76555 research outputs found
Sort by
Exploring Abnormal Neurofilament Aggregates at the Neuromuscular Junction of a Charcot-Marie- Tooth Disease Type 2E Mouse Model
No full textCharcot-Marie-Tooth Disease (CMT) type 2 is a rare inherited neuropathy that results in the degeneration of peripheral nerves caused by mutations in genes that affect the peripheral axon. CMT patients suffer from sensorimotor problems, such as gait anomaly, reduced tendon reflexes, and leg weakness. Previous studies have reported neurofilament aggregates in the spinal cord and dorsal root ganglia (DRG) of a CMT disease type 2E (CMT2E) mouse model, which is caused by mutations in neurofilament light chain (NEFL) and results in a severe form of CMT. Moreover, transfected human motor neurons have shown abnormal neuronal intermediate filament networks associated with defects in axonal transport; however, the neuromuscular junction (NMJ) remains to be assessed. In the present study, we report abnormal NMJ morphologies with presynaptic varicosities at the NMJ of Nefl N98S/+ mice using high resolution light microscopy. Additionally, wire hang tests revealed reduced grip strength in mutant mice, while electromyography (EMG) measurements showed decreased nerve conduction velocities. Together, these observations shed light on plausible pathomechanisms affecting the NMJ in CMT2E, highlighting abnormalities in synaptic ultrastructure and aggregations that could impact synaptic functionality
Defining the Role of TOP1MT in mtDNA Stability, Release, and Innate Immunity
No full textTOP1MT is a mitochondria-localized topoisomerase that is encoded in the nuclear genome. Cells and animals lacking TOP1MT are viable, but exhibit reduced mitochondrial DNA (mtDNA) copy number, increased mtDNA instability, and an enhanced susceptibility to mtDNA damaging agents like the chemotherapy drug Doxorubicin. Loss of TOP1MT has also been linked to the release of mtDNA from mitochondria into the cytoplasm, leading to activation of innate immune signaling1,2 . Our hypothesis was depletion of TOP1MT will result in an increased susceptibility to mtDNA instability, which will lead to the release of mtDNA into the cytosol and sensing via the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. Dox treatment was shown to induce the release of mitochondrial DNA into the cytoplast, due to the aggregation of nucleoids and changing mitochondrial network phenotype. Ifit3 levels were seen to be raised in Top1mt-/- mouse embryonic fibroblast (MEF) cells, but they showed no increased expression of ZBP1 when exposed to doxorubicin for 24 hours, whereas exposure to LPS heightened ZBP1 expression. In future studies this period should be extended to see if the effects of doxorubicin exposure on mtDNA damage and release into the cytoplasm could be seen
Effects of Tryptophan Metabolites on Polarization of CD8+ Tfh-like T Cells
No full textCD8+ cytotoxic T cells are critical to a successful anti-tumor response, but their different phenotypes have distinct functions and gene expression. Previous studies have demonstrated a connection between L-tryptophan metabolites and polarization of CD4+ T cell differentiation, with preliminary data from the Chang Lab showing markers characteristic of CXCR5+ CD4+ Tfh cells, as well as some indication that CXCR5+ CD8+ Tfh-like cells may also be induced by the metabolites. Continued investigation of this relationship could produce more effective and precise cancer treatments by engineering cell populations. In this study, we probe the connections between the L-tryptophan metabolic pathway, CD8+ Tfh-like cell differentiation, and the capacity of these cells to support melanoma tumor clearance. To this end, we further defined the capacity of L-Tryptophan metabolites in the promotion of CXCR5+ CD8+ T cell differentiation via in vitro treatment of CD8+ T cells with L-Tryptophan metabolites. Furthermore, the extent of potential anti-tumor functionality conferred by treatment of the cells was determined through challenge of melanoma in tumor-bearing mice
Classifying intracranial germ cell tumors using miRNA and methylation sequencing of cerebrospinal fluid
No full textNew Insights into Cellular Senescence and Oxygen Effects on Trophoblast Differentiation
No full textTrophoblast cell functions are important for healthy decidualization and placentation throughout mammalian embryonic development. Trophoblast stem cells (TS) in the placental villi differentiate into extravillous trophoblasts (EVT), which invade uterine spiral arteries along the endometrium, establishing maternal blood flow into the placenta, enabling nutrient, gas and waste exchange between the mother and the fetus. EVT migration and invasion, crucial for healthy placentation and defects, result in placental disorders. Similarly, placental senescence features are known to be critical in placental aging and have been linked to trophoblasts. However, knowledge about specific developmental senescence in trophoblast is very limited. Oxygen gradients are important in EVT differentiation and are established by spiral artery remodeling. Similarly, classic features of senescence have also been described in TS and EVT cells. Defects in establishing the oxygen gradient and aberrant senescence expression have both been independently linked with placental disorders (e.g. preeclampsia and fetal growth restriction).
This study investigates the effects of senolytic drug on differentiating EVT to explore important senescence features important in development. We utilized transcriptomic, antibody-based biomolecular, and functional assays to investigate how Dasatinib+ Quercetin (D+Q) senolytic drug and oxygen level affected the morphology and biomarkers critical in EVT differentiation. Our data reveals that EVT has optimal differentiation in 3% oxygen conditions compared to higher oxygen; suggesting that oxygen inherently modulates senescence differentiation features. Immunofluorescence data revealed that EVT expressing Ki67 and LAMINB1 are less vulnerable to the effect of removed senescence. EVT expressing p21+, p57+ are targeted by senolytic drug. Additionally, we found that EVT precursors are prone to D+Q treatment, unlike mature EVT which continue proliferating despite the exposure to drug.
These findings provide a valuable understanding of important senescent markers in trophoblast cells critical for the continuation of a healthy pregnancy which is valuable to understand placental disorders. Moreover, these same features of developmental senescence are valuable to understand the senescence features associated with aging in disease later in human life
Cross-sectional association between blood cholesterol and calcium levels in genetically diverse strains of mice.
Get PDFGenetically diverse outbred mice allow for the study of genetic variation in the context of high dietary and environmental control. Using a machine learning approach, we investigated clinical and morphometric factors that associate with serum cholesterol levels in 840 genetically unique Diversity Outbred mice of both sexes (n = 417 male and 423 female), and on both a control chow (% kcals in diet: protein 22%, carbohydrate 62%, fat 16%, no cholesterol) and high fat high sucrose (% kcals in diet: protein 15%, carbohydrate 41%, fat 45%, 0.05% cholesterol). We find expected elevations of cholesterol in male mice, as well as in mice with elevated serum triglycerides and/or fed a high fat high sucrose diet. The third strongest predictor was serum calcium which correlated with serum cholesterol across both diets and sexes (r = 0.39-0.48) in both Diversity Outbred (P = 3.0 × 1
A machine learning approach for quantifying age-related histological changes in the mouse kidney.
Get PDFThe ability to quantify aging-related changes in histological samples is important, as it allows for evaluation of interventions intended to effect health span. We used a machine learning architecture that can be trained to detect and quantify these changes in the mouse kidney. Using additional held out data, we show validation of our model, correlation with scores given by pathologists using the Geropathology Research Network aging grading scheme, and its application in providing reproducible and quantifiable age scores for histological samples. Aging quantification also provides the insights into possible changes in image appearance that are independent of specific geropathology-specified lesions. Furthermore, we provide trained classifiers for H&E-stained slides, as well as tutorials on how to use these and how to create additional classifiers for other histological stains and tissues using our architecture. This architecture and combined resources allow for the high throughput quantification of mouse aging studies in general and specifically applicable to kidney tissues
Folate Deficiency and/or the Genetic Variant Mthfr(677C \u3eT) Can Drive Hepatic Fibrosis or Steatosis in Mice, in a Sex-Specific Manner
Get PDFScope: Disturbances in one-carbon metabolism contribute to nonalcoholic fatty liver disease (NAFLD) which encompasses steatosis, steatohepatitis, fibrosis, and cirrhosis. The goal is to examine impact of folate deficiency and the Mthfr677C \u3eT variant on NAFLD. Methods and results: This study uses the new Mthfr677C \u3eT mouse model for the human MTHFR677C \u3eT variant. Mthfr677CC and Mthfr677TT mice were fed control diet (CD) or folate-deficient (FD) diets for 4 months. FD and Mthfr677TT alter choline/methyl metabolites in liver and/or plasma (decreased S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio, methyltetrahydrofolate, and betaine; increased homocysteine [Hcy]). FD, with contribution from Mthfr677TT, provokes fibrosis in males. Studies of normal livers reveal alterations in plasma markers and gene expression that suggest an underlying predisposition to fibrosis induced by FD and/or Mthfr677TT in males. These changes are absent or reverse in females, consistent with the sex disparity of fibrosis. Sex-based differences in methylation potential, betaine, sphingomyelin, and trimethylamine-N-oxide (TMAO) levels may prevent fibrogenesis in females. In contrast, Mthfr677TT alters choline metabolism, dysregulates expression of lipid metabolism genes, and promotes steatosis in females. Conclusion: This study suggests that folate deficiency predisposes males to fibrosis, which is exacerbated by Mthfr677TT, whereas Mthfr677TT predisposes females to steatosis, and reveal novel contributory mechanisms for these NAFLD-related disorders
3D convolutional neural networks predict cellular metabolic pathway use from fluorescence lifetime decay data.
Get PDFFluorescence lifetime imaging of the co-enzyme reduced nicotinamide adenine dinucleotide (NADH) offers a label-free approach for detect- ing cellular metabolic perturbations. However, the relationships between variations in NADH lifetime and metabolic pathway changes are complex, preventing robust interpretation of NADH lifetime data relative to metabolic phenotypes. Here, a three-dimensional convolutional neural network (3D CNN) trained at the cell level with 3D NAD(P)H lifetime decay images (two spatial dimensions and one time dimension) was developed to identify metabolic pathway usage by cancer cells. NADH fluorescence lifetime images of MCF7 breast cancer cells with three isolated metabolic pathways, glycolysis, oxidative phosphorylation, and glutaminolysis were obtained by a multiphoton fluorescence lifetime microscope and then segmented into individual cells as the input data for the classification models. The 3D CNN models achieved over 90% accuracy in identifying cancer cells reliant on glycolysis, oxidative phosphorylation, or glutaminolysis. Furthermore, the model trained with human breast cancer cell data successfully predicted the differences in metabolic phenotypes of macrophages from control and POLG-mutated mice. These results suggest that the integration of autofluorescence lifetime imaging with 3D CNNs enables intracellular spatial patterns of NADH intensity and temporal dynamics of the lifetime decay to discriminate multiple metabolic phenotypes. Furthermore, the use of 3D CNNs to identify metabolic phenotypes from NADH fluorescence lifetime decay images eliminates the need for time- and expertise-demanding exponential decay fitting procedures. In summary, metabolic-prediction CNNs will enable live-cell and in vivo metabolic measurements with single-cell resolution, filling a current gap in metabolic measurement technologies
An in vitro neurogenetics platform for precision disease modeling in the mouse.
Get PDFThe power and scope of disease modeling can be markedly enhanced through the incorporation of broad genetic diversity. The introduction of pathogenic mutations into a single inbred mouse strain sometimes fails to mimic human disease. We describe a cross-species precision disease modeling platform that exploits mouse genetic diversity to bridge cell-based modeling with whole organism analysis. We developed a universal protocol that permitted robust and reproducible neural differentiation of genetically diverse human and mouse pluripotent stem cell lines and then carried out a proof-of-concept study of the neurodevelopmental gene DYRK1A. Results in vitro reliably predicted the effects of genetic background on Dyrk1a loss-of- function phenotypes in vivo. Transcriptomic comparison of responsive and unresponsive strains identified molecular pathways conferring sensitivity or resilience to Dyrk1a1A loss and highlighted differential messenger RNA isoform usage as an important determinant of response. This cross-species strategy provides a powerful tool in the functional analysis of candidate dis- ease variants identified through human genetic studies