ETS1 variants confer susceptibility to ankylosing spondylitis in Han Chinese

Introduction

ETS1 is a negative regulator of the Th17 differentiation gene and plays a central role in the pathogenesis of autoimmune diseases. We aimed to investigate whether polymorphisms in ETS1 confer susceptibility to ankylosing spondylitis (AS) in Han Chinese.

Methods

We selected seven single nucleotide polymorphisms (SNPs) within ETS1 based on HapMap data and previous genome-wide association study. Genotyping involved the TaqMan method in 1,015 patients with AS and 1,132 healthy controls from Shandong Province, and 352 AS patients and 400 healthy controls from Ningxia, a northwest region in China. Gene expression was determined by real-time PCR.

Results

The SNP rs1128334 was strongly associated with AS (odds ratio 1.204, 95% confidence interval 1.06-1.37; P = 0.005). This association was confiexrmed in the Ningxia population (P = 0.015). Carriers of the haplotype TAT for rs12574073, rs1128334 and rs4937333 were associated with increased risk of AS and haplotype CGC with reduced risk as compared to controls. In addition, ETS1 expression was lower in AS patients than controls. The risk allele A of rs1128334 and haplotype A-T of rs1128334 and rs4937333 were associated with decreased expression of ETS1.

Conclusions

Common variants in ETS1 may contribute to AS susceptibility in Han Chinese people.     

Enhancement of keratinolytic activity of a thermophilic subtilase by improving its autolysis resistance and thermostability under reducing conditions

WF146 protease, a thermophilic subtilase from thermophile Bacillus sp. WF146, suffers excessive autolysis in the presence of reducing agents. In this report, two autolytic sites of WF146 protease were modified by site-directed mutagenesis. The introduction of prolines into the autolytic sites increased the autolysis resistance of the enzyme under reducing conditions. The double mutant N63P/A66P displayed a 2.8-fold longer half-life at 80°C and higher hydrolytic activities than wild-type enzyme toward soluble (casein) and insoluble (keratin azure) substrates at high temperatures. In the presence of reducing agents, N63P/A66P was able to degrade feather at 80°C (∼3 h), with hydrolysis efficiency comparable to that of proteinase K at 50°C (∼24 h). Meanwhile, the mutant N63P/A66P had the ability to hydrolyze PrP^Sc-like prion protein at high temperatures. In virtue of these properties, N63P/A66P is of great interest to be used in recycling of keratinous wastes, such as feather, and disinfection of medical apparatus. In addition, our study may provide useful information needed to explore keratinolytic potential of thermophilic subtilases, even if they are produced by non-keratinolytic microorganisms.     

Immune protection induced on day 10 following administration of the 2009 A/H1N1 pandemic influenza vaccine

Background

The 2009 swine-origin influenza virus (S-OIV) H1N1 pandemic has caused more than 18,000 deaths worldwide. Vaccines against the 2009 A/H1N1 influenza virus are useful for preventing infection and controlling the pandemic. The kinetics of the immune response following vaccination with the 2009 A/H1N1 influenza vaccine need further investigation.

Methodology/Principal Findings

58 volunteers were vaccinated with a 2009 A/H1N1 pandemic influenza monovalent split-virus vaccine (15 µg, single-dose). The sera were collected before Day 0 (pre-vaccination) and on Days 3, 5, 10, 14, 21, 30, 45 and 60 post vaccination. Specific antibody responses induced by the vaccination were analyzed using hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). After administration of the 2009 A/H1N1 influenza vaccine, specific and protective antibody response with a major subtype of IgG was sufficiently developed as early as Day 10 (seroprotection rate: 93%). This specific antibody response could maintain for at least 60 days without significant reduction. Antibody response induced by the 2009 A/H1N1 influenza vaccine could not render protection against seasonal H1N1 influenza (seroconversion rate: 3% on Day 21). However, volunteers with higher pre-existing seasonal influenza antibody levels (pre-vaccination HI titer ≥1∶40, Group 1) more easily developed a strong antibody protection effect against the 2009 A/H1N1 influenza vaccine as compared with those showing lower pre-existing seasonal influenza antibody levels (pre-vaccination HI titer <1∶40, Group 2). The titer of the specific antibody against the 2009 A/H1N1 influenza was much higher in Group 1 (geometric mean titer: 146 on Day 21) than that in Group 2 (geometric mean titer: 70 on Day 21).

Conclusions/Significance

Recipients could gain sufficient protection as early as 10 days after vaccine administration. The protection could last at least 60 days. Individuals with a stronger pre-existing seasonal influenza antibody response may have a relatively higher potential for developing a stronger humoral immune response after vaccination with the 2009 A/H1N1 pandemic influenza vaccine.     

Signal Regulatory Protein α Negatively Regulates β2 Integrin-Mediated Monocyte Adhesion,Transendothelial Migration and Phagocytosis

Background

Signal regulate protein α (SIRPα) is involved in many functional aspects of monocytes. Here we investigate the role of SIRPα in regulating β₂ integrin-mediated monocyte adhesion, transendothelial migration (TEM) and phagocytosis.

Methodology/Principal Findings

THP-1 monocytes/macropahges treated with advanced glycation end products (AGEs) resulted in a decrease of SIRPα expression but an increase of β₂ integrin cell surface expression and β₂ integrin-mediated adhesion to tumor necrosis factor-α (TNFα)–stimulated human microvascular endothelial cell (HMEC-1) monolayers. In contrast, SIRPα overexpression in THP-1 cells showed a significant less monocyte chemotactic protein-1 (MCP-1)–triggered cell surface expression of β₂ integrins, in particular CD11b/CD18. SIRPα overexpression reduced β₂ integrin-mediated firm adhesion of THP-1 cells to either TNFα–stimulated HMEC-1 monolayers or to immobilized intercellular adhesion molecule-1 (ICAM-1). SIRPα overexpression also reduced MCP-1–initiated migration of THP-1 cells across TNFα–stimulated HMEC-1 monolayers. Furthermore, β₂ integrin-mediated THP-1 cell spreading and actin polymerization in response to MCP-1, and phagocytosis of bacteria were both inhibited by SIRPα overexpression.

Conclusions/Significance

SIRPα negatively regulates β₂ integrin-mediated monocyte adhesion, transendothelial migration and phagocytosis, thus may serve as a critical molecule in preventing excessive activation and accumulation of monocytes in the arterial wall during early stage of atherosclerosis.     

Cadmium Induces PC12 Cells Apoptosis via an Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase-Mediated Mitochondrial Apoptotic Pathway

To investigate the role of mitogen-activated protein kinase (MAPK) and downstream events in cadmium (Cd)-induced neuronal apoptosis executed via the mitochondrial apoptotic pathway, this study used the PC-12 cell line as a neuronal model. The result showed that Cd significantly decreased cell viability and the Bcl-2?/?Bax ratio and increased the percentage of apoptotic cells, release of cytochrome c, caspase-3, and poly(ADP-ribose) polymerase cleavage, and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G. In addition, exposure to Cd-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2?/?Bax ratio and cytochrome c release and suppressed caspase-3 and poly(ADP-ribose) polymerase cleavage and AIF and endonuclease G nuclear translocation. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathway played an important role in Cd-induced PC12 cells apoptosis.     

Real Time Measures of Prestin Charge and Fluorescence during Plasma Membrane Trafficking Reveal Sub-Tetrameric Activity

Prestin (SLC26a5) is the outer hair cell integral membrane motor protein that drives cochlear amplification, and has been described as an obligate tetramer. We studied in real time the delivery of YFP-prestin to the plasma membrane of cells from a tetracycline-inducible cell line. Following the release of temperature block to reinstate trans Golgi network delivery of the integral membrane protein, we measured nonlinear capacitance (NLC) and membrane fluorescence during voltage clamp. Prestin was delivered exponentially to the plasma membrane with a time constant of less than 10 minutes, with both electrical and fluorescence methods showing high temporal correlation. However, based on disparity between estimates of prestin density derived from either fluorescence or NLC, we conclude that sub-tetrameric forms of prestin contribute to our electrical and fluorescence measures. Thus, in agreement with previous observations we find that functional prestin is not an obligate tetramer.     

Activation In Vitro of Mutant MoFe Proteins from Azotobacter vinelandii by Reconstituent Solutions

nifB-MoFe protein （nifB-Av1）, AnifE MoFe protein （△nifE Av1） and AnifZ MoFe protein （△nifZ Av1） were obtained by chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from nifB point-mutated, nifE deleted and nifZ deleted mutant stains （UW45, DJ35 and DJ194） of Azotobacter vinelandii Llpmann, respectively. When complemented with nltrogenase Fe protein （Av2）, AnifZ Av1 had partial activity and both nifB-Avl and △nifE Av1 had hardly any activity, but could be obviously activated by FeMoco extracted from wild-type MoFe protein （OP Av1） or △nifZ Av1. After being Incubated with excess O-phenanthrollne （O-phen） for 150 mln at 30 ℃ and subjected to chromatography on a Sephadex G-25 column In an Ar atmosphere, nifB- Av1C, △nifE Av1C and △nifZ Av1C were obtained, respectively. Based on a calculation of Fe atoms In the Ophen-Fe compound with ε 512nm = 11 100, lost Fe atoms of nifB-Av1, △nifE Av1 and △nifZ Av1 were estimated to be 1.35, 2.89 and 8.44 per molecule of protein, respectively. As a result of the Fe loss, △nifZ Av1 loses Its original activity. In the presence of both MgATP and Av2, these Fe-loslng proteins, but not the original proteins untreated with O-phen, could be significantly activated by reconstltuent solution （RS） composed of dlthlothreltol, ferric homocltrate, Na2S and Na2MoO4, or K2CrO4, or KMnO4. But In the absence of MgATP or Av2, the activation did not occur, with the exception that △nifZ AvlC was partially activated, and the activity was only 17%. These findings Indicate that： （I） △nifZ Avl with half P-cluster content Is somewhat different from FeMoco-deflclent nifB-Avl and ,△nifE Av1 with respect to protein conformation either before or after treatment with O-phen; （11） full activation of these proteins with RS requires pretreatment with O-phen and the simultaneous presence of MgATP and Av2.     

Differentially Expressed Genes in EEC and LMS Syndromes

Objectives

Ectrodactyly ectodermal dysplasia cleft lip/palate (EEC) syndrome and limb-mammary syndrome (LMS) share a similar phenotype and the same pathogenic gene, which complicates the ability to distinguish between these diagnoses. The current study aims to identify a potential and practical clinical biomarker to distinguish EEC from LMS.

Methods

Two EEC pedigrees and one LMS pedigree that have been previously reported were reanalyzed. After confirmation of the causative mutations for these new patients, whole-genome expression microarray analysis was performed to assess the molecular genetic changes in these families.

Results

Five new patients with classic symptoms were reported, and these individuals exhibited the same mutation as their relatives (c.812 G>C; c.611G>A; and c.680G>A). According to the whole genome expression results, the EEC patients exhibited different gene expression characteristics compared with the LMS patients. More than 5,000 genes were differentially expressed (changes >2 or <0.5-fold) among the EEC patients, LMS patients and healthy individuals. The top three altered pathways have been implicated in apoptosis, the hematopoietic cell lineage and the Toll-like receptor signaling pathway.

Conclusion

Our results provide additional clinical and molecular information regarding EEC and LMS and suggest that peripheral blood cytokines may represent a promising clinical biomarker for the diagnosis of these syndromes.     

A N‐(2‐hydroxyethyl)piperazine dangled 2,5‐diphenyl‐1,3,4‐oxadiazole‐based fluorescent sensor for selective relay recognition of Cu2+ and sulfide in water

A new 2,5‐diphenyl‐1,3,4‐oxadiazole‐based derivative (L) was synthesized and applied as a highly selective and sensitive fluorescent sensor for relay recognition of Cu²⁺ and S^2? in water (Tris–HCl 10 mM, pH = 7.0) solution. L exhibits an excellent selectivity to Cu²⁺ over other examined metal ions with a prominent fluorescence ‘turn‐off’ at 392 nm. L interacts with Cu²⁺ through a 1:2 binding stoichiometry with a detection limit of 4.8 × 10^–7 M. The on‐site formed L–2Cu²⁺ complex exhibits excellent selectivity to S^2? with a fluorescence ‘off–on’ response via a Cu²⁺ displacement approach. Copyright © 2016 John Wiley & Sons, Ltd.