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LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

University of Miami Leonard M. Miller School of Medicine

The development of RNA interference (RNAi)-based gene silencing approaches has revolutionized biomedical research. These technologies have been applied for functional genomic studies in a variety of areas, including cancer research, by facilitating a better understanding of the mechanisms that underlie tumorigenicity and the identification of novel factors that either promote or inhibit oncogenic transformation.

RNA interference as an anticancer therapy: a patent perspective.

The development of metastases involves the dissociation of cells from the primary tumor to penetrate the basement membrane, invade and then exit the vasculature to seed, and colonize distant tissues. The last step, establishment of macroscopic tumors at distant sites, is the least well understood. Four isogenic mouse breast cancer cell lines (67NR, 168FARN, 4TO7, and 4T1) that differ in their ability to metastasize when implanted into the mammary fat pad are used to model the steps of metastasis. Only 4T1 forms macroscopic lung and liver metastases. Because some miRNAs are dysregulated in cancer and affect cellular transformation, tumor formation, and metastasis, we examined whether changes in miRNA expression might explain the differences in metastasis of these cells.

miR-200 enhances mouse breast cancer cell colonization to form distant metastases.

RNAi-based gene silencing mechanisms have emerged as indispensible tools for experimentally induced, sequence-specific inhibition of gene expression. The translation of these technologies for therapeutic applications has required the development of clinically feasible delivery strategies that facilitate the uptake of the siRNAs by specific cell type(s) and tissues. This feature review focuses on the progress made in the targeted delivery of siRNAs and the challenges encountered in the development of safe and effective therapeutic silencing agents.

Advances in cell-type specific delivery of RNAi-based therapeutics.

MicroRNAs (miRNA) are key regulators of many important biological processes from insulin secretion and fat metabolism to cellular proliferation and differentiation. Given the critical role that these small regulatory RNAs play in biology, it is not surprising that the alteration of miRNA expression patterns can have pathogenic consequences. The association between miRNA dysregulation and pathogenesis has been most widely studied in tumorigenesis, and a large number of miRNAs have been identified whose expression levels are changed in various tumor types. Although the role that miRNAs play in the development of metastasis is more poorly defined, recent studies have begun to identify miRNAs that can regulate key steps in the metastatic cascade. This review focuses on two emerging stories, the regulation of the epithelial-to-mesenchymal transition by members of the miR-200 family, and the pleiotropic nature of the metastasis suppressor miR-31.

MicroRNAs and metastasis: little RNAs go a long way.

All viruses require host cell factors to replicate. A large number of host factors have been identified that participate at numerous points of the human immunodeficiency virus 1 (HIV-1) life cycle. Recent evidence supports a role for components of the trans-Golgi network (TGN) in mediating early steps in the HIV-1 life cycle. The conserved oligomeric Golgi (COG) complex is a heteroctamer complex that functions in coat protein complex I (COPI)-mediated intra-Golgi retrograde trafficking and plays an important role in the maintenance of Golgi structure and integrity as well as glycosylation enzyme homeostasis. The targeted silencing of components of lobe B of the COG complex, namely COG5, COG6, COG7 and COG8, inhibited HIV-1 replication. This inhibition of HIV-1 replication preceded late reverse transcription (RT) but did not affect viral fusion. Silencing of the COG interacting protein the t-SNARE syntaxin 5, showed a similar defect in late RT product formation, strengthening the role of the TGN in HIV replication. 

Target silencing of components of the conserved oligomeric Golgi complex impairs HIV-1 replication.

GABAergic synaptic transmission is known to play a critical role in the assembly of neuronal circuits during development and is responsible for maintaining the balance between excitatory and inhibitory signaling in the brain during maturation into adulthood. Importantly, defects in GABAergic neuronal function and signaling have been linked to a number of neurological diseases, including autism spectrum disorders, schizophrenia, and epilepsy. With patient-specific induced pluripotent stem cell (iPSC)-based models of neurological disease, it is now possible to investigate the disease mechanisms that underlie deficits in GABAergic function in affected human neurons. To that end, tools that enable the labeling and purification of viable GABAergic neurons from human pluripotent stem cells would be of great value.

hVGAT-mCherry: A novel molecular tool for analysis of GABAergic neurons derived from human pluripotent stem cells.

Understanding the molecular mechanisms that underlie neurodegenerative disorders has been hampered by a lack of readily available model systems that replicate the complexity of the human disease. Recent advances in stem cell technology have facilitated the derivation of patient-specific stem cells from a variety of differentiated cell types. These induced pluripotent stem cells (iPSCs) are attractive disease models since they can be grown and differentiated to produce large numbers of disease-relevant cell types. However, most iPSC lines are derived in advance of, and without the benefit of, neuropathological confirmation of the donor - the gold standard for many disease classifications and measurement of disease severity. While others have reported the generation of autopsy-confirmed iPSC lines from patient explants, these methods require outgrowth of cadaver tissue, which require additional time and is often only successful ∼50% of the time. Here we report the rapid generation of autopsy-confirmed iPSC lines from peripheral blood mononuclear cells (PBMCs) drawn postmortem. Since this approach doesn't require the propagation of previously frozen cadaver tissue, iPSC can be rapidly and efficiently produced from patients with autopsy-confirmed pathology. These matched iPSC-derived patient-specific neurons and postmortem brain tissue will support studies of specific mechanisms that drive the pathogenesis of neurodegenerative diseases.

Generation of disease-specific autopsy-confirmed iPSCs lines from postmortem isolated Peripheral Blood Mononuclear Cells.

Potentially pathogenic alterations have been identified in individuals with autism spectrum disorders (ASDs) within a variety of key neurodevelopment genes. While this hints at a common ASD molecular etiology, gaps persist in our understanding of the neurodevelopmental mechanisms impacted by genetic variants enriched in ASD patients. Induced pluripotent stem cells (iPSCs) can model neurodevelopment in vitro, permitting the characterization of pathogenic mechanisms that manifest during corticogenesis. Taking this approach, we examined the transcriptional differences between iPSC-derived cortical neurons from patients with idiopathic ASD and unaffected controls over a 135-day course of neuronal differentiation. Our data show ASD-specific misregulation of genes involved in neuronal differentiation, axon guidance, cell migration, DNA and RNA metabolism, and neural region patterning. Furthermore, functional analysis revealed defects in neuronal migration and electrophysiological activity, providing compelling support for the transcriptome analysis data. This study reveals important and functionally validated insights into common processes altered in early neuronal development and corticogenesis and may contribute to ASD pathogenesis.

Derek M. Dykxhoorn

Dr. John T. Macdonald Foundation Department of Human Genetics,
John P. Hussman Institute for Human Genomics

University of Miami Leonard M. Miller School of Medicine

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

Derek M. Dykxhoorn

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Dr. John T. Macdonald Foundation Department of Human Genetics,John P. Hussman Institute for Human Genomics

University of Miami Leonard M. Miller School of Medicine

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

Dr. John T. Macdonald Foundation Department of Human Genetics,
John P. Hussman Institute for Human Genomics