and administering the digestive enzyme, separation of the islets from acinar tissue, and culture of islets are all things that must be considered. The purpose of this review is to provide a history of the development of islet isolation procedures and to serve as a practical guide to rodent islet research for newcomers to islet biology. We discuss key elements of mouse islet isolation including choosing collagenase, the digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews techniques for assessing islet viability and function such as visual assessment, glucose-stimulated insulin secretion and intracellular calcium measurements. A detailed protocol is provided that describes a common method our laboratory uses to obtain viable and functional mouse islets for in vitro study. This review thus provides a strong foundation for successful procurement and purification of high-quality mouse islets for research purposes.
RESUMEN
Pancreatic islet cells develop mature physiological responses to glucose and other fuels postnatally. In this study, we used fluorescence imaging techniques to measure changes in intracellular calcium ([Ca2+]i) to compare islets isolated from mice on postnatal days 0, 4, and 12 with islets from adult CD-1 mice. In addition, we used publicly available RNA-sequencing data to compare expression levels of key genes in ß-cell physiology with [Ca2+]i data across these ages. We show that islets isolated from mice on postnatal day 0 displayed elevated [Ca2+]i in basal glucose (≤4â¯mM) but lower [Ca2+]i responses to stimulation by 12-20â¯mM glucose compared to adult. Neonatal islets displayed more adult-like [Ca2+]i in basal glucose by day 4 but continued to show lower [Ca2+]i responses to 16 and 20â¯mM glucose stimulation up to at least day 12. A right shift in glucose sensing (EC50) correlated with lower fragment-per-kilobase-of-transcript-per-million-reads-mapped (FPKM) of Slc2a2 (glut2) and Actn3 and increased FPKM for Galk1 and Nupr1. Differences in [Ca2+]i responses to additional stimuli were also observed. Calcium levels in the endoplasmic reticulum were elevated on day 0 but became adult-like by day 4, which corresponded with reduced expression in Atp2a2 (SERCA2) and novel K+-channel Ktd17, increased expression of Pml, Wfs1, Thada, and Herpud1, and basal [Ca2+]i maturing to adult levels. Ion-channel activity also matured rapidly, but RNA sequencing data mining did not yield strong leads. In conclusion, the maturation of islet [Ca2+]i signaling is complex and multifaceted; several possible gene targets were identified that may participate in this process.
Asunto(s)
Señalización del Calcio , Islotes Pancreáticos/metabolismo , Envejecimiento/fisiología , Animales , Animales Recién Nacidos , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/efectos de los fármacos , Diabetes Mellitus/tratamiento farmacológico , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Glucólisis/efectos de los fármacos , Glucólisis/genética , Homeostasis/efectos de los fármacos , Homeostasis/genética , Islotes Pancreáticos/efectos de los fármacos , Ratones , Nifedipino/farmacología , Cloruro de Potasio/farmacologíaRESUMEN
Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , ADN Helicasas/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Esclerosis Tuberosa/metabolismo , Secuencia de Aminoácidos , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/química , Evolución Molecular , Femenino , Humanos , Insulina/farmacología , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fenotipo , Proteínas de Unión a Poli-ADP-Ribosa/química , ARN Helicasas/química , Proteínas con Motivos de Reconocimiento de ARN/química , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Pez Cebra/metabolismoAsunto(s)
Pólipos Adenomatosos/patología , ADN Glicosilasas/genética , Neoplasias Duodenales/epidemiología , Pólipos Adenomatosos/diagnóstico , Pólipos Adenomatosos/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Duodenales/diagnóstico , Neoplasias Duodenales/genética , Neoplasias Duodenales/patología , Duodenoscopía/estadística & datos numéricos , Duodeno/diagnóstico por imagen , Duodeno/patología , Femenino , Humanos , Mucosa Intestinal/diagnóstico por imagen , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Prevalencia , Estudios Prospectivos , Adulto JovenRESUMEN
Inherited renal cell carcinoma (RCC) is associated with multiple familial cancer syndromes but most individuals with features of non-syndromic inherited RCC do not harbor variants in the most commonly tested renal cancer predisposition genes (CPGs). We investigated whether undiagnosed cases might harbor mutations in CPGs that are not routinely tested for by testing 118 individuals with features suggestive of inherited RCC (family history of RCC, two or more primary RCC aged <60 years, or early onset RCC ≤46 years) for the presence of pathogenic variants in a large panel of CPGs. All individuals had been prescreened for pathogenic variants in the major RCC genes. We detected pathogenic or likely pathogenic (P/LP) variants of potential clinical relevance in 16.1% (19/118) of individuals, including P/LP variants in BRIP1 (n = 4), CHEK2 (n = 3), MITF (n = 1), and BRCA1 (n = 1). Though the power to detect rare variants was limited by sample size the frequency of truncating variants in BRIP1, 4/118, was significantly higher than in controls (P = 5.92E-03). These findings suggest that the application of genetic testing for larger inherited cancer gene panels in patients with indicators of a potential inherited RCC can increase the diagnostic yield for P/LP variants. However, the clinical utility of such a diagnostic strategy requires validation and further evaluation and in particular, confirmation of rarer RCC genotype-phenotype associations is required.
Asunto(s)
Carcinoma de Células Renales/genética , Heterogeneidad Genética , Mutación de Línea Germinal , Neoplasias Renales/genética , Adolescente , Adulto , Anciano , Proteína BRCA1/genética , Carcinoma de Células Renales/patología , Quinasa de Punto de Control 2/genética , Niño , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Femenino , Pruebas Genéticas/métodos , Pruebas Genéticas/normas , Humanos , Neoplasias Renales/patología , Masculino , Factor de Transcripción Asociado a Microftalmía/genética , Persona de Mediana Edad , ARN Helicasas/genéticaRESUMEN
Constitutional translocations, typically involving chromosome 3, have been recognized as a rare cause of inherited predisposition to renal cell carcinoma (RCC) for four decades. However, knowledge of the molecular basis of this association is limited. We have characterized the breakpoints by genome sequencing (GS) of constitutional chromosome abnormalities in five individuals who presented with RCC. In one individual with constitutional t(10;17)(q11.21;p11.2), the translocation breakpoint disrupted two genes: the known renal tumor suppressor gene (TSG) FLCN (and clinical features of Birt-Hogg-Dubé syndrome were detected) and RASGEF1A. In four cases, the rearrangement breakpoints did not disrupt known inherited RCC genes. In the second case without chromosome 3 involvement, the translocation breakpoint in an individual with a constitutional t(2;17)(q21.1;q11.2) mapped 12 Kb upstream of NLK. Interestingly, NLK has been reported to interact indirectly with FBXW7 and a previously reported RCC-associated translocation breakpoint disrupted FBXW7. In two cases of constitutional chromosome 3 translocations, no candidate TSGs were identified in the vicinity of the breakpoints. However, in an individual with a constitutional chromosome 3 inversion, the 3p breakpoint disrupted the FHIT TSG (which has been reported previously to be disrupted in two apparently unrelated families with an RCC-associated t(3;8)(p14.2;q24.1). These findings (a) expand the range of constitutional chromosome rearrangements that may be associated with predisposition to RCC, (b) confirm that chromosome rearrangements not involving chromosome 3 can predispose to RCC, (c) suggest that a variety of molecular mechanisms are involved the pathogenesis of translocation-associated RCC, and (d) demonstrate the utility of GS for investigating such cases.