FXR, a multipurpose nuclear receptor.

The farnesoid X receptor (FXR) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. In the past six years, remarkable inroads have been made into determining the functional importance of FXR. This receptor has been shown to have crucial roles in controlling bile acid homeostasis, lipoprotein and glucose metabolism, hepatic regeneration, intestinal bacterial growth and the response to hepatotoxins. Thus, the development of FXR agonists might prove useful for the treatment of diabetes, cholesterol gallstones, and hepatic and intestinal toxicity.

Longitudinal SARS-CoV-2 antibody study using the Easy Check COVID-19 IgM/IgG™ lateral flow assay.

Since the initial identification of the novel coronavirus SARS-CoV-2 in December of 2019, researchers have raced to understand its pathogenesis and begun devising vaccine and treatment strategies. An accurate understanding of the body's temporal immune response against SARS-CoV-2 is paramount to successful vaccine development and disease progression monitoring. To provide insight into the antibody response against SARS-CoV-2, plasma samples from 181 PCR-confirmed COVID-19 patients collected at various timepoints post-symptom onset (PSO) were tested for the presence of anti-SARS-CoV-2 IgM and IgG antibodies via lateral flow. Additionally, 21 donors were tracked over time to elucidate patient-specific immune responses. We found sustained levels of anti-SARS-CoV-2 antibodies past 130 days PSO, with 99% positivity observed at 31-60 days PSO. By 61-90 days PSO, the percentage of IgM-/IgG+ results were nearly equal to that of IgM+/IgG+ results, demonstrating a shift in the immune response with a decrease in IgM antibody levels. Results from this study not only provide evidence that the antibody response to COVID-19 can persist for over 4 months, but also demonstrates the ability of Easy Check™ to monitor seroconversion and antibody response of patients. Easy Check was sufficiently sensitive to detect antibodies in patient samples as early as 1-4 days PSO with 86% positivity observed at 5-7 days PSO. Further studies are required to determine the longevity and efficacy of anti-SARS-CoV-2 antibodies, and whether they are protective against re-infection.

The neonatal ventromedial hypothalamus transcriptome reveals novel markers with spatially distinct patterning.

The ventromedial hypothalamus (VMH) is a distinct morphological nucleus involved in feeding, fear, thermoregulation, and sexual activity. It is essentially unknown how VMH circuits underlying these innate responses develop, in part because the VMH remains poorly defined at a cellular and molecular level. Specifically, there is a paucity of cell-type-specific genetic markers with which to identify neuronal subgroups and manipulate development and signaling in vivo. Using gene profiling, we now identify approximately 200 genes highly enriched in neonatal (postnatal day 0) mouse VMH tissue. Analyses of these VMH markers by real or virtual (Allen Brain Atlas; http://www.brain-map.org) experiments revealed distinct regional patterning within the newly formed VMH. Top neonatal markers include transcriptional regulators such as Vgll2, SF-1, Sox14, Satb2, Fezf1, Dax1, Nkx2-2, and COUP-TFII, but interestingly, the highest expressed VMH transcript, the transcriptional coregulator Vgll2, is completely absent in older animals. Collective results from zebrafish knockdown experiments and from cellular studies suggest that a subset of these VMH markers will be important for hypothalamic development and will be downstream of SF-1, a critical factor for normal VMH differentiation. We show that at least one VMH marker, the AT-rich binding protein Satb2, was responsive to the loss of leptin signaling (Lep(ob/ob)) at postnatal day 0 but not in the adult, suggesting that some VMH transcriptional programs might be influenced by fetal or early postnatal environments. Our study describing this comprehensive "VMH transcriptome" provides a novel molecular toolkit to probe further the genetic basis of innate neuroendocrine behavioral responses.

Regulation of hepatic Insig-2 by the farnesoid X receptor.

Activation of the farnesoid X receptor (FXRalpha) affects genes controlling many pathways, including those involved in bile acid and glucose homeostasis. Here we report that a critical gene involved in cholesterol homeostasis, Insig-2, was induced when mice or cultured cells were treated with FXRalpha agonists or infected with constitutively active FXRalpha. No such induction was observed in agonist-treated FXRalpha-/- mice. Further analysis, which included EMSAs, reporter gene activation, and chromatin immunoprecipitation, identified two functional FXRalpha response elements within intron 2 of the mouse Insig-2 gene. In addition to increasing hepatic Insig-2 protein levels in wild-type mice, FXRalpha activation also reduced lanosterol 14alpha-demethylase mRNA levels and 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein levels. Together, these changes likely account for the decrease in cholesterol synthesis observed after activation of FXR in primary hepatocytes. In conclusion, the current study links hepatic FXRalpha activation to regulation of genes involved in cholesterol synthesis.

Detection and Characterization of Circulating Tumour Cells in Multiple Myeloma.

Multiple myeloma (MM) remains an incurable disease despite recent therapeutic improvements. The ability to detect and characterize MM circulating tumour cells (CTCs) in peripheral blood provides an alternative to replace or augment invasive bone marrow (BM) biopsies with a simple blood draw, providing real-time, clinically relevant information leading to improved disease management and therapy selection. Here we have developed and qualified an enrichment-free, cell-based immunofluorescence MM CTC assay that utilizes an automated digital pathology algorithm to distinguish MM CTCs from white blood cells (WBCs) on the basis of CD138 and CD45 expression levels, as well as a number of morphological parameters. These MM CTCs were further characterized for expression of phospho-ribosomal protein S6 (pS6) as a readout for PI3K/AKT pathway activation. Clinical feasibility of the assay was established by testing blood samples from a small cohort of patients, where we detected populations of both CD138pos and CD138neg MM CTCs. In this study, we developed an immunofluorescent cell-based assay to detect and characterize CTCs in MM.

Eliminating SF-1 (NR5A1) sumoylation in vivo results in ectopic hedgehog signaling and disruption of endocrine development.

Sumoylation is generally considered a repressive mark for many transcription factors. However, the in vivo importance of sumoylation for any given substrate remains unclear and is questionable because the extent of sumoylation appears exceedingly low for most substrates. Here, we permanently eliminated SF-1/NR5A1 sumoylation in mice (Sf-1(K119R, K194R, or 2KR)) and found that Sf-1(2KR/2KR) mice failed to phenocopy a simple gain of SF-1 function or show elevated levels of well-established SF-1 target genes. Instead, mutant mice exhibited marked endocrine abnormalities and changes in cell fate that reflected an inappropriate activation of hedgehog signaling and other potential SUMO-sensitive targets. Furthermore, unsumoylatable SF-1 mutants activated Shh and exhibited preferential recruitment to Shh genomic elements in cells. We conclude that the sumoylation cycle greatly expands the functional capacity of transcription factors such as SF-1 and is leveraged during development to achieve cell-type-specific gene expression in multicellular organisms.

FXR regulates organic solute transporters alpha and beta in the adrenal gland, kidney, and intestine.

Expression of the farnesoid X receptor (FXR; NR1H4) is limited to the liver, intestine, kidney, and adrenal gland. However, the role of FXR in the latter two organs is unknown. In the current study, we performed microarray analysis using RNA from H295R cells infected with constitutively active FXR. Several putative FXR target genes were identified, including the organic solute transporters alpha and beta (OSTalpha and OSTbeta). Electromobility shift assays and promoter-reporter studies identified functional farnesoid X receptor response elements (FXREs) in the promoters of both human genes. These FXREs are conserved in both mouse genes. Treatment of wild-type mice with 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl-isoxazole (GW4064), a synthetic FXR agonist, induced OSTalpha and OSTbeta mRNAs in the intestine and kidney. Both mRNAs were also induced when wild-type, but not FXR-deficient (FXR-/-), adrenals were cultured in the presence of GW4064. OSTalpha and OSTbeta mRNA levels were also induced in the adrenals and kidneys of wild-type, but not FXR-/-, mice after the increase of plasma bile acids in response to the hepatotoxin alpha-naphthylisothiocyanate. Finally, overexpression of human OSTalpha and OSTbeta facilitated the uptake of conjugated chenodeoxycholate and the activation of FXR target genes. These results demonstrate that OSTalpha and OSTbeta are novel FXR target genes that are expressed in the adrenal gland, kidney, and intestine.

Activation of the nuclear receptor FXR induces fibrinogen expression: a new role for bile acid signaling.

Three genes, fibrinogen-alpha (FBGalpha), -beta, and -gamma, encode proteins that make up the mature FBG protein complex. This complex is secreted from the liver and plays a key role in coagulation in response to vascular disruption. We identified all three FBG genes in a screen designed to isolate genes that are regulated by the farnesoid X receptor (FXR; NR1H4). Treatment of human hepatoma cells with either naturally occurring or synthetic [3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl-isoxazole] FXR ligands resulted in the induction of transcripts for all three genes. The induction of FBGbeta mRNA in response to activated FXR appears to be a primary transcriptional response, as it is blocked by actinomycin D but not by cycloheximide. Four FXR isoforms were recently identified that differ either at their N termini and/or by the presence of four amino acids in the hinge region. Interestingly, the activities of the human FBGbeta promoter-reporter constructs were highly induced by FXR isoforms that lack the four amino acid insert. The observation that all three FBG subunits are induced by specific FXR isoforms, in response to FXR ligands, suggests that bile acids and FXR modulate fibrinolytic activity.

Alpha-crystallin is a target gene of the farnesoid X-activated receptor in human livers.

Alpha-crystallins comprise 35% of soluble proteins in the ocular lens and possess chaperone-like functions. Furthermore, the alphaA subunit (alphaA-crystallin) of alpha crystallin is thought to be "lens-specific" as only very low levels of expression were detected in a few non-lenticular tissues. Here we report that human alphaA-crystallin is expressed in human livers and is regulated by farnesoid X-activated receptor (FXR) in response to FXR agonists. AlphaA-crystallin was identified in a microarray screen as one of the most highly induced genes after treatment of HepG2 cells with the synthetic FXR ligand GW4064. Northern blot and quantitative real-time PCR analyses confirmed that alphaA-crystallin expression was induced in HepG2-derived cell lines and human primary hepatocytes and hepatic stellate cells in response to either natural or synthetic FXR ligands. Transient transfection studies and electrophoretic mobility shift assays revealed a functional FXR response element located in intron 1 of the human alphaA-crystallin gene. Importantly, immunohistochemical staining of human liver sections showed increased alphaA-crystallin expression in cholangiocytes and hepatocytes. As a member of the small heat shock protein family possessing chaperone-like activity, alphaA-crystallin may be involved in protection of hepatocytes from the toxic effects of high concentrations of bile acids, as would occur in disease states such as cholestasis.