RESUMEN
The global incidence of invasive fungal infections (IFIs) has increased over the past few decades, mainly in immunocompromised patients, and is associated with high mortality and morbidity. Aspergillus fumigatus is one of the most common and deadliest IFI pathogens. Major hurdles to treating fungal infections remain the lack of rapid and definitive diagnosis, including the frequent need for invasive procedures to provide microbiological confirmation, and the lack of specificity of structural imaging methods. To develop an Aspergillus-specific positron emission tomography (PET) imaging agent, we focused on fungal-specific sugar metabolism. We radiolabeled cellobiose, a disaccharide known to be metabolized by Aspergillus species, and synthesized 2-deoxy-2-[18F]fluorocellobiose ([18F]FCB) by enzymatic conversion of 2-deoxy-2-[18F]fluoroglucose ([18F]FDG) with a radiochemical yield of 60 to 70%, a radiochemical purity of >98%, and 1.5 hours of synthesis time. Two hours after [18F]FCB injection in A. fumigatus pneumonia as well as A. fumigatus, bacterial, and sterile inflammation myositis mouse models, retained radioactivity was only seen in foci with live A. fumigatus infection. In vitro testing confirmed production of ß-glucosidase enzyme by A. fumigatus and not by bacteria, resulting in hydrolysis of [18F]FCB into glucose and [18F]FDG, the latter being retained by the live fungus. The parent molecule was otherwise promptly excreted through the kidneys, resulting in low background radioactivity and high target-to-nontarget ratios at A. fumigatus infectious sites. We conclude that [18F]FCB is a promising and clinically translatable Aspergillus-specific PET tracer.
Asunto(s)
Aspergillus fumigatus , Celobiosa , Tomografía de Emisión de Positrones , Animales , Tomografía de Emisión de Positrones/métodos , Celobiosa/metabolismo , Aspergillus fumigatus/metabolismo , Ratones , Aspergilosis/diagnóstico por imagen , Fluorodesoxiglucosa F18/química , Aspergillus/metabolismo , Distribución Tisular , Radiofármacos/química , Radiofármacos/metabolismoRESUMEN
Previous studies of natural variants in Drosophila melanogaster implicated the Wnt signaling receptor frizzled in sleep. Given that the Wnt signaling pathway is highly conserved across species, we hypothesized that frizzled class receptor 1 (Fzd1), the murine homolog of frizzled, would also have a role in sleep. Using a CRISPR transgenic approach, we removed most of the Fzd1 coding region from C57BL/6N mice. We used a video assay to measure sleep characteristics in Fzd1-deficient mice. As Wnt signaling is known to affect visuospatial memory, we also examined the impact of the deletion on learning and memory using the novel object recognition (NOR) paradigm. Fzd1-deficient mice had altered sleep compared to littermate controls. The mice did not respond differently to the NOR paradigm compared to controls but did display anxiety-like behavior. Our strategy demonstrates that the study of natural variation in Drosophila sleep translates into candidate genes for sleep in vertebrate species such as the mouse.
RESUMEN
Aims: Patients with ADP-ribose-acceptor hydrolase 3 ( ARH3 ) deficiency exhibit stress-induced childhood-onset neurodegeneration with ataxia and seizures (CONDSIAS). ARH3 degrades protein-linked poly(ADP- ribose) (PAR) synthesized by poly(ADP-ribose)polymerase (PARP)-1 during oxidative stress, leading to cleavage of the ADP-ribose linked to protein. ARH3 deficiency leads to excess accumulation of PAR, resulting in PAR-dependent cell death or parthanatos. Approximately one-third of patients with homozygous mutant ARH3 die from cardiac arrest, which has been described as neurogenic, suggesting that ARH3 may play an important role in maintaining myocardial function. To address this question, cardiac function was monitored in Arh3 -knockout (KO) and - heterozygous (HT) mice. Methods and results: Arh3 -KO male mice displayed cardiac hypertrophy by histopathology and decreased cardiac contractility assessed by MRI. In addition, both genders of Arh3 -KO and -HT mice showed decreased cardiac contractility by dobutamine stress test assessed by echocardiography. A direct role of ARH3 on myocardial function was seen with a Langendorff-perfused isolated heart model . Arh3 -KO male mouse hearts showed decreased post-ischemic rate pressure products, increased size of ischemia-reperfusion (IR) infarcts, and elevated PAR levels. Consistently, in vivo IR injury showed enhanced infarct size in Arh3 -KO mice in both genders. In addition, Arh3 -HT male mice showed increased size of in vivo IR infarcts. Treatment with an FDA-approved PARP inhibitor, rucaparib, improved cardiac contractility during dobutamine-induced stress and exhibited reduced size of in vivo IR infarcts. To understand better the role of ARH3, CRISPR-Cas9 was used to generate different Arh3 genotypes of myoblasts and myotubes. Incubation with H2O2 decreased viability of Arh3 -KO and -HT myoblasts and myotubes, resulting in PAR-dependent cell death that was reduced by PARP inhibitors or by transfection with the Arh3 gene. Conclusion: ARH3 regulates PAR homeostasis in myocardium to preserve function and protect against oxidative stress; PARP inhibitors reduce the myocardial dysfunction seen with Arh3 mutations.
RESUMEN
ADP-ribosylation is a reversible reaction with ADP-ribosyltransferases catalyzing the forward reaction and ADP-ribose-acceptor hydrolases (ARHs) hydrolyzing the ADP-ribose acceptor bond. ARH2 is a member of the 39-kDa ARH family (ARH1-3), which is expressed in heart and skeletal muscle. ARH2 failed to exhibit any in vitro enzymatic activity. To determine its possible in vivo activities, Arh2 -knockout (KO) and - heterozygous (Het) mice were generated using CRISPR-Cas9. Arh2 -KO mice exhibited decreased cardiac contractility by MRI, echocardiography and dobutamine stress with cardiomegaly and abnormal motor function. Arh2 -Het mice showed results similar to those seen in Arh2 -KO mice except for cardiomegaly. Arh2 -KO and -Het mice and mouse embryonic fibroblasts (MEFs) developed spontaneous tumors and subcutaneous tumors in nude mice. We identified 13 mutations in Arh2 -Het MEFs and heterozygous tumors, corresponding to human ARH2 mutations in cancers obtained from COSMIC. Of interest, the L116R mutation in Arh2 gene plays a critical role in aggressive tumorigenesis in nude mice, corresponding to human ARH2 mutations in stomach adenocarcinoma. Both genders of Arh2 -KO and -Het mice showed increased unexpectedly deaths and decreased survival rate during a 24-month observation, caused by tumor, inflammation, non-inflammation (e.g., cardiomegaly, dental dysplasia), and congenital diseases. Thus, Arh2 plays a pivotal role in cardiac function, tumorigenesis, inflammation, and overall survival.
RESUMEN
GM1 gangliosidosis is a rare lysosomal storage disorder affecting multiple organ systems, primarily the central nervous system, and is caused by functional deficiency of ß-galactosidase (GLB1). Using CRISPR/Cas9 genome editing, we generated a mouse model to evaluate characteristics of the disease in comparison to GM1 gangliosidosis patients. Our Glb1-/- mice contain small deletions in exons 2 and 6, producing a null allele. Longevity is approximately 50 weeks and studies demonstrated that female Glb1-/- mice die six weeks earlier than male Glb1-/- mice. Gait analyses showed progressive abnormalities including abnormal foot placement, decreased stride length and increased stance width, comparable with what is observed in type II GM1 gangliosidosis patients. Furthermore, Glb1-/- mice show loss of motor skills by 20 weeks assessed by adhesive dot, hanging wire, and inverted grid tests, and deterioration of motor coordination by 32 weeks of age when evaluated by rotarod testing. Brain MRI showed progressive cerebellar atrophy in Glb1-/- mice as seen in some patients. In addition, Glb1-/- mice also show significantly increased levels of a novel pentasaccharide biomarker in urine and plasma which we also observed in GM1 gangliosidosis patients. Glb1-/- mice also exhibit accumulation of glycosphingolipids in the brain with increases in GM1 and GA1 beginning by 8 weeks. Surprisingly, despite being a null variant, this Glb1-/- mouse most closely models the less severe type II disease and will guide the development of new therapies for patients with the disorder.