Neergaardcarlsson0110

Human skeletal muscle is a heterogeneous tissue composed of multiple fiber types that express unique contractile and metabolic properties. While analysis of mixed fiber samples predominates and holds value, increasing attention has been directed toward studying proteins segregated by fiber type, a methodological distinction termed "fiber type-specific." Fiber type-specific protein studies have the advantage of uncovering key molecular effects that are often missed in mixed fiber homogenate studies but also require greater time and resource-intensive methods, particularly when applied to human muscle. This review summarizes and compares current methods used for fiber type-specific protein analysis, highlighting their advantages and disadvantages for human muscle studies, in addition to recent advances in these techniques. These methods can be grouped into three categories based on the initial processing of the tissue 1) muscle-specific fiber homogenates, 2) cross sections of fiber bundles, and 3) isolated single fibers, with various subtechniques for performing fiber type identification and protein quantification. The relative implementation for each unique methodological approach is analyzed from 83 fiber type-specific studies of proteins in live human muscle found in the literature to date. These studies have investigated several proteins involved in a wide range of cellular functions that are important to muscle tissue. The second half of this review summarizes key findings from this ensemble of fiber type-specific human protein studies. We highlight examples of where this analytical approach has helped to improve understanding of important physiological topics such as insulin sensitivity, muscle hypertrophy, muscle fatigue, and adaptation to different exercise programs.The ability to replace defective cells in an airway with cells that can engraft, integrate, and restore a functional epithelium could potentially cure a number of lung diseases. Progress toward the development of strategies to regenerate the adult lung by either in vivo or ex vivo targeting of endogenous stem cells or pluripotent stem cell derivatives is limited by our fundamental lack of understanding of the mechanisms controlling human lung development, the precise identity and function of human lung stem and progenitor cell types, and the genetic and epigenetic control of human lung fate. In this review, we intend to discuss the known stem/progenitor cell populations, their relative differences between rodents and humans, their roles in chronic lung disease, and their therapeutic prospects. Additionally, we highlight the recent breakthroughs that have increased our understanding of these cell types. These advancements include novel lineage-traced animal models and single-cell RNA sequencing of human airway cells, which have provided critical information on the stem cell subtypes, transition states, identifying cell markers, and intricate pathways that commit a stem cell to differentiate or to maintain plasticity. As our capacity to model the human lung evolves, so will our understanding of lung regeneration and our ability to target endogenous stem cells as a therapeutic approach for lung disease.MicroRNAs (miRNAs) are short, noncoding RNAs that are evolutionarily conserved across many different species. miRNA regulation of gene expression, specifically in the context of the mammalian brain, has been well characterized; however, the regulation of miRNA degradation is still a focus of ongoing research. This review focuses on recent findings concerning the cellular mechanisms that govern miRNA degradation, with an emphasis on target-mediated miRNA degradation and how this phenomenon is uniquely poised to maintain homeostasis in neuronal systems.Hypertension (HTN) is a polyfactorial disease that can manifest severe cardiovascular pathologies such as heart failure or stroke. Genome-wide association studies (GWAS) of HTN indicate that single-nucleotide polymorphisms (SNPs) contribute to increased risk for HTN and resistance to some HTN drug regimens (Hiltunen TP et al., J Am Heart Assoc 4 e001521, 2015; Le MT et al., PLoS One 8 e52062, 2013; McDonough CW et al., J Hypertens 31 698-704, 2013; Vandell AG et al., Hypertension 60 957-964, 2012). However, cellular mechanistic insights of such SNPs remain largely unknown. Using a bank of induced pluripotent stem cells (iPSCs) derived from patients with HTN and CRISPR/Cas9-mediated gene-editing approach, we investigated the effects of a female HTN risk-associated SNP (rs1154431) of the G protein-coupled estrogen receptor (GPER) (Bassuk SS, Manson JE., Clin Chem 60 68-77, 2014) in vascular endothelial cells. Although GPER1 deletion reduced endothelial nitric oxide synthase (eNOS) activation in iPSC-derived endothelial cells (iECs), the polymorphism itself did not significantly affect eNOS and NO production in a comparison of isogenic hemizygous iECs expressing either normal (P16) or HTN-associated (L16) GPER. Interestingly, we demonstrate for the first time that GPER plays a role in regulation of adhesion molecule expression and monocyte adhesion to iECs. Moreover, the L16 iECs had higher expression of inflammation genes than P16 iECs, implying that the risk variant may affect carrier individuals through increased inflammatory activity. This study further indicates that iPSCs are a useful platform for exploring mechanistic insights underlying hypertension GWAS endeavors.Epileptic seizures are the manifestation of hypersynchronous and excessive neuronal excitation. While the glutamatergic and GABAergic neurons play major roles in shaping fast neuronal excitation/inhibition homeostasis, it is well illustrated that astrocytes profoundly regulate neuronal excitation by controlling glutamate, GABA, cannabinoids, adenosine, and concentration of K+ around neurons. However, little is known about whether microglia take part in the regulation of acute neuronal excitation and ongoing epileptic behaviors. We proposed that if microglia are innately ready to respond to epileptic overexcitation, depletion of microglia might alter neuronal excitability and severity of acute epileptic seizures. We found that microglia depletion by plx3397, an inhibitor of CSF1R, exacerbates seizure severity and excitotoxicity-induced neuronal degeneration, indicating that microglia are rapidly responsive to the change of excitation/inhibition homeostasis and participate in the protection of neurons from overexcitation.Human flap endonuclease 1 (FEN1) is a structure-specific, multifunctional endonuclease essential for DNA replication and repair. Our previous study showed that in response to DNA damage, FEN1 interacts with the PCNA-like Rad9-Rad1-Hus1 complex instead of PCNA to engage in DNA repair activities, such as stalled DNA replication fork repair, and undergoes SUMOylation by SUMO-1. Here, we report that succinylation of FEN1 was stimulated in response to DNA replication fork-stalling agents, such as ultraviolet (UV) irradiation, hydroxyurea, camptothecin, and mitomycin C. K200 is a key succinylation site of FEN1 that is essential for gap endonuclease activity and could be suppressed by methylation and stimulated by phosphorylation to promote SUMO-1 modification. Succinylation at K200 of FEN1 promoted the interaction of FEN1 with the Rad9-Rad1-Hus1 complex to rescue stalled replication forks. Impairment of FEN1 succinylation led to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of FEN1 succinylation in regulating its roles in DNA replication and repair, thus maintaining genome stability.Generation of neurons from human induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain samples and greatly facilitates the progress of research in neurological diseases. However, it is still a challenge to generate a particular neuronal subtype with high purity and yield for determining the pathogenesis of diseased neurons using biochemical approaches. Motor neurons (MNs) are a specialized neuronal subtype responsible for governing both autonomic and volitional movement. Dysfunctions in MNs are implicated in a variety of movement diseases, such as amyotrophic lateral sclerosis (ALS). In this study, we generated functional MNs from human iPSCs via lentiviral delivery of transcription factors. Moreover, we optimized induction conditions by using different combinations of transcription factors and found that a single lentiviral vector expressing three factors [neurogenin-2 (NGN2), insulin gene enhancer 1 (ISL1), and LIM/homeobox 3 (LHX3)] is necessary and sufficient to induce iPSC-derived MNs (iPSC-MNs). These MNs robustly expressed general neuron markers [microtubule-associated protein 2 (MAP2), neurofilament protein (SMI-32), and tubulin β-3 class III (TUBB3)] and MN-specific markers [HB9 and choline acetyltransferase (ChAT)] and showed electrical maturation and firing of action potentials within 3 wk. This approach significantly improved the neuronal survival, yield, and purity, making it feasible to obtain abundant materials for biochemical studies in modeling movement diseases.The endoplasmic reticulum protein Jagunal (JAGN1) was first identified as a requirement for Drosophila melanogaster oocyte development. Subsequent studies in human patients linked mutations in JAGN1 to severe congenital neutropenia, as well as a broad range of additional symptoms, suggesting that JAGN1 function is required in many tissues. Moreover, JAGN1 orthologs are found throughout animal and plant phylogeny, suggesting that JAGN1 supports fundamental cellular processes not restricted to egg development or neutrophil function. JAGN1 lacks sequence similarity or recognizable domains other than a coatomer protein complex I-binding motif, and its cellular function is currently unknown. JAGN1 shares a tetraspanning membrane topology with two families of known cargo transporters the tetraspanins and the endoplasmic reticulum vesicle (Erv) proteins. Herein, we discuss the similarities between JAGN1, tetraspanins, and Ervs and, based on those, suggest a role for JAGN1 in facilitating the traffic of cell-restricted and ubiquitously expressed proteins at the endoplasmic reticulum-Golgi interface.Muscle-specific E3 ubiquitin ligases have been identified in muscle atrophy-inducing conditions. The purpose of the current study was to explore the functional role of F-box and leucine-rich protein 22 (Fbxl22), and a newly identified splice variant (Fbxl22-193), in skeletal muscle homeostasis and neurogenic muscle atrophy. In mouse C2C12 muscle cells, promoter fragments of the Fbxl22 gene were cloned and fused with the secreted alkaline phosphatase reporter gene to assess the transcriptional regulation of Fbxl22. The tibialis anterior muscles of male C57/BL6 mice (12-16 wk old) were electroporated with expression plasmids containing the cDNA of two Fbxl22 splice variants and tissues collected after 7, 14, and 28 days. Gastrocnemius muscles of wild-type and muscle-specific RING finger 1 knockout (MuRF1 KO) mice were electroporated with an Fbxl22 RNAi or empty plasmid and denervated 3 days posttransfection, and tissues were collected 7 days postdenervation. The full-length gene and novel splice variant are transcriptionally induced early (after 3 days) during neurogenic muscle atrophy.