👤 Nancy Hernandez

Various cancer stem cell (CSC) biomarkers have been identified for hepatocellular carcinoma (HCC), but little is known about the implications of heterogeneity and shared molecular networks within the CSC population. Through miRNA profile analysis in an HCC cohort ( Show less

Kir7.1 is an inwardly rectifying potassium channel with important roles in the regulation of the membrane potential in retinal pigment epithelium, uterine smooth muscle, and hypothalamic neurons. Regulation of G protein-coupled inwardly rectifying potassium (GIRK) channels by G protein-coupled receptors (GPCRs) via the G protein βγ subunits has been well characterized. However, how Kir channels are regulated is incompletely understood. We report here that Kir7.1 is also regulated by GPCRs, but through a different mechanism. Using Western blotting analysis, we observed that multiple GPCRs tested caused a striking reduction in the complex glycosylation of Kir7.1. Further, GPCR-mediated reduction of Kir7.1 glycosylation in HEK293T cells did not alter its expression at the cell surface but decreased channel activity. Of note, mutagenesis of the sole Kir7.1 glycosylation site reduced conductance and open probability, as indicated by single-channel recording. Additionally, we report that the L241P mutation of Kir7.1 associated with Lebers congenital amaurosis (LCA), an inherited retinal degenerative disease, has significantly reduced complex glycosylation. Collectively, these results suggest that Kir7.1 channel glycosylation is essential for function, and this activity within cells is suppressed by most GPCRs. The melanocortin-4 receptor (MC4R), a GPCR previously reported to induce ligand-regulated activity of this channel, is the only GPCR tested that does not have this effect on Kir7.1. Show less

Genome-wide association studies have struggled to identify functional genes and variants underlying complex phenotypes. We recruited a multi-ethnic cohort of healthy volunteers (n = 91) and used their tissue to generate induced pluripotent stem cells (iPSCs) and hepatocyte-like cells (HLCs) for genome-wide mapping of expression quantitative trait loci (eQTLs) and allele-specific expression (ASE). We identified many eQTL genes (eGenes) not observed in the comparably sized Genotype-Tissue Expression project's human liver cohort (n = 96). Focusing on blood lipid-associated loci, we performed massively parallel reporter assays to screen candidate functional variants and used genome-edited stem cells, CRISPR interference, and mouse modeling to establish rs2277862-CPNE1, rs10889356-DOCK7, rs10889356-ANGPTL3, and rs10872142-FRK as functional SNP-gene sets. We demonstrated HLC eGenes CPNE1, VKORC1, UBE2L3, and ANGPTL3 and HLC ASE gene ACAA2 to be lipid-functional genes in mouse models. These findings endorse an iPSC-based experimental framework to discover functional variants and genes contributing to complex human traits. Show less

Recent genome-wide association studies (GWAS) and pathway analyses supported long-standing observations of an association between immune-mediated diseases and Parkinson disease (PD). The post-GWAS era provides an opportunity for cross-phenotype analyses between different complex phenotypes. To test the hypothesis that there are common genetic risk variants conveying risk of both PD and autoimmune diseases (ie, pleiotropy) and to identify new shared genetic variants and their pathways by applying a novel statistical framework in a genome-wide approach. Using the conjunction false discovery rate method, this study analyzed GWAS data from a selection of archetypal autoimmune diseases among 138 511 individuals of European ancestry and systemically investigated pleiotropy between PD and type 1 diabetes, Crohn disease, ulcerative colitis, rheumatoid arthritis, celiac disease, psoriasis, and multiple sclerosis. NeuroX data (6927 PD cases and 6108 controls) were used for replication. The study investigated the biological correlation between the top loci through protein-protein interaction and changes in the gene expression and methylation levels. The dates of the analysis were June 10, 2015, to March 4, 2017. The primary outcome was a list of novel loci and their pathways involved in PD and autoimmune diseases. Genome-wide conjunctional analysis identified 17 novel loci at false discovery rate less than 0.05 with overlap between PD and autoimmune diseases, including known PD loci adjacent to GAK, HLA-DRB5, LRRK2, and MAPT for rheumatoid arthritis, ulcerative colitis and Crohn disease. Replication confirmed the involvement of HLA, LRRK2, MAPT, TRIM10, and SETD1A in PD. Among the novel genes discovered, WNT3, KANSL1, CRHR1, BOLA2, and GUCY1A3 are within a protein-protein interaction network with known PD genes. A subset of novel loci was significantly associated with changes in methylation or expression levels of adjacent genes. The study findings provide novel mechanistic insights into PD and autoimmune diseases and identify a common genetic pathway between these phenotypes. The results may have implications for future therapeutic trials involving anti-inflammatory agents. Show less

Depression symptoms following immune response to a challenge have been reported after the recovery from sickness. A RNA-Seq study of the dysregulation of the microglia transcriptome in a model of inflammation-associated depressive behavior was undertaken. The transcriptome of microglia from mice at day 7 after Bacille Calmette Guérin (BCG) challenge was compared to that from unchallenged Control mice and to the transcriptome from peripheral macrophages from the same mice. Among the 562 and 3,851 genes differentially expressed between BCG-challenged and Control mice in microglia and macrophages respectively, 353 genes overlapped between these cells types. Among the most differentially expressed genes in the microglia, serum amyloid A3 (Saa3) and cell adhesion molecule 3 (Cadm3) were over-expressed and coiled-coil domain containing 162 (Ccdc162) and titin-cap (Tcap) were under-expressed in BCG-challenged relative to Control. Many of the differentially expressed genes between BCG-challenged and Control mice were associated with neurological disorders encompassing depression symptoms. Across cell types, S100 calcium binding protein A9 (S100A9), interleukin 1 beta (Il1b) and kynurenine 3-monooxygenase (Kmo) were differentially expressed between challenged and control mice. Immune response, chemotaxis, and chemokine activity were among the functional categories enriched by the differentially expressed genes. Functional categories enriched among the 9,117 genes differentially expressed between cell types included leukocyte regulation and activation, chemokine and cytokine activities, MAP kinase activity, and apoptosis. More than 200 genes exhibited alternative splicing events between cell types including WNK lysine deficient protein kinase 1 (Wnk1) and microtubule-actin crosslinking factor 1(Macf1). Network visualization revealed the capability of microglia to exhibit transcriptome dysregulation in response to immune challenge still after resolution of sickness symptoms, albeit lower than that observed in macrophages. The persistent transcriptome dysregulation in the microglia shared patterns with neurological disorders indicating that the associated persistent depressive symptoms share a common transcriptome basis. Show less

Autosomal-recessive early-onset parkinsonism is clinically and genetically heterogeneous. The genetic causes of approximately 50% of autosomal-recessive early-onset forms of Parkinson disease (PD) remain to be elucidated. Homozygozity mapping and exome sequencing in 62 isolated individuals with early-onset parkinsonism and confirmed consanguinity followed by data mining in the exomes of 1,348 PD-affected individuals identified, in three isolated subjects, homozygous or compound heterozygous truncating mutations in vacuolar protein sorting 13C (VPS13C). VPS13C mutations are associated with a distinct form of early-onset parkinsonism characterized by rapid and severe disease progression and early cognitive decline; the pathological features were striking and reminiscent of diffuse Lewy body disease. In cell models, VPS13C partly localized to the outer membrane of mitochondria. Silencing of VPS13C was associated with lower mitochondrial membrane potential, mitochondrial fragmentation, increased respiration rates, exacerbated PINK1/Parkin-dependent mitophagy, and transcriptional upregulation of PARK2 in response to mitochondrial damage. This work suggests that loss of function of VPS13C is a cause of autosomal-recessive early-onset parkinsonism with a distinctive phenotype of rapid and severe progression. Show less

The kidney is the organ most commonly involved in systemic amyloidosis. This study reports the largest clinicopathologic series of renal amyloidosis. This study provides characteristics of 474 renal amyloidosis cases evaluated at the Mayo Clinic Renal Pathology Laboratory from 2007 to 2011, including age, sex, serum creatinine, proteinuria, type of amyloid, and tissue distribution according to type. The type of amyloid was Ig amyloidosis in 407 patients (85.9%), AA amyloidosis in 33 (7.0%), leukocyte chemotactic factor 2 amyloidosis in 13 (2.7%), fibrinogen A α chain amyloidosis in 6 (1.3%), Apo AI, Apo AII, or Apo AIV amyloidosis in 3 (0.6%), combined AA amyloidosis/Ig heavy and light chain amyloidosis in 1 (0.2%), and unclassified in 11 (2.3%). Laser microdissection/mass spectrometry, performed in 147 cases, was needed to determine the origin of amyloid in 74 of the 474 cases (16%), whereas immunofluorescence failed to diagnose 28 of 384 light chain amyloidosis cases (7.3%). Leukocyte chemotactic factor 2 amyloidosis and Apo AI, Apo AII, or Apo AIV amyloidosis were characterized by diffuse interstitial deposition, whereas fibrinogen A α chain amyloidosis showed obliterative glomerular involvement. Compared with other types, Ig amyloidosis was associated with lower serum creatinine, higher degree of proteinuria, and amyloid spicules. In the authors' experience, the vast majority of renal amyloidosis cases are Ig derived. The newly identified leukocyte chemotactic factor 2 amyloidosis form was the most common of the rarer causes of renal amyloidosis. With the advent of laser microdissection/mass spectrometry for amyloid typing, the origin of renal amyloidosis can be determined in >97% of cases. Show less

Macrophages are professional phagocytic cells that orchestrate innate immune responses and have considerable phenotypic diversity at different anatomical locations. However, the mechanisms that control the heterogeneity of tissue macrophages are not well characterized. Here we found that the nuclear receptor LXRα was essential for the differentiation of macrophages in the marginal zone (MZ) of the spleen. LXR-deficient mice were defective in the generation of MZ and metallophilic macrophages, which resulted in abnormal responses to blood-borne antigens. Myeloid-specific expression of LXRα or adoptive transfer of wild-type monocytes restored the MZ microenvironment in LXRα-deficient mice. Our results demonstrate that signaling via LXRα in myeloid cells is crucial for the generation of splenic MZ macrophages and identify an unprecedented role for a nuclear receptor in the generation of specialized macrophage subsets. Show less

Differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes is regulated by the interplay between extrinsic signals and intrinsic epigenetic determinants. In this study, we analyze the effect that the extracellular ligands sonic hedgehog (Shh) and bone morphogenetic protein 4 (BMP4), have on histone acetylation and gene expression in cultured OPCs. Shh treatment favored the progression toward oligodendrocytes by decreasing histone acetylation and inducing peripheral chromatin condensation. BMP4 treatment, in contrast, inhibited the progression toward oligodendrocytes and favored astrogliogenesis by favoring global histone acetylation and retaining euchromatin. Pharmacological treatment or silencing of histone deacetylase 1 (Hdac1) or histone deacetylase 2 (Hdac2) in OPCs did not affect BMP4-dependent astrogliogenesis, while it prevented Shh-induced oligodendrocyte differentiation and favored the expression of astrocytic genes. Transcriptional profiling of treated OPCs, revealed that BMP4-inhibition of oligodendrocyte differentiation was accompanied by increased levels of Wnt (Tbx3) and Notch-target genes (Jag1, Hes1, Hes5, Hey1, and Hey2), decreased recruitment of Hdac and increased histone acetylation at these loci. Similar upregulation of Notch-target genes and increased histone acetylation were observed in the corpus callosum of mice infused with BMP4 during cuprizone-induced demyelination. We conclude that Shh and Bmp4 differentially regulate histone acetylation and chromatin structure in OPCs and that BMP4 acts as a potent inducer of gene expression, including Notch and Wnt target genes, thereby enhancing the crosstalk among signaling pathways that are known to inhibit myelination and repair. Show less

Retinal Müller (glial) cells undergo "reactive gliosis", a stress response that is accompanied by changes in their morphology and upregulation of various cellular markers. Reactive gliosis is seen in many retinal diseases and conditions; however, it is not known whether it is a common, stereotypic response or the nature of the response varies with the type of retinal stress. To address this question, we have examined gene expression changes in Müller cells exposed to elevated pressure. Rat Müller cells (rMC-1) were exposed to elevated pressure, and RNA was extracted and analyzed using Affymetrix GeneChip microarrays to identify pressure-responsive genes. Analysis of microarray data showed that at 6 h, 186 genes had > 1.5-fold change with FDR < 0.01. Of these, 62 genes were up-regulated while 124 genes were down-regulated. At 24 h, 73 genes changed > 1.5-fold. Of these, 37 genes were up-regulated while 36 genes were down-regulated. Ingenuity canonical pathway analysis showed that several signaling and metabolic pathways were significantly changed in Müller cells under high pressure. In addition, among up- and down-regulated genes, we identified eight genes-areg, bmp4, cyp1b1, gpnmb, herc2, msh2, heph, and selenbp1, that have been directly or indirectly associated with elevated intraocular pressure. Two genes, areg and gpnmb, further showed time-dependent changes in mRNA and protein expression. The results show that Müller cells in vitro respond to elevated pressure by differential regulation of expressed genes. The transcriptional profile is different from that seen with hypoxia, which indicates that Müller cells respond differentially to different microenvironmental changes in the retina. Show less

Peroxisome proliferator-activated receptor (PPAR) γ coactivator-1β (PGC-1β) is a transcriptional coactivator that induces hypertriglyceridemia in response to dietary fats through activating hepatic lipogenesis and lipoprotein secretion. The expression of PGC-1β is regulated by free fatty acids. Here we show that PGC-1β regulates plasma triglyceride metabolism through stimulating apolipoprotein C3 (APOC3) expression and elevating APOC3 levels in circulation. Remarkably, liver-specific knockdown of APOC3 significantly ameliorates PGC-1β-induced hypertriglyceridemia in mice. Hepatic expression of PGC-1β and APOC3 is reduced in response to acute and chronic treatments with nicotinic acid, a widely prescribed drug for lowering plasma triglycerides. Adenoviral-mediated knockdown of PGC-1β or APOC3 in the liver recapitulates the hypolipidemic effect of nicotinic acid. Proteomic analysis of hepatic PGC-1β transcriptional complex indicates that it stimulates APOC3 expression through coactivating orphan nuclear receptor ERRα and recruiting chromatin-remodeling cofactors. Together, these studies identify PGC-1β as an important regulator of the APOC3 gene cluster and reveal a mechanism through which nicotinic acid achieves its therapeutic effects. Show less

The sleep-deprivation-induced changes in delta power, an electroencephalographical correlate of sleep need, and brain transcriptome profiles have importantly contributed to current hypotheses on sleep function. Because sleep deprivation also induces stress, we here determined the contribution of the corticosterone component of the stress response to the electrophysiological and molecular markers of sleep need in mice. N/A SETTINGS: Mouse sleep facility. C57BL/6J, AKR/J, DBA/2J mice. Sleep deprivation, adrenalectomy (ADX). Sleep deprivation elevated corticosterone levels in 3 inbred strains, but this increase was larger in DBA/2J mice; i.e., the strain for which the rebound in delta power after sleep deprivation failed to reach significance. Elimination of the sleep-deprivation-associated corticosterone surge through ADX in DBA/2J mice did not, however, rescue the delta power rebound but did greatly reduce the number of transcripts affected by sleep deprivation. Genes no longer affected by sleep deprivation cover pathways previously implicated in sleep homeostasis, such as lipid, cholesterol (e.g., Ldlr, Hmgcs1, Dhcr7, -24, Fkbp5), energy and carbohydrate metabolism (e.g., Eno3, G6pc3, Mpdu1, Ugdh, Man1b1), protein biosynthesis (e.g., Sgk1, Alad, Fads3, Eif2c2, -3, Mat2a), and some circadian genes (Per1, -3), whereas others, such as Homer1a, remained unchanged. Moreover, several microRNAs were affected both by sleep deprivation and ADX. Our findings indicate that corticosterone contributes to the sleep-deprivation-induced changes in brain transcriptome that have been attributed to wakefulness per se. The study identified 78 transcripts that respond to sleep loss independent of corticosterone and time of day, among which genes involved in neuroprotection prominently feature, pointing to a molecular pathway directly relevant for sleep function. Show less

To identify loci for age at menarche, we performed a meta-analysis of 32 genome-wide association studies in 87,802 women of European descent, with replication in up to 14,731 women. In addition to the known loci at LIN28B (P = 5.4 × 10⁻⁶⁰) and 9q31.2 (P = 2.2 × 10⁻³³), we identified 30 new menarche loci (all P < 5 × 10⁻⁸) and found suggestive evidence for a further 10 loci (P < 1.9 × 10⁻⁶). The new loci included four previously associated with body mass index (in or near FTO, SEC16B, TRA2B and TMEM18), three in or near other genes implicated in energy homeostasis (BSX, CRTC1 and MCHR2) and three in or near genes implicated in hormonal regulation (INHBA, PCSK2 and RXRG). Ingenuity and gene-set enrichment pathway analyses identified coenzyme A and fatty acid biosynthesis as biological processes related to menarche timing. Show less

Investigate the effect of hydrostatic pressure (HP) on 3', 5'-cyclic adenosine monophosphate (cAMP) levels and downstream signaling in cultures of normal optic nerve head (ONH) astrocytes from Caucasian American (CA) and African American (AA) donors. Intracellular cAMP levels were assayed after exposing ONH astrocytes to HP for varying times. Quantitative RT-PCR was used to determine the expression levels of selected cAMP pathway genes in human ONH astrocytes after HP treatment. Western blots were used to measure changes in the phosphorylation state of cAMP response element binding protein (CREB) in astrocytes subjected to HP, ATP, and phosphodiesterase or kinase inhibitors. The basal intracellular cAMP level is similar among AA and CA astrocytes. After exposure to HP for 15 min and 30 min in the presence of a phosphodiesterase inhibitor a further increase of intracellular cAMP was observed in AA astrocytes, but not in CA astrocytes. Consistent with activation of the cAMP-dependent protein kinase pathway, CREB phosphorylation (Ser-133) was increased to a greater extent in AA than in CA astrocytes after 3 h of HP. Exposure to elevated HP for 3-6 h differentially altered the expression levels of selected cAMP pathway genes (ADCY3, ADCY9, PTHLH, PDE7B) in AA compared to CA astrocytes. Treatment with ATP increased more CREB phosphorylation in CA than in AA astrocytes, suggesting differential Ca(2+) signaling in these populations. Activation of the cAMP-dependent signaling pathway by pressure may be an important contributor to increased susceptibility to elevated intraocular pressure and glaucoma in AA, a population at higher risk for the disease. Show less

Plasma liver-enzyme tests are widely used in the clinic for the diagnosis of liver diseases and for monitoring the response to drug treatment. There is considerable evidence that human genetic variation influences plasma levels of liver enzymes. However, such genetic variation has not been systematically assessed. In the present study, we performed a genome-wide association study of plasma liver-enzyme levels in three populations (total n = 7715) with replication in three additional cohorts (total n = 4704). We identified two loci influencing plasma levels of alanine-aminotransferase (ALT) (CPN1-ERLIN1-CHUK on chromosome 10 and PNPLA3-SAMM50 on chromosome 22), one locus influencing gamma-glutamyl transferase (GGT) levels (HNF1A on chromosome 12), and three loci for alkaline phosphatase (ALP) levels (ALPL on chromosome 1, GPLD1 on chromosome 6, and JMJD1C-REEP3 on chromosome 10). In addition, we confirmed the associations between the GGT1 locus and GGT levels and between the ABO locus and ALP levels. None of the ALP-associated SNPs were associated with other liver tests, suggesting intestine and/or bone specificity. The mechanisms underlying the associations may involve cis- or trans-transcriptional effects (some of the identified variants were associated with mRNA transcription in human liver or lymphoblastoid cells), dysfunction of the encoded proteins (caused by missense variations at the functional domains), or other unknown pathways. These findings may help in the interpretation of liver-enzyme tests and provide candidate genes for liver diseases of viral, metabolic, autoimmune, or toxic origin. The specific associations with ALP levels may point to genes for bone or intestinal diseases. Show less

The liver X receptors (LXR) alpha and beta are ligand-induced transcription factors that regulate the expression of genes important for cholesterol metabolism, lipogenesis, and other metabolic pathways. Despite their high degree of similarity, LXRs have redundant as well as nonredundant functions. The regulation of LXRs' intranuclear mobility most likely plays a major role in the regulation of their transcriptional activities. In order to elucidate how ligand binding, receptor-protein and receptor-DNA interactions affect intranuclear receptor mobility, we expressed transcriptionally active yellow fluorescent protein (YFP)-LXR alpha and YFP-LXR beta in Cos-7 cells. We used the fluorescence recovery after photobleaching (FRAP) technique and confocal laser scanning microscopy as well as Triton X-100 permeabilization experiments and fluorescence microscopy to measure differences in the intranuclear mobility between LXR alpha and LXR beta. The image analyses revealed that after agonist binding, LXR alpha exhibits slower intranuclear trafficking and greater intranuclear immobilization compared with LXR beta. In addition, mutational analysis showed that the integrity of the Activation Function (AF)-2 region of LXR alpha is essential for its immobilization whereas the integrity of the DNA binding domain is not. These findings imply that specific protein interactions with the AF-2 region of LXR alpha play a role in its intranuclear immobilization. Show less

The spliceosomal U1C protein is critical to the initiation and regulation of precursor messenger RNA (pre-mRNA) splicing, as part of the U1 small nuclear ribonucleoprotein particle (snRNP). We have produced full-length and 61 residue constructs of human U1C in soluble form in Escherichia coli. Atomic absorption spectroscopy and mass spectrometry show that both constructs contain one Zn atom and are monomeric. Gelmobility-shift assays showed that one molecule of recombinant U1C, either full-length or 61 residue construct, can be incorporated into the U1 snRNP core domain in the presence of U1 70k. This result is in perfect agreement with the previous experiment with U1C isolated from the HeLa U1 snRNP showing that the recombinant U1C is functionally active. We have determined the solution structure of the N-terminal 61 residue construct of U1C by NMR. A Cys(2)His(2)-type zinc finger, distinct from the TFIIIA-type, is extended at its C terminus by two additional helices. The two Zn-coordinating histidine residues are separated by a five residue loop. The conserved basic residues in the first two helices and the intervening loop may be involved in RNA binding. The opposite beta-sheet face with two surface-exposed Tyr residues may be involved in protein contacts. Both the full-length and 61 residue constructs of human U1C fail to bind RNA containing the 5' splice site sequence, in contrast to what has been reported for the Saccharomyces cerevisiae orthologue. Show less

A modified method of differential display was employed to identify a novel gene (named PSZA11q14), the expression of which was reduced in brains from patients with schizophrenia. Decreased expression of PSZA11q14 was identified initially in Brodmann's area (BA) 21 from a small group of patients with schizophrenia (n = 4) and normal controls (n = 6) and was confirmed subsequently using independent RT-PCR assay in BA 21, 22, and 9, and in hippocampus from a larger group of patients with schizophrenia (n = 36) and controls (n = 35). PSZA11q14 is located on chromosome 11q14, an area shown previously to co-segregate with schizophrenia and related disorders in several families. Decreased expression of PSZA11q14 in patients with schizophrenia and its location on 11q14 provide converging lines of evidence indicating that PSZA11q14 may be involved in at least some cases of schizophrenia. PSZA11q14 shows no significant homology with any known gene. It has no introns and produces two RNA transcripts of approximately 4.5 and approximately 7.0 kb. The largest open reading frame (ORF) in the PSZA11q14 transcripts may potentially encode for a short polypeptide of 71 amino acids. High frequency of rare codons, the short size of this ORF, and low homology with mouse sequences, however, indicate that PSZA11q14 may instead represent a novel member of a family of nonprotein-coding RNA genes that are not translated and that function at the RNA level. PSZA11q14 is located within the first intron of the DLG-2 gene and transcribed in the opposite direction to DLG-2. These results suggest that PSZA11q14 may be considered a candidate gene for schizophrenia acting as an antisense regulator of DLG-2, which controls assembling functional N-methyl-D-aspartate (NMDA) receptors. Show less