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Summary: Preeclampsia (PE) is a leading cause of maternal and perinatal death. Accumulated evidence suggests that many long non‐coding RNAs (lncRNAs) are abnormally expressed in placentas from PE patients, and they may play functional roles in the development of PE. The present study aimed to investigate the functional role TUG1 in PE and explore the potential molecular mechanisms. Clinically, the expression of TUG1 in placental tissues from PE and normal controls was determined. In vitro, human trophoblast HTR‐8/SVneo and JAR cells were employed for loss or gain‐of‐function assays. Our results demonstrate that TUG1 was downregulated in PE placental tissues compared with normal controls. Moreover, downregulation of TUG1 inhibited the migration and invasion of trophoblast‐like cells. Bioinformatics analysis and functional assays showed that TUG1 interacted with miR‐204‐5p and negatively regulated the expression and function of miR‐204‐5p in trophoblast cells. Furthermore, TUG1‐mediated migration and invasion of trophoblast cells was regulated by miR‐204‐5p. Together, our results suggested that TUG1 regulates trophoblast migration and invasion partly through sponging miR‐204‐5p, and the TUG1/miR‐204‐5p axis could be a potential therapeutic target for the treatment of PE. [ABSTRACT FROM AUTHOR]

Miscarriage is the spontaneous loss of a pregnancy before the fetus reaching viability, including all pregnancy losses from the time of conception until 24 weeks of gestation. Three or more times of consecutive spontaneous miscarriages is defined as recurrent miscarriage (RM). The underlying causes cannot be confirmed in nearly 50% of RM patients. We investigated the differential expression of long noncoding RNAs (lncRNAs) in chorionic villi tissues of RM patients compared with normal women, and their possible involvement in the pathogenic pathways leading to RM. A total number of 1449 differentially expressed lncRNAs were identified from chorionic villi tissues of RM patients compared to normal women. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis revealed that these differentially expressed lncRNAs were involved in 26 biological pathways including 11 up-regulated and 15 down-regulated ones. Functional analysis showed that pathways of endocrine, immunity, ECM-receptor interactions and apoptosis are the major pathogenic mechanisms involved in the development of RM. Characterization of these lncRNAs in different pathways opened a new starting point for further investigating the epigenetic regulation mechanisms of lncRNAs in RM. [ABSTRACT FROM AUTHOR]

Background: PTPRD, encoding protein tyrosine phosphatases receptor type D, is located at chromosome 9p23–24.1, a loci frequently lost in many types of tumors. Recently, PTPRD has been proposed to function as a tumor suppressor gene. The current study aimed to investigate PTPRD expression and its prognostic significance in primary gastric adenocarcinoma. Methods and Results: Quantitative real time reverse transcription PCR (qRT-PCR) and western blotting were used to examine PTPRD expression in paired gastric tumourous and paracancerous tissues. Compared with the matched normal gastric mucosa tissues, both the mRNA (P = 0.0138) and protein (P = 0.0093) expression of PTPRD in fresh surgical specimens were significantly reduced. Clinicopathological and prognostic roles of PTPRD in gastric adenocarcinoma were investigated using immunohistochemistry with 513 paraffin-embedded gastric adenocarcinoma tissue blocks. Statistical analysis revealed that reduced PTPRD expression was significantly associated with T stage (P = 0.004), TNM stage (P<0.001) and tumor size (P = 0.003). Furthermore, Kaplan-Meier survival analysis revealed that low expression of PTPRD significantly correlated with poor survival of gastric cancer patients (P<0.001). Cox regression analysis confirmed PTPRD expression as independent predictor of the overall survival of gastric cancer patients. The MTT assay determined the effects of PTPRD on cell proliferation of MGC803 and GES1 cell lines. Restoring PTPRD expression in MGC803 cells significantly inhibited their growth rate. Silencing PTPRD expression by siRNA treatment in GES1 significantly enhanced cell proliferation compared with mock siRNA treatment. Methylation analysis of PTPRD promoter CpG island in 3 primary GC samples showed one case with partial methylation. Conclusions: These results indicated that PTPRD is a candidate tumour suppressor in gastric cancer. Thus, PTPRD may play an important role in gastric tumorigenesis and serve as a valuable prognostic marker of gastric adenocarcinoma. [ABSTRACT FROM AUTHOR]

• We identified mcr-1 gene in fifteen clinical isolates of Enterobacteriaceae. • The mcr-1 -harboring plasmids belonged to different incompatibility group (IncI2, IncX4 and IncP1). • One mcr-1- bearing isolate EC09 was susceptible to colistin with MIC of 0.5 mg/L. • EC09 demonstrated lowest transcription level of mcr-1 gene. Colistin resistance mediated by plasmids for their rapid dissemination in Enterobacteriaceae is alarming. We aimed to characterize the genetic features of mcr-1 gene as well as the role of promoters in gene expression and levels of colistin resistance among clinical isolates of Enterobacteriaceae. Methods: Clinical isolates of Enterobacteriaceae were collected in thirteen cities in China and screened for mcr-1 gene using polymerase chain reaction (PCR) amplification and sequencing. Antimicrobial susceptibility testing, transformation assay and plasmid sequencing, quantitative real-time PCR were performed for mcr -1-positive isolates. Promoter-probe vector pKK232-8 was utilized to assess the activity of the mcr-1 promoters. This study identified the mcr-1 gene in 15 clinical isolates of Enterobacteriaceae, among which 14 were resistant to colistin, with MICs of 4–8 mg/L, while one mcr-1- bearing isolate EC09 was susceptible to colistin, with an MIC of 0.5 mg/L. Moreover, mcr-1 -harbouring plasmids from 10 clinical isolates were transferrable via transformation and belonged to different incompatibility groups (IncI2 and IncX4). Plasmid pEC09 failed to transform and belonged to IncP1. A genetic structure containing the mcr-1-pap2 element was detected in these plasmids. EC09 demonstrated the lowest transcription level of mcr-1 gene, as determined by quantitative real-time PCR, which was in accordance with its susceptibility to colistin. Furthermore, the promoter activity of mcr-1 in pEC09 was the lowest, as determined by promoter-probe vector pKK232-8. Promoter variations were associated with expression of the mcr-1 gene and ultimately the levels of colistin resistance. [ABSTRACT FROM AUTHOR]

Preterm birth (PTB) is a live birth delivered before 37 weeks of gestation (GW). About one-third of PTBs result from the preterm premature rupture of membranes (PPROM). Up to the present, the pathogenic mechanisms underlying PPROM are not clearly understood. Here, we investigated the differential expression of long chain non-coding RNAs (lncRNAs) in placentas of PTBs with PPROM, and their possible involvement in the pathogenic pathways leading to PPROM. A total number of 1954, 776, and 1050 lncRNAs were identified with a microarray from placentas of PPROM (group A), which were compared to full-term birth (FTB) (group B), PTB (group C), and premature rupture of membrane (PROM) (group D) at full-term, respectively. Instead of investigating the individual pathogenic role of each lncRNA involved in the molecular mechanism underlying PPROM, we have focused on investigating the metabolic pathways and their functions to explore what is the likely association and how they are possibly involved in the development of PPROM. Six groups, including up-regulation and down-regulation in the comparisons of A vs. B, A vs. C, and A vs. D, of pathways were analyzed. Our results showed that 22 pathways were characterized as up-regulated 7 down-regulated in A vs. C, 18 up-regulated and 15 down-regulated in A vs. D, and 33 up-regulated and 7 down-regulated in A vs. B. Functional analysis showed pathways of infection and inflammatory response, ECM-receptor interactions, apoptosis, actin cytoskeleton, and smooth muscle contraction are the major pathogenic mechanisms involved in the development of PPROM. Characterization of these pathways through identification of lncRNAs opened new avenues for further investigating the epigenomic mechanisms of lncRNAs in PPROM as well as PTB. [ABSTRACT FROM AUTHOR]

Abstract: C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins consisting of at least one carbohydrate-recognition domain (CRD), which participate in nonself-recognition and clearance of invaders. In invertebrate, some multidomain C-type lectins have been identified, but their relative functions and binding mechanism are still meager. In the present study, A C-type lectin (CfLec-4) with four CRDs from Chlamys farreri was selected to investigate its possible function in innate immunity. The mRNA expression of CfLec-4 in hemocytes was significantly up-regulated (P <0.01) after the stimulations of β-glucan, LPS or PGN, and reached the highest expression level at 3, 6, 12h post-stimulation, which was 27.9-, 22.6- or 47.9-fold of that in blank group, respectively. Immunohistochemistry assay with polyclonal antibody specific for CfLec-4 revealed that the endogenous CfLec-4 was mainly located in the hepatopancreas, kidney and gonad of the scallops. The recombinant CfLec-4 (rCflec-4) could bind LPS, PGN, glucan and mannose in vitro, but could not bind LTA. Furthermore, rCflec-4 displayed a broader bacteria binding spectrum towards Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus as well as Gram-negative bacteria Escherichia coli, Vibrio anguillarum and fungi Pichia pastoris. Meanwhile, rCfLec-4 could significantly (P <0.01) enhance the phagocytosis of hemocytes in vitro. The results clearly suggested that four-CRD containing CfLec-4 not only served as PRR with wider recognition spectrum, but also functioned as an opsonin participating in the clearance of invaders in scallops. It could be inferred that the diversity and complexity of CRDs in C-type lectins endowed these receptors with comprehensive recognition spectrum and multiple immune functions against complex living environment. [Copyright &y& Elsevier]

Abstract: Iodothyronine deiodinase is responsible for the deiodination of thyroxine T4 to T3, and involved in the complex neuroendocrine-immune regulatory network to optimize the immune response in vertebrate. In this study, the full-length cDNA of an iodothyronine deiodinase (designated as CfDx) was cloned from scallop Chlamys farreri. The complete cDNA sequence of CfDx was of 1404 bp and contained an open reading frame of 900 bp encoding a polypeptide of 299 amino acids. The deduced amino acid sequence of CfDx contained an in-frame TGA stop codon probably encoding an essential selenocysteine (SeC), and there was a conserved region of about 15 amino acids surrounding the SeC residue. The CfDx mRNA transcripts were detected in all the tested tissues, including haemocytes, hepatopancreas, kidney, adductor muscle, gonad, gill and mantle, with the higher expression level in hepatopancreas and kidney. After LPS stimulation, the CfDx mRNA expression level in haemocytes increased significantly at 12 h (25.35-fold, P < 0.05) and 24 h (7.62-fold, P < 0.05), and the concentration of T3 in haemolymph increased significantly at 12 h (3.62 ng dL−1, P < 0.05) even the concentration of T4 did not change significantly. After scallop received an injection of 50 μg CfDx dsRNA, the expression level of CfDx mRNA in haemocytes began to decrease significantly at 36 h and maintained the relative low level (about 0.3-fold of the PBS control group) from 36 to72 h, but the ratio of T4/T3 in haemolymph began to increase at 36 h (2.31-fold, P < 0.05) and kept increasing from 36 to 72 h comparing with that in the PBS control group. These results indicated that CfDx was a homologue of iodothyronine deiodinase in scallop C. farreri, and it might be involved in the immunomodulation via regulating the concentration of thyroid hormones T3 and T4 in the haemolymph of scallop. [Copyright &y& Elsevier]

Abstract: Monoamine oxidase (MAO) is an essential enzyme in the catabolism of monoamines, and implicated in the immune response of vertebrates. In the present study, the full-length cDNA encoding monoamine oxidase (designated CfMAO) was cloned from Chlamys farreri by using rapid amplification of cDNA ends (RACE) approaches and expression sequence tag (EST) analysis. The open reading frame of CfMAO cDNA encoded 519 amino acids, which shared 73.9% similarity with that from oyster Crassostrea gigas, and 64.5–66.3% similarity with those from vertebrates. A conserved Amino_oxidase domain and a transmembrane domain were identified in the deduced CfMAO protein. The mRNA transcripts of CfMAO could be detected in all the tested tissues, including haemocytes, hepatopancreas, kidney, adductor muscle, mantle, gill and gonad. The mRNA expression of CfMAO was up-regulated significantly in haemocytes of scallops during 6–48h after bacteria Vibrio anguillarum challenge, and it reached the peak (25.9-fold, P <0.05) at 12h. The cDNA fragment encoding the mature peptide of CfMAO was expressed in the prokaryotic expression system, and 1mg of the recombinant protein (rCfMAO) could catalyze the deamination of 3665.59nmol serotonin, 2061.89nmol norepinephrine, 2104.85nmol epinephrine or 3040.34nmol dopamine within 1min (nmolmin−1 mg−1) in vitro. When the reaction mixture was coincubated with 0.1mmolL−1 MAO inhibitor clorgyline, its catalyzing activity to deaminize serotonin and dopamine was decreased significantly to 1603.69 and 955.39nmolmin−1 mg−1 (P <0.05) respectively. These results indicated that CfMAO, as the homologue of monoamine oxidase in scallop C. farreri, could modulate the immune response of scallops through the deamination of monoamines. [Copyright &y& Elsevier]

Abstract: As a transcription factor, Interleukin-2 enhancer binding factor 2 (ILF2) regulates IL-2 gene at level of transcription, splicing and translation in vertebrates and plays significant roles in immune system. In this study, an ILF2 homolog was identified from Chinese mitten crab Eriocheir sinensis (designated as EsILF) by expressed sequence tag (EST) analysis. The full-length cDNA of EsILF was of 2159bp, containing a 5′ untranslated region (UTR) of 90bp, a 3′ UTR of 866bp with a poly (A) tail, and an open reading frame (ORF) of 1203bp encoding a polypeptide of 400 amino acids with the predicted molecular weight of 44.3kDa, which shared 59.6–64.5% identities with vertebrate ILF2. There were a conserved N-terminal RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain in the primary structure, strongly suggesting that EsILF was a homolog of vertebrate ILF2. The mRNA of EsILF was constitutively expressed in all tested tissues of untreated crabs, including hepatopancreas, gill, gonad, muscle, heart and hemocytes, with highest expression in muscle and relative lower levels in hemocytes and gonad. The mRNA expression of EsILF in hemocytes was regulated differently after the crabs were stimulated by bacteria Listonella anguillarum and fungi Pichia pastoris GS115. The expression level was significantly (P <0.05) down-regulated to 0.35- and 0.29-fold compared with blank group at 6h and 12h after the stimulation of L. anguillarum, while P. pastoris significantly (P <0.05) up-regulated the expression level to 3.2-fold compared with the blank group at 6h post treatment. The results indicated that EsILF was involved in the immune response of crab toward both L. anguillarum and P. pastoris. [Copyright &y& Elsevier]

Abstract: C-type lectins are a superfamily of proteins that can bind pathogen-associated molecular patterns (PAMPs) and microorganisms through the recognition of carbohydrates, thus they are directly involved in innate defense mechanisms as part of the acute-phase response to infection. In this study, the cDNA of a novel C-type lectin (designated as AiCTL-7) was cloned from bay scallop Argopecten irradians by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of AiCTL-7 was of 651 bp containing a 525 bp open reading frame which encoded a signal peptide of 15 residues and a conserved carbohydrate-recognition domain (CRD) of 174 residues with the EPD and WSD motifs instead of the invariant EPN and WND motifs for determining the carbohydrate-binding specificity and constructing Ca2+-binding site 2 in vertebrates. The deduced amino acid sequence of AiCTL-7 CRD shared homology not only with the CRDs of C-type lectins in mollusks, but also with the fish lectin CRDs. The mRNA transcripts of AiCTL-7 were mainly detected in the tissue of hepatopancreas and also marginally detectable in kidney, gonad, hemocytes, heart and adductor of health scallop. After challenge with fungi Pichia pastoris GS115 and Gram-negative bacteria Listonella anguillarum, the relative expression level of AiCTL-7 was up-regulated significantly in hepatopancreas and hemocytes. The CRD of AiCTL-7 was recombined and expressed in Escherichia coli, and the recombinant protein (rAiCTL-7) aggregated P. pastoris remarkably in a Ca2+-dependent manner, and this agglutination could be inhibited by d-mannose, but not by d-galactose or β-1,3-glucan. However, rAiCTL-7 displayed no obvious agglutinating activity against L. anguillarum. These results collectively indicated that AiCTL-7 was involved in the primitive acute-phase response to microbial invasion as an important pattern recognition receptor (PRR) in the innate immune system of scallops. [Copyright &y& Elsevier]

Abstract: C-type lectins are a superfamily of Ca2+ dependent carbohydrate-recognition proteins which play significant diverse roles in nonself-recognition and clearance of invaders. In the present study, a C-type lectin (CfLec-2) from Zhikong scallop Chlamys farreri was selected to investigate its functions in innate immunity. The mRNA expression of CfLec-2 in hemocytes was significantly up-regulated (P <0.01) after scallops were stimulated by LPS, PGN or β-glucan, and reached the highest expression level at 12h post-stimulation, which was 72.5-, 23.6- or 43.8-fold compared with blank group, respectively. The recombinant CfLec-2 (designated as rCfLec-2) could bind LPS, PGN, mannan and zymosan in vitro, but it could not bind β-glucan. Immunofluorescence assay with polyclonal antibody specific for CfLec-2 revealed that CfLec-2 was mainly located in the mantle, kidney and gonad. Furthermore, rCfLec-2 could bind to the surface of scallop hemocytes, and then initiated cellular adhesion and recruited hemocytes to enhance their encapsulation in vitro, and this process could be specifically blocked by anti-rCfLec-2 serum. These results collectively suggested that CfLec-2 from the primitive deuterostome C. farreri could perform two distinct immune functions, pathogen recognition and cellular adhesion synchronously, while these functions were performed by collectins and selectins in vertebrates, respectively. The synchronous functions of pathogen recognition and cellular adhesion performed by CfLec-2 tempted us to suspect that CfLec-2 was an ancient form of C-type lectin, and apparently the differentiation of these two functions mediated by C-type lectins occurred after mollusk in phylogeny. [Copyright &y& Elsevier]

A C-type lectin (CfLec-3) from Chlamys farreri with three carbohydrate-recognition domains (CRDs) was selected to dissect the possible mechanisms of PAMP binding and functional differentiation of invertebrate lectins. CfLec-3 distributed broadly, and its mRNA expression in hemocytes increased significantly after stimulations with LPS, PGN or β-glucan, but not poly(I:C). The recombinant CfLec-3 (rCfLec-3) could bind PAMPs and several microbes. rCfLec-3 mediated hemocytes phagocytosis against Escherichia coli and encapsulation towards agarose beads. Obvious functional differentiation occurred among the three CRDs, as CRD1 exhibited higher activity to bind PAMPs, while CRD2/3 were expert in promoting hemocyte mediated opsonisation. The tertiary structural differences were suspected to be associated with such functional differentiation. PAMP binding abilities of CfLec-3 were determined by Ca2+-binding site 2 motif. When Pro in this motif of each CRD was mutated into Ser, their PAMP binding abilities were deprived absolutely. rCRD2 acquired mannan binding capability when its EPD was replaced by EPN, but lost when EPN in rCRD3 was changed into EPD. The Pro in Ca2+-binding site 2 was indispensable for PAMPs binding, while Asn was determinant for specific binding to mannan. It shed new insight into PAMPs binding mechanism of invertebrate C-type lectins and their functional differentiation. [ABSTRACT FROM AUTHOR]