Construction of Adipogenic ceRNA Network Based on lncRNA Expression Profile of Adipogenic Differentiation of Human MSC Cells

Biochemical Genetics - The Long non-coding RNA (lncRNA) expression profile data of ten samples including human Mesenchymal Stem Cell (MSC) adipogenic differentiation 0, 3, and 6 days from...     

Reduction of 8-iso-prostaglandin F2α in the first week after Roux-en-Y gastric bypass surgery

Obesity is associated with increased markers of oxidative stress. We examined whether oxidative stress is reduced within the first week after Roux‐en‐Y gastric bypass (RYGB) surgery and could be related to changes in adipose tissue depots. The reactive oxygen species (ROS) marker 8‐iso‐prostaglandin F2α (8‐iso‐PGF2α) and activity of antioxidant glutathione peroxidases (GPX) in plasma were compared before and ～1 week after RYGB. The effects of RYGB on subcutaneous adipose tissue and interstitial fluid 8‐iso‐PGF2α levels and subcutaneous adipose tissue expression of GPX‐3 were also assessed. Levels of 8‐iso‐PGF2α in subcutaneous and visceral adipose tissue were determined. Plasma 8‐iso‐PGF2α levels decreased (122 ± 75 to 56 ± 15 pg/ml, P = 0.001) and GPX activity increased (84 ± 18 to 108 ± 25 nmol/min/ml, P = 0.003) in the first week post‐RYGB. RYGB also resulted in reductions of 8‐iso‐PGF2α in subcutaneous adipose tissue (1,742 ± 931 to 1,132 ± 420 pg/g fat, P = 0.046) and interstitial fluid (348 ± 118 to 221 ± 83 pg/ml, P = 0.046) that were comparable to plasma (26–33%, P = 0.74). Adipose GPX‐3 expression was increased (6.7 ± 4.7‐fold, P = 0.004) in the first postoperative week. The improvements in oxidative stress occurred with minimal weight loss (2.4 ± 3.4%, P = 0.031) and elevations in plasma interleukin‐6 (18.0 ± 46.8 to 28.0 ± 58.9 pg/ml, P = 0.004). Subcutaneous and visceral adipose tissues express comparable 8‐iso‐PGF2α levels (1,204 ± 470 and 1,331 ± 264 pg/g fat, respectively; P = 0.34). These data suggest that RYGB affects adipose tissue leading to the restoration of adipose redox balance within the first postoperative week and that plasma 8‐iso‐PGF2α is primarily derived from subcutaneous adipose tissue.     

Probing the limits of aptamer affinity with a microfluidic SELEX platform

Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (K_d) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a K_d of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer K_d.     

Prediction of transposable element derived enhancers using chromatin modification profiles

Experimentally characterized enhancer regions have previously been shown to display specific patterns of enrichment for several different histone modifications. We modelled these enhancer chromatin profiles in the human genome and used them to guide the search for novel enhancers derived from transposable element (TE) sequences. To do this, a computational approach was taken to analyze the genome-wide histone modification landscape characterized by the ENCODE project in two human hematopoietic cell types, GM12878 and K562. We predicted the locations of 2,107 and 1,448 TE-derived enhancers in the GM12878 and K562 cell lines respectively. A vast majority of these putative enhancers are unique to each cell line; only 3.5% of the TE-derived enhancers are shared between the two. We evaluated the functional effect of TE-derived enhancers by associating them with the cell-type specific expression of nearby genes, and found that the number of TE-derived enhancers is strongly positively correlated with the expression of nearby genes in each cell line. Furthermore, genes that are differentially expressed between the two cell lines also possess a divergent number of TE-derived enhancers in their vicinity. As such, genes that are up-regulated in the GM12878 cell line and down-regulated in K562 have significantly more TE-derived enhancers in their vicinity in the GM12878 cell line and vice versa. These data indicate that human TE-derived sequences are likely to be involved in regulating cell-type specific gene expression on a broad scale and suggest that the enhancer activity of TE-derived sequences is mediated by epigenetic regulatory mechanisms.     

NF-kappaB and not the MAPK signaling pathway regulates GADD45beta expression during acute inflammation

The GADD45 (growth arrest and DNA damage-inducible) family of genes is involved in the regulation of cell cycle progression and apoptosis. To study signaling pathways affecting GADD45beta expression and to examine systematically in vivo the GADD45beta expression in tissues following various toxic stresses, we created a transgenic mouse by fusing the GADD45beta promoter to firefly luciferase (Gadd45beta-luc). In vivo GADD45beta expression was assessed by measuring the luciferase activity in the Gadd45beta-luc transgenic mouse using a non-invasive imaging system (IVIS Imaging System, Xenogen Corporation). We found that a number of agents that induce oxidative stress, such as sodium arsenite, CCl4, lipopolysaccharide (LPS), or tumor necrosis factor-alpha, are able to induce luciferase expression throughout the entire animal. In liver, spleen, lung, intestine, kidney, and heart, we observed an induction of luciferase activity after LPS treatment, which correlates with an increase of GADD45beta mRNA in these tissues. Processes that induce DNA damage activate the NF-kappaB signaling pathway. Several inhibitors of the NF-kappaB signaling pathway, including dexamethasone, thalidomide, and a proteasome inhibitor, bortezomib, showed inhibitory effects on LPS-induced GADD45beta expression as indicated by a decrease of the luciferase activity. Northern blot analysis confirmed a broad inhibitory effect of bortezomib on LPS-induced GADD45beta mRNA expression in spleen, lung, and intestine. In liver of bortezomib-treated mice, we observed a reverse correlation between the luciferase activity and the GADD45beta mRNA level. We speculate that such a discrepancy could be due to severe liver toxicity caused by bortezomib and LPS co-treatment. MAPK inhibitors had transient and inconsistent effects on LPS-induced luciferase expression. Our data are consistent with the notion that NF-kappaB, but not the MAPK signaling pathways, is involved in the in vivo regulation of GADD45beta expression. Thus, NF-kappaB signaling involves induction of GADD45beta expression, which supports the proposed role of GADD45beta in protecting cells against DNA damaged under various stress conditions.     

Transcriptional status of known and novel genes tagged with consensus of 33.15 repeat loci employing minisatellite-associated sequence amplification (MASA) and real-time PCR in water buffalo, Bubalus bubalis

We conducted minisatellite-associated sequence amplification (MASA) with an oligo (5' CACCTCTCCACCTGCC 3') based on consensus of 33.15 repeat loci using cDNA from the testis, ovary, spleen, kidney, heart, liver, and lung of water buffalo Bubalus bubalis and uncovered 25 amplicons of six different sizes (1,263, 846/847, 602, 576, 487, and 324 base pairs). These fragments, cloned and sequenced, were found to represent several functional, regulatory, and structural genes. Blast search of all the 25 amplicons showed homologies with 43 transcribing genes across the species. Of these, the 846/847-bp fragment, having homology with the adenylate kinase gene, showed nucleotide changes at six identical places in the ovary and testis. The 1,263; 324; and 487-bp fragments showed homology with the secreted modular calcium binding protein (SMOC-1), leucine-rich repeat neuronal 6A (LRRN6A) mRNA, and human TTTY5 mRNA, respectively. Real-time PCR showed maximum expression of AKL, LRRN6A, and T-cell receptor gamma (TCR-gamma)-like genes in the testis, SMOC-1 in the liver, and the T-cell receptor-like (TCRL) gene in the spleen compared to those used as endogenous control. We construe that these genes have evolved from a common progenitor and conformed to various biological functions during the course of evolution. MASA approach coupled with real-time PCR has potentials to uncover accurate expression of a large number of genes within and across the species circumventing the screening of cDNA library.     

Investigation of an Anomalously Accelerating Substitution in the Folding of a Prototypical Two-State Protein

The folding rates of two-state single-domain proteins are generally resistant to small-scale changes in amino acid sequence. For example, having surveyed here over 700 single-residue substitutions in 24 well-characterized two-state proteins, we find that the majority (55%) of these substitutions affect folding rates by less than a factor of 2, and that only 9% affect folding rates by more than a factor of 8. Among those substitutions that significantly affect folding rates, we find that accelerating substitutions are an order of magnitude less common than those that decelerate the process. One of the most extreme outliers in this data set, an arginine-to-phenylalanine substitution at position 48 (R48F) of chymotrypsin inhibitor 2 (CI2), accelerates the protein's folding rate by a factor of 36 relative to that of the wild-type protein and is the most accelerating substitution reported to date in a two-state protein. In order to better understand the origins of this anomalous behavior, we have characterized the kinetics of multiple additional substitutions at this position. We find that substitutions at position 48 in CI2 fall into two distinct classes. The first, comprising residues that ablate the charge of the wild-type arginine but retain the hydrophobicity of its alkane chain, accelerate folding by at least 10-fold. The second class, comprising all other residues, produces folding rates within a factor of two of the wild-type rate. A significant positive correlation between hydrophobicity and folding rate across all of the residues we have characterized at this position suggests that the hydrophobic methylene units of the wild-type arginine play a significant role in stabilizing the folding transition state. Likewise, studies of the pH dependence of the histidine substitution indicate a strong correlation between folding rate and charge state. Thus, mutations that ablate the arginine's positive charge while retaining the hydrophobic contacts of its methylene units tend to dramatically accelerate folding. Previous studies have suggested that arginine 48 plays an important functional role in CI2, which may explain why it is highly conserved despite the anomalously large deceleration it produces in the folding of this protein.     

Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus

We previously described a renal protective effect of factor B deficiency in MRL/lpr mice. Factor B is in the MHC cluster; thus, the deficient mice were H2b, the haplotype on which the knockout was derived, whereas the wild-type littermates were H2k, the H2 of MRL/lpr mice. To determine which protective effects were due to H2 vs factor B deficiency, we derived H2b congenic MRL/lpr mice from the 129/Sv (H2b) strain. Autoantibody profiling using autoantigen microarrays revealed that serum anti-Smith and anti-small nuclear ribonucleoprotein complex autoantibodies, while present in the majority of H2k/k MRL/lpr mice, were absent in the H2b/b MRL/lpr mice. Surprisingly, 70% of MRL/lpr H2b/b mice were found to be serum IgG3 deficient (with few to no IgG3-producing B cells). In addition, H2b/b IgG3-deficient MRL/lpr mice had significantly less proteinuria, decreased glomerular immune complex deposition, and absence of glomerular subepithelial deposits compared with MRL/lpr mice of any H2 type with detectable serum IgG3. Despite these differences, total histopathologic renal scores and survival were similar among the groups. These results indicate that genes encoded within or closely linked to the MHC region regulate autoantigen selection and isotype switching to IgG3 but have minimal effect on end-organ damage or survival in MRL/lpr mice.     

Hydrophilic anilinogeranyl diphosphate prenyl analogues are Ras function inhibitors

Sequential processing of H-Ras by protein farnesyl transferase (FTase), Ras converting enzyme (Rce1), and protein-S-isoprenylcysteine O-methyltransferase (Icmt) to give H-Ras C-terminal farnesyl-S-cysteine methyl ester is required for appropriate H-Ras membrane localization and function, including activation of the mitogen-activated protein kinase (MAPK) cascade. We employed a Xenopus laevis oocyte whole-cell model system to examine whether anilinogeranyl diphosphate analogues of similar shape and size, but with a hydrophobicity different from that of the FTase substrate farnesyl diphosphate (FPP), could ablate biological function of H-Ras. Analysis of oocyte maturation kinetics following microinjection of in vitro analogue-modified H-Ras into isoprenoid-depleted oocytes revealed that analogues with a hydrophobicity near that of FPP supported H-Ras biological function, while the analogues p-nitroanilinogeranyl diphosphate (p-NO2-AGPP), p-cyanoanilinogeranyl diphosphate (p-CN-AGPP), and isoxazolaminogeranyl diphosphate (Isox-GPP) with hydrophobicities 2-5 orders of magnitude lower than that of FPP did not. We found that although H-Ras modified with FPP analogues p-NO2-AGPP, p-CN-AGPP, and Isox-GPP was an efficient substrate for C-terminal postprenylation processing by Rce1 and Icmt, co-injection of H-Ras with analogues p-NO2-AGPP, p-CN-AGPP, or Isox-GPP could not activate MAPK. We propose that H-Ras biological function requires a minimum lipophilicity of the prenyl group to allow important interactions downstream of the C-terminal processed H-Ras protein. The hydrophilic FPP analogues p-NO2-AGPP, p-CN-AGPP, and Isox-GPP are H-Ras function inhibitors (RFIs) and serve as lead compounds for a unique class of potential anticancer therapeutics.