Long non-coding RNA expression profiles in neutrophils revealed potential biomarker for prediction of renal involvement in SLE patients.

OBJECTIVE: The long non-coding RNA plays an important role in inflammation and autoimmune diseases. The aim of this study is to screen and identify abnormally expressed lncRNAs in peripheral blood neutrophils of SLE patients as novel biomarkers and to explore the relationship between lncRNAs levels and clinical features, disease activity and organ damage. METHODS: RNA-seq technology was used to screen differentially expressed lncRNAs in neutrophils from SLE patients and healthy donors. Based on the results of screening, candidate lncRNA levels in neutrophils of 88 SLE patients, 35 other connective disease controls, and 78 healthy controls were qualified by real-time quantitative polymerase chain reaction. RESULTS: LncRNA expression profiling revealed 360 up-regulated lncRNAs and 224 down-regulated lncRNAs in neutrophils of SLE patients when compared with healthy controls. qPCR assay validated that the expression of Lnc-FOSB-1:1 was significantly decreased in neutrophils of SLE patients when compared with other CTD patients or healthy controls. It correlated negatively with SLE Disease Activity Index 2000 (SLEDAI-2K) score (r = -0.541, P < 0.001) and IFN scores (r = -0.337, P = 0.001). More importantly, decreased Lnc-FOSB-1:1 expression was associated with lupus nephritis. Lower baseline Lnc-FOSB-1:1 level was associated with higher risk of future renal involvement (within an average of 2.6 years) in patients without renal disease at baseline (P = 0.019). CONCLUSION: LncRNA expression profile in neutrophils of SLE patients revealed differentially expressed lncRNAs. Validation study on Lnc-FOSB-1:1 suggest that it is a potential biomarker for prediction of near future renal involvement.

Puerarin inhibits titanium particle-induced osteolysis and RANKL-induced osteoclastogenesis via suppression of the NF-κB signaling pathway.

Osteolysis around the prosthesis and subsequent aseptic loosening are the main causes of prosthesis failure. Inflammation due to wear particles and osteoclast activation are the key factors in osteolysis and are also potential targets for the treatment of osteolysis. However, it is not clear whether puerarin can inhibit chronic inflammation and alleviate osteolysis. In this study, we investigated the effect of puerarin on Ti particle-induced inflammatory osteolysis in vivo in rat femoral models and in vitro in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast activation models. Our in vivo results showed that puerarin significantly inhibited Ti particle-induced osteolysis and the expression of matrix metallopeptidase 9 (MMP-9), nuclear factor of activated T cells 1 (NFATc1), tumour necrosis factor (TNF)-α and interleukin (IL)-6. In vitro, puerarin prevented RANKL-induced osteoclast differentiation, bone resorption and F-actin ring formation in a concentration-dependent manner. Furthermore, puerarin decreased the phosphorylation of p65 and prevented p65 moving from the cytoplasm to the nucleus. Puerarin also reduced the expression of osteoclast-specific factors and inhibited the inflammatory response. In conclusion, our study proves that puerarin can block the NF-κB signalling pathway to inhibit osteoclast activation and inflammatory processes, which provides a new direction for the treatment of osteolysis-related diseases.

Fluorometric titration assay of affinity of tight-binding nonfluorescent inhibitor of glutathione S-transferase.

To determine inhibition constant (K(i)) of tight-binding inhibitor, the putative method estimated an apparent K(i) from the response of initial rates to total concentrations of the inhibitor considering its depletion during binding for conversion into the true K(i), but was impractical with glutathione S-transferase of sophisticated kinetics. A fluorometric titration assay of dissociation constant (K(d)) was thus proposed. Schistosoma japonicum glutathione S-transferase (SjGST) action on a nonfluorescent divalent pro-inhibitor and glutathione yielded a divalent product in active site to act as a tight-binding inhibitor, whose binding quenched fluorescence of SjGST at 340 nm under the excitation at 280 nm. K(d) was estimated from the response of fluorescence of SjGST at 340 nm to total concentrations of the divalent product considering its depletion during binding. By fluorometric titration assay, K(d) of two tested nonfluorescent divalent products varied from subnanomolar to nanomolar, but both were resistant to change of SjGST levels and consistent with their apparent K(i) estimated via the putative method. Hence, fluorometric titration assay of K(d) of nonfluorescent tight-binding inhibitors/ligands was effective to GST and may be universally applicable to common enzymes/proteins; affinities of tight-binding inhibitors of GST can be approximated by their apparent K(i) estimated via the putative method.

Fluorometric titration approach for calibration of quantity of binding site of purified monoclonal antibody recognizing epitope/hapten nonfluorescent at 340 nm.

A fluorometric titration approach was proposed for the calibration of the quantity of monoclonal antibody (mcAb) via the quench of fluorescence of tryptophan residues. It applied to purified mcAbs recognizing tryptophan-deficient epitopes, haptens nonfluorescent at 340 nm under the excitation at 280 nm, or fluorescent haptens bearing excitation valleys nearby 280 nm and excitation peaks nearby 340 nm to serve as Förster-resonance-energy-transfer (FRET) acceptors of tryptophan. Titration probes were epitopes/haptens themselves or conjugates of nonfluorescent haptens or tryptophan-deficient epitopes with FRET acceptors of tryptophan. Under the excitation at 280 nm, titration curves were recorded as fluorescence specific for the FRET acceptors or for mcAbs at 340 nm. To quantify the binding site of a mcAb, a universal model considering both static and dynamic quench by either type of probes was proposed for fitting to the titration curve. This was easy for fitting to fluorescence specific for the FRET acceptors but encountered nonconvergence for fitting to fluorescence of mcAbs at 340 nm. As a solution, (a) the maximum of the absolute values of first-order derivatives of a titration curve as fluorescence at 340 nm was estimated from the best-fit model for a probe level of zero, and (b) molar quantity of the binding site of the mcAb was estimated via consecutive fitting to the same titration curve by utilizing such a maximum as an approximate of the slope for linear response of fluorescence at 340 nm to quantities of the mcAb. This fluorometric titration approach was proved effective with one mcAb for six-histidine and another for penicillin G.

Dexmedetomidine Reverses Postoperative Spatial Memory Deficit by Targeting Surf1 and Cytochrome c.

Anesthesia and surgery are associated with perioperative neurocognitive disorders (PND). Dexmedetomidine is known to improve PND in rats; however, little is known about the mechanisms. Male Sprague-Dawley rats were subjected to resection of the hepatic apex under propofol anesthesia to clinically mimic human abdominal surgery. The rats were divided into four groups: control group (C), anesthesia group (A), model group (M), and model + dex group (D). Cognitive function was evaluated with the Morris water maze (MWM). Neuronal morphology was observed with H&E staining, Nissl's staining and immunohistochemistry. Transcriptome analysis and quantitative real-time PCR were performed to investigate functional mitochondrial mRNA changes in the hippocampus. Protein levels were measured by Western blotting at 1, 3, and 7 days after surgery. Surgery-induced cognitive decline lasted for three days, but not seven days after surgery in the M group; however, rats in the D group were significantly improved by dexmedetomidine. No significant differences in the number of neurons were observed between the groups after surgery. Rats from the M group showed significantly greater expression levels of Iba-1 and GFAP compared with the C group and the D group. Rats in the M group demonstrated increased Surf1 and Cytochrome c expression on days 1 and 3, but not day 7; similar changes were not induced in rats in the D group. Dexmedetomidine appears to reverse surgery-induced behavior, mitigate the higher density of Iba-1 and GFAP, and downregulate the expression of Surf1 and Cytochrome c protein in the hippocampus of rats in a PND model.

Effects of LPS on the Secretion of Gonadotrophin Hormones and Expression of Genes in the Hypothalamus-Pituitary-Ovary (HPG) Axis in Laying Yangzhou Geese.

Lipopolysaccharide (LPS) from gram-negative bacteria was found to be involved in the decrease in laying performance in goose flocks with high stocking density during summer months. LPS injection delayed the increase in the laying rate and altered hierarchical follicle morphology. While there is evidence that LPS exerts suppressive effects on goose reproduction, the time course effects of LPS on the hypothalamus-pituitary-ovary (HPG) axis remain elusive. In this study, we investigated the expression of genes in the HPG axis and the plasma gonadotrophin hormone concentrations in breeding geese at 0, 6, 12, 24, and 36 h after intravenous injection with LPS. The results showed that LPS treatment enhanced and suppressed expression of hypothalamic gonadotropin-inhibiting hormone (GnIH) and gonadotrophin-releasing hormone (GnRH) mRNA, respectively, and similar effects were observed on the mRNA expression of their receptors, GnIHR and GnRHR, in the pituitary. LPS treatment transiently increased follicle FSHß mRNA expression at 12 h and exerted no significant effect on LHß mRNA expression in the pituitary. Regardless of the expression of FSHß and LHß, plasma follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations were significantly increased during 24-36 h after LPS treatment. In the ovary, StAR and Cyp11a1 were mainly expressed in the granulosa layer (GL) of hierarchical follicles, while Cyp17a1 and Cyp19a1 were mainly expressed in white follicles (WFs) and yellowish follicles (YFs), and to a lesser extent in the theca layer (TL). After LPS treatment, the mRNA levels of Cyp11a1 in the GLs, Cyp17a1 in the WFs and TL, and Cyp19a1 in the WFs, YFs, and TL were significantly decreased. However, LPS treatment transiently upregulated StAR expression at 12 h. These results indicate that the exposure of laying geese to LPS may impair the HPG axis and disturb ovarian steroidogenesis. Our research provides new insights into reproductive dysfunction caused by LPS and the immune challenge in birds.

Identification of LncRNA Linc00513 Containing Lupus-Associated Genetic Variants as a Novel Regulator of Interferon Signaling Pathway.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by augmented type I interferon signaling. High-throughput technologies have identified plenty of SLE susceptibility single-nucleotide polymorphisms (SNPs) yet the exact roles of most of them are still unknown. Functional studies are principally focused on SNPs in the coding regions, with limited attention paid to the SNPs in non-coding regions. Long non-coding RNAs (lncRNAs) are important players in shaping the immune response and show relationship to autoimmune diseases. In order to reveal the role of SNPs located near SLE related lncRNAs, we performed a transcriptome profiling of SLE patients and identified linc00513 as a significantly over expressed lncRNA containing functional SLE susceptibility loci in the promoter region. The risk-associated G allele of rs205764 and A allele of rs547311 enhanced linc00513 promoter activity and related to increased expression of linc00513 in SLE. We also identified linc00513 to be a novel positive regulator of type I interferon pathway by promoting the phosphorylation of STAT1 and STAT2. Elevated linc00513 expression positively correlated with IFN score in SLE patients. Linc00513 expression was higher in active disease patients than those inactive ones. In conclusion, our data identify two functional promoter variants of linc00513 that contribute to increased level of linc00513 and confer susceptibility on SLE. The study provides new insights into the genetics of SLE and extends the role of lncRNAs in the pathogenesis of SLE.

Selective and sensitive homogenous assay of serum albumin with 1-anilinonaphthalene-8-sulphonate as a biosensor.

Homogenous selective assay of albumin (ALB) in clinical sera was tested with 1-anilinonaphthalene-8-sulphonate (ANS) as Förster-resonance-energy-transfer (FRET) acceptor of tryptophan residues and biosensor of ALB. Between the excitation at 280 and 350 nm, the ratio of the fluorescence at 470 nm of free ANS in ethanol was about 1.9 while that of the complexes of ALB and ANS was about 3.9, supporting FRET in complexes of ANS and ALB. ANS below 1.0 mM saturated one site of ALB with Kd of about 0.13 µM in 20 mM sodium phosphate buffer at pH 7.0. For selective assay of ALB, 0.30 µM ANS was used to quantify fluorescence of the complexes at 470 nm under the excitation at 280 nm. ALB from 1.8 to 25 nM was quantified, whose lower limit was below 1% than that by bromocresol green assay while one-third than that by immunoturbidimetric assay. Globular proteins at comparable levels gave negligible signals. This new method showed reasonable resistance to other interfering substances in clinical sera. Quantities of ALB in clinical sera by this method were consistent with those by bromocresol green assay and immunoturbidimetric assay. Hence, homogenous assay of ALB with ANS as FRET biosensor was effective.

Facile characterization of the immobilization of streptavidin on magnetic submicron particles with a fluorescent probe of streptavidin.

To characterize streptavidin immobilization on magnetic submicron particles (MSPs), residual streptavidin after magnetic removal of immobilized streptavidin was quantified with N-biotinyl-N'-(1-naphthyl)-ethylenediamine (BNEDA) based on Förster resonance energy transfer. Residual BNEDA after magnetic removal of bound BNEDA was measured by its own fluorescence. Streptavidin was immobilized at about 12 mg per gram of MSPs and easily retained over 50% of its original activity. These assays facilitated optimized streptavidin immobilization on MSPs.