ABSTRACT
Loss-of-function mutations in hepatocyte nuclear factor 1A (HNF1A) are known to cause rare forms of diabetes and alter hepatic physiology through unclear mechanisms. In the general population, 1:100 individuals carry a rare, protein-coding HNF1A variant, most of unknown functional consequence. To characterize the full allelic series, we performed deep mutational scanning of 11,970 protein-coding HNF1A variants in human hepatocytes and clinical correlation with 553,246 exome-sequenced individuals. Surprisingly, we found that â¼1:5 rare protein-coding HNF1A variants in the general population cause molecular gain of function (GOF), increasing the transcriptional activity of HNF1A by up to 50% and conferring protection from type 2 diabetes (odds ratio [OR] = 0.77, p = 0.007). Increased hepatic expression of HNF1A promoted a pro-atherogenic serum profile mediated in part by enhanced transcription of risk genes including ANGPTL3 and PCSK9. In summary, â¼1:300 individuals carry a GOF variant in HNF1A that protects carriers from diabetes but enhances hepatic secretion of atherogenic lipoproteins.
ABSTRACT
Cis-regulatory elements (CREs) can be classified by the shapes of their transcription start site (TSS) profiles, which are indicative of distinct regulatory mechanisms. Massively parallel reporter assays (MPRAs) are increasingly being used to study CRE regulatory mechanisms, yet the degree to which MPRAs replicate individual endogenous TSS profiles has not been determined. Here, we present a new low-input MPRA protocol (TSS-MPRA) that enables measuring TSS profiles of episomal reporters as well as after lentiviral reporter chromatinization. To sensitively compare MPRA and endogenous TSS profiles, we developed a novel dissimilarity scoring algorithm (WIP score) that outperforms the frequently used earth mover's distance on experimental data. Using TSS-MPRA and WIP scoring on 500 unique reporter inserts, we found that short (153 bp) MPRA promoter inserts replicate the endogenous TSS patterns of â¼60% of promoters. Lentiviral reporter chromatinization did not improve fidelity of TSS-MPRA initiation patterns, and increasing insert size frequently led to activation of extraneous TSS in the MPRA that are not active in vivo. We discuss the implications of our findings, which highlight important caveats when using MPRAs to study transcription mechanisms. Finally, we illustrate how TSS-MPRA and WIP scoring can provide novel insights into the impact of transcription factor motif mutations and genetic variants on TSS patterns and transcription levels.
Subject(s)
Gene Expression Regulation , Regulatory Sequences, Nucleic Acid , Transcription Initiation Site , Promoter Regions, Genetic , Transcription Factors/genetics , High-Throughput Nucleotide SequencingABSTRACT
Advances in stem cell technology, including the use of induced pluripotent stem cells (iPSC) to produce neurons and glial cells, offer new hope for patients with neurological disease and injuries. Pet dogs with spinal cord injuries provide an important spontaneous animal model for evaluating new approaches to stem cell therapy. Therefore, studies were conducted to identify optimal conditions for generating neural progenitor cells (NPC) from canine induced pluripotent stem cells (iPSC) for preliminary evaluation in animals with spinal cord injury. We found that canine NPC could be induced to differentiate into mature neural cells, including glia and neurons. In addition, canine NPC did not form teratomas when injected in NOD/SCID mice. In a pilot study, two dogs with chronic spinal cord injury underwent fluoroscopically guided intrathecal injections of canine NPC. In follow-up MRI evaluations, tumor formation was not observed at the injection sites. However, none of the animals experienced meaningful clinical or electrophysiological improvement following NPC injections. These studies provide evidence that canine iPSC can be used to generate NPC for evaluation in cellular therapy of chronic spinal cord injury in the dog spontaneous injury model. Further refinements in the cell implantation procedure are likely required to enhance stem cell treatment efficacy.
ABSTRACT
The use of magnetic nanoparticles (MNPs) in drug delivery vehicles must address issues such as drugloading capacity, desired release profile, aqueous dispersion stability, biocompatibility with cells and tissue, and retention of magnetic properties after interaction with macromolecules or modification via chemical reactions. Amphotericin B (AmB) is still the first choice for the treatment of severe paracoccidioidomycosis, an important systemic fungal infection caused by Paracoccidoides brasiliensis. Unfortunately, AmB causes acute side effects (mainly urinary problems) following intravenous administration, which limits its clinical use. The use of magnetic nanoparticles stabilized with biocompatible substances, together with the possibility of their conjugation with drugs has become a new nanotechnological strategy in the treatment of diseases for drug delivery to specific locations, such as the lungs in paracoccidoidiodomycosis. This review provides an overview of the disease, its etiologic agent and treatment with emphasis on the main strategies to improve the use of AmB in nanoformulations.
El uso de nanopartículas magnéticas (MNPS) en los vehículos de suministro de fármacos debe abordar cuestiones como la capacidad de carga de las drogas, el perfil deseado de liberación, estabilidad de la dispersión acuosa, biocompatibilidad con las células, tejidos y la conservación o la modificación de las propiedades magnéticas después de la interacción con macromoléculas y/o reacciones químicas. La anfotericina B (AnB) continua siendo la primera opción para el tratamiento de la paracoccidioidomicosis grave, una importante infección sistémica causada por el hongo Paracoccidioides brasiliensis. Sin embargo, la AnB causa efectos secundarios agudos (principalmente problemas urinarios) tras la administración intravenosa, limitando su uso clínico. El uso de nanopartículas magnéticas estabilizadas con sustancias biocompatibles y conjugadas con fármacos, se ha convertido en una nueva estrategia nanotecnológica para el tratamiento de enfermedades en sitios específicos, como los pulmones en paracoccidoidiodomycosis. En esta revisión se hace una descripción general de la enfermedad, su agente etiológico y su tratamiento con énfasis en la principales estrategias para mejorar el uso de AnB en nanoformulaciones.
