Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific ...differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.
Toolsets available for in-depth analysis of scRNA-seq datasets by biologists with little informatics experience is limited. Here, we describe an informatics tool (PyMINEr) that fully automates cell ...type identification, cell type-specific pathway analyses, graph theory-based analysis of gene regulation, and detection of autocrine-paracrine signaling networks in silico. We applied PyMINEr to interrogate human pancreatic islet scRNA-seq datasets and discovered several features of co-expression graphs, including concordance of scRNA-seq-graph structure with both protein-protein interactions and 3D genomic architecture, association of high-connectivity and low-expression genes with cell type enrichment, and potential for the graph structure to clarify potential etiologies of enigmatic disease-associated variants. We further created a consensus co-expression network and autocrine-paracrine signaling networks within and across islet cell types from seven datasets. PyMINEr correctly identified changes in BMP-WNT signaling associated with cystic fibrosis pancreatic acinar cell loss. This proof-of-principle study demonstrates that the PyMINEr framework will be a valuable resource for scRNA-seq analyses.
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•PyMINEr automates advanced scRNA-seq analyses without coding•Data integration of T2D-associated genes into co-expression graph networks•Consensus catalog of human pancreatic autocrine-paracrine signaling networks•BMP and WNT pathways are induced in human cystic fibrosis pancreata
Tyler et al. create PyMINEr, an open-source program (https://www.sciencescott.com/pyminer) that automates analyses of expression datasets without coding. These analyses include clustering, differential expression, pathway analyses, co-expression networks, marker gene identification, and autocrine-paracrine signaling prediction. Integration of seven datasets shows elevated BMP-WNT signaling in cystic fibrosis pancreata.
The domestic ferret (Mustela putorius furo) is an important animal model for multiple human respiratory diseases. It is considered the 'gold standard' for modeling human influenza virus infection and ...transmission. Here we describe the 2.41 Gb draft genome assembly of the domestic ferret, constituting 2.28 Gb of sequence plus gaps. We annotated 19,910 protein-coding genes on this assembly using RNA-seq data from 21 ferret tissues. We characterized the ferret host response to two influenza virus infections by RNA-seq analysis of 42 ferret samples from influenza time-course data and showed distinct signatures in ferret trachea and lung tissues specific to 1918 or 2009 human pandemic influenza virus infections. Using microarray data from 16 ferret samples reflecting cystic fibrosis disease progression, we showed that transcriptional changes in the CFTR-knockout ferret lung reflect pathways of early disease that cannot be readily studied in human infants with cystic fibrosis disease.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to ...reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene-deficient model in the domestic ferret using recombinant adeno-associated virus-mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases.
Diabetes is prevalent among people with CF (PwCF) and associated with worse clinical outcomes. CFTR modulators are highly effective in improving the disease course of CF. However, the effects of ...elexacaftor/tezacaftor/ivacaftor (ETI) on glucose metabolism in PwCF are unclear.
Twenty youth and adults with CF underwent frequently sampled oral glucose tolerance tests (fsOGTT) before and after ETI initiation. Glucose, insulin, and C-peptide were collected at 0, 10, 30, 60, 90, and 120 min after 1.75 g/kg (max 75 g) of dextrose. HbA1c and continuous glucose monitoring (CGM) were collected in a subset. Estimates of insulin secretion (C-peptide index), insulin resistance (HOMA2 IR and IS(OGTT Cpep)), and β-cell function (C-peptide oral disposition index, oDIcoeo), were compared before and after ETI.
Participants were a median (IQR) of 20.4 (14.1, 28.6) years old, 75 % male. Follow-up occurred 10.5 (10.0, 12.3) months after ETI initiation. BMI z-score increased from 0.3 (-0.3, 0.8) to 0.8 (0.4, 1.5), p = 0.013 between visits. No significant differences were observed in glucose tolerance, glucose area under the curve, nor fsOGTT glucose concentrations before and after ETI. Median (IQR) C-peptide index increased from 5.7 (4.1, 8.3) to 8.8 (5.5, 10.8) p = 0.013 and HOMA2 IR increased (p < 0.001), while oDIcoeo was unchanged (p = 0.67). HbA1c decreased from 5.5 % (5.5, 5.8) to 5.4 % (5.2, 5.6) (p = 0.003) while CGM variables did not change.
BMI z-score and measures of both insulin resistance and insulin secretion increased within the first year of ETI initiation. β-cell function adjusted for insulin sensitivity (oDIcoeo) did not change.
Alpha-1 antitrypsin deficiency (AATD) is the most common genetic cause and risk factor for chronic obstructive pulmonary disease, but the field lacks a large-animal model that allows for longitudinal ...assessment of pulmonary function. We hypothesized that ferrets would model human AATD-related lung and hepatic disease. AAT-knockout (AAT-KO) and PiZZ (E342K, the most common mutation in humans) ferrets were generated and compared with matched controls using custom-designed flexiVent modules to perform pulmonary function tests, quantitative computed tomography (QCT), bronchoalveolar lavage (BAL) proteomics, and alveolar morphometry. Complete loss of AAT (AAT-KO) led to increased pulmonary compliance and expiratory airflow limitation, consistent with obstructive lung disease. QCT and morphometry confirmed emphysema and airspace enlargement, respectively. Pathway analysis of BAL proteomics data revealed inflammatory lung disease and impaired cellular migration. The PiZ mutation resulted in altered AAT protein folding in the liver, hepatic injury, and reduced plasma concentrations of AAT, and PiZZ ferrets developed obstructive lung disease. In summary, AAT-KO and PiZZ ferrets model the progressive obstructive pulmonary disease seen in AAT-deficient patients and may serve as a platform for preclinical testing of therapeutics including gene therapy.
Oxidative stress and impaired insulin secretion in cystic fibrosis pig pancreas O'Malley, Yunxia; Coleman, Mitchell C.; Sun, Xingshen ...
Advances in redox research : an official journal of the Society for Redox Biology and Medicine and the Society for Free Radical Research-Europe,
07/2022, Letnik:
5
Journal Article
Recenzirano
Odprti dostop
•Pancreatic CuZnSOD activity and glutathione levels are significantly diminished, oxidatively modified proteins are upregulated in a well-established cystic fibrosis (CF) pig model.•CF pig pancreas ...and cultured islets have increased superoxide and peroxide production.•An SOD-mimetic, GC4401inhibits superoxide production in CF pig pancreas.•CF pig islets have diminished insulin secretion in response to high glucose, but acute treatment with a Nox inhibitor or an SOD mimetic does not restore insulin secretion.•Oxidative stress may be a chronic contributing factor to insulin secretory defects in people with CF.
Cystic fibrosis-related diabetes (CFRD) is one the most common comorbidities in cystic fibrosis (CF). Pancreatic oxidative stress has been postulated in the pathogenesis of CFRD, but no studies have been done to show an association. The main obstacle is the lack of suitable animal models and no immediate availability of pancreas tissue in humans. In the CF porcine model, we found increased pancreatic total glutathione (GSH), glutathione disulfide (GSSG), 3-nitrotyrosine- and 4-hydroxynonenal-modified proteins, and decreased copper zinc superoxide dismutase (CuZnSOD) activity, all indicative of oxidative stress. CF pig pancreas demonstrated increased DHE oxidation (as a surrogate marker of superoxide) in situ compared to non-CF and this was inhibited by a SOD-mimetic (GC4401). Catalase and glutathione peroxidase activities were not different between CF and non-CF pancreas. Isolated CF pig islets had significantly increased DHE oxidation, peroxide production, reduced insulin secretion in response to high glucose and diminished secretory index compared to non-CF islets. Acute treatment with apocynin or an SOD mimetic failed to restore insulin secretion. These results are consistent with the hypothesis that CF pig pancreas is under significant oxidative stress as a result of increased O2●− and peroxides combined with reduced antioxidant defenses against reactive oxygen species (ROS). We speculate that insulin secretory defects in CF may be due to oxidative stress.
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Ferrets are an important emerging model of cystic fibrosis related diabetes. However, there is little documented experience in the use of advanced techniques to quantify aspects of diabetes ...pathophysiology in the ferret. Glycemic clamps are the gold standard technique to assess both insulin sensitivity and insulin secretion in humans and animal models of diabetes. We therefore sought to develop techniques for glycemic clamps in ferrets. To assess insulin sensitivity, we performed euglycemic hyperinsulinemic clamps in 5-6 week old ferrets in the anesthetized and conscious states. To assess insulin secretion, we performed hyperglycemic clamps in conscious ferrets. To evaluate responsiveness of ferret islet and entero-insular hormones to low glucose, a portion of the hyperglycemic clamps were followed by a hypoglycemic clamp. The euglycemic hyperinsulinemic clamps demonstrated insulin responsiveness in ferrets similar to that previously observed in humans and rats. The anesthetic isoflurane induced marked insulin resistance, whereas lipid emulsion induced mild insulin resistance. In conscious ferrets, glucose appearance was largely suppressed at 4 mU/kg/min insulin infusion, whereas glucose disposal was progressively increased at 4 and 20 mU/kg/min insulin. Hyperglycemic clamp induced first phase insulin secretion. Hypoglycemia induced a rapid diminishment of insulin, as well as a rise in glucagon and pancreatic polypeptide levels. The incretins GLP-1 and GIP were affected minimally by hyperglycemic and hypoglycemic clamp. These techniques will prove useful in better defining the pathophysiology in ferrets with cystic fibrosis related diabetes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Development of an efficient cryopreservation technique for the domestic ferret is key for the long-term maintenance of valuable genetic specimens of this species and for the conservation of related ...endangered species. Unfortunately, current cryopreservation procedures, such as slow-rate freezing and vitrification with open pulled straws, are inefficient. In this report, we describe a pipette tip-based vitrification method that significantly improves the development of thawed ferret embryos following embryo transfer (ET). Ferret embryos at the morula (MR), compact morula (CM), and early blastocyst (EB) stages were vitrified using an Eppendorf microloader pipette tip as the chamber vessel. The rate of in vitro development was significantly (P < 0.05) higher among embryos vitrified at the CM (93.6%) and EB (100%) stages relative to those vitrified at the MR stages (58.7%). No significant developmental differences were observed when comparing CM and EB vitrified embryos with nonvitrified control CM (100%) and EB (100%) embryos. In addition, few differences in the ultrastructure of intracellular lipid droplets or in microfilament structure were observed between control embryos and embryos vitrified at any developmental stage. Vitrified-thawed CM/EB embryos cultured for 2 or 16 h before ET resulted in live birth rates of 71.3% and 77.4%, respectively. These rates were not significantly different from the control live birth rate (79.2%). However, culture for 32 h (25%) or 48 h (7.8%) after vitrification significantly reduced the rate of live births. These data indicate that the pipette chamber vitrification technique significantly improves the live birth rate of transferred ferret embryos relative to current state-of-the-art methods..
Removing undesired reflection from an image captured through a glass surface is a very challenging problem with many practical applications. For improving reflection removal, cascaded deep models ...have been usually adopted to estimate the transmission in a progressive manner. However, most existing methods are still limited in exploiting the result in prior stage for guiding transmission estimation. In this paper, we present a novel two-stage network with reflection-aware guidance (RAGNet) for single image reflection removal (SIRR). To be specific, the reflection layer is firstly estimated due to that it generally is much simpler and is relatively easier to estimate. Reflection-aware guidance (RAG) module is then elaborated for better exploiting the estimated reflection in predicting transmission layer. By incorporating feature maps from the estimated reflection and observation, RAG can be used (i) to mitigate the effect of reflection from the observation, and (ii) to generate mask in soft partial convolution for mitigating the effect of deviating from linear combination hypothesis. A dedicated mask loss is further presented for reconciling the contributions of encoder and decoder features. Experiments on five commonly used datasets demonstrate the quantitative and qualitative superiority of our RAGNet in comparison to the state-of-the-art SIRR methods. The source code and pre-trained model are available at
https://github.com/liyucs/RAGNet
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