The glycine arginine‐rich domain of the RNA‐binding protein nucleolin regulates its subcellular localization

Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein‐protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid‐liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA‐RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.     

Near-infrared chemiluminescence from the oxidation of ammonia in aqueous alkaline solution.

The chemiluminescent oxidation of ammonia with hypobromite in aqueous alkaline solution evokes a broadly distributed emission in the near-infrared region, with intensity maxima at 1055 nm and 1270 nm.     

New dinucleoside analogues via cross-coupling metathesis

Synthesis of 3'-3', 5-5', and 3'-5' dimeric thymidine, linked by an olefinic chain between glycosidic moieties is described. Cross metathesis reaction of 3' or 5' O-allyl analogues of thymidine led to the expected 3'-3' and 5'-5' dimeric compounds, respectively. In order to obtain the 3'-5' dimer, 5'-O-allyl and 3'-O-allyl monomers were first linked by their free 3' OH and 5' OH groups through a glutaryl spacer; ring closing metathesis was then operated upon this temporary dimer, followed by glutaryl removal.     

The relationship between Y chromosome DNA haplotypes and Y chromosome deletions leading to male infertility

Microdeletions on the short arm of the Y chromosome have defined three non-overlapping regions (AZFa, b, c) recurrently deleted among infertile males. These regions contain several genes or gene families involved in male germ-cell development and maintenance. Even though a meiotic origin for these microdeletions is assumed, the mechanisms and causes leading to microdeletion formation are largely unknown. In order to assess whether some Y chromosome groups (or haplogroups) are predisposed to, or protected against, deletion formation during male meiosis, we have defined and compared Y chromosome haplogroup distribution in a group of infertile/subfertile males harbouring Yq deletions and in a relevant Northwestern European control population. Our analyses suggest that Y chromosome deletion formation is, at least in the study populations, a stochastic event independent of the Y chromosome background on which they arise and may be caused by other genetic and/or environmental factors.     

Accessibility of antigenic sites recognized by AUA1, HMFG1 and HMFG2 monoclonal antibodies: its influence on antibody binding of live cells

We assessed the immunoreactivity of live and alcohol-fixed monolayers of HRA-19, a rectal adenocarcinoma cell line, to the monoclonal antibodies AUA1, HMFG1 and HMFG2. Differences in staining patterns between live and alcohol-fixed colonies were found. The well-polarized cells forming the centers of the monolayer colonies showed strong membrane staining when the cells were alcohol-fixed prior to AUA1 incubation, but showed no staining when the cells were alive during the incubation. When AUA1 incubation was done both before and after alcohol fixation, membrane staining was again seen, ruling out the possibility of antigenic modulation. Incubation of live cells with AUA1 together with EDTA showed strong staining of dissociating cells. It is concluded that AUA1 antigenic sites, which on polarized cells are basolateral in location, are inaccessible to the antibody-containing culture fluid, which bathes the apical aspects of the cells, but they become accessible after alcohol fixation, or treatment with EDTA. HMFG1 antigenic sites are located on the apical cell membrane, and accordingly, no differences were seen between incubation of live and alcohol-fixed cells when incubated with HMFG1. The antigenic sites of HMFG2 are partly intracellular, and in our monolayer model, the staining of live cells was weaker and more scarce than on alcohol-fixed cells. It is concluded that immunostaining of cytological and histological material of tumours may not adequately predict antibody binding on live cells, and thus, these findings are of importance in the context of selection of monoclonal antibodies for clinical radio-immunotargeting.     

UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model

Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine, were background-dependent. Elevation of ethyl-β-d-glucuronide and N-acetylglycine was found to be a common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.     

Implication of intestinal VDR deficiency in inflammatory bowel disease

Background

To investigate the function of the intestinal Vdr gene in inflammatory bowel disease (IBD), in conjunction with the discovery of possible metabolic markers for IBD using intestine-specific Vdr knockout mice.

Methods

Vdr^ΔIEpC mice were generated, phenotyped and treated with a time-course of 3% dextran sulfate sodium (DSS) to induce colitis. Colitis was diagnosed by evaluating clinical symptoms and intestinal histopathology. Gene expression analysis was carried out. In addition, metabolic markers of IBD were explored by metabolomics.

Results

Vdr^ΔIEpC mice showed abnormal body size, colon structures and feces color. Calcium, collagen, and intestinal proliferation-related gene expression were all decreased, and serum alkaline phosphatase was highly increased. In the acute model which was treated with 3% DSS for six days, Vdr^ΔIEpC mice showed a high score of IBD symptoms; enlarged mucosal layer and damaged muscularis layer. In the recovery experiment model, where mice were treated with 3% DSS for four days and water for three days, Vdr^ΔIEpC mice showed a high score of IBD symptoms; severe damage of mucosal layer and increased expression of genes encoding proinflammatory cytokines. Feces metabolomics revealed decreased concentrations of taurine, taurocholic acid, taurodeoxycholic acid and cholic acid in Vdr^ΔIEpC mice.

Conclusions

Disruption of the intestinal Vdr gene showed phenotypical changes that may exacerbate IBD. These results suggest that VDR may play an important role in IBD.General significanceVDR function has been implicated in IBD. This is of value for understanding the etiology of IBD and for development of diagnostic biomarkers for IBD.     

Metabolomics reveals attenuation of the SLC6A20 kidney transporter in nonhuman primate and mouse models of type 2 diabetes mellitus

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na(+)-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.     

Site-specific effects of peptide lipidation on beta-amyloid aggregation and cytotoxicity

Beta-amyloid (Abeta) aggregates at low concentrations in vivo, and this may involve covalently modified forms of these peptides. Modification of Abeta by 4-hydroxynonenal (4-HNE) initially increases the hydrophobicity of these peptides and subsequently leads to additional reactions, such as peptide cross-linking. To model these initial events, without confounding effects of subsequent reactions, we modified Abeta at each of its amino groups using a chemically simpler, close analogue of 4-HNE, the octanoyl group: K16-octanoic acid (OA)-Abeta, K28-OA-Abeta, and Nalpha-OA-Abeta. Octanoylation of these sites on Abeta-(1-40) had strikingly different effects on fibril formation. K16-OA-Abeta and K28-OA-Abeta, but not Nalpha-OA-Abeta, had increased propensity to aggregate. The type of aggregate (electron microscopic appearance) differed with the site of modification. The ability of octanoyl-Abeta peptides to cross-seed solutions of Abeta was the inverse of their ability to form fibrils on their own (i.e. Abeta approximately Nalpha-OA-Abeta>K16-OA-Abeta>K28-OA-Abeta). By CD spectroscopy, K16-OA-Abeta and K28-OA-Abeta had increased beta-sheet propensity compared with Abeta-(1-40) or Nalpha-OA-Abeta. K16-OA-Abeta and K28-OA-Abeta were more amphiphilic than Abeta-(1-40) or Nalpha-OA-Abeta, as shown by lower "critical micelle concentrations" and higher monolayer collapse pressures. Finally, K16-OA-Abeta and K28-OA-Abeta are much more cytotoxic to N2A cells than Abeta-(1-40) or Nalpha-OA-Abeta. The greater cytotoxicity of K16-OA-Abeta and K28-OA-Abeta may reflect their greater amphiphilicity. We conclude that lipidation can make Abeta more prone to aggregation and more cytotoxic, but these effects are highly site-specific.