Liraglutide improves atherosclerosis by regulating long non-coding RNA RMRP/miR-128-1-5P/Gadd45g axis.

OBJECTIVE: Recent studies indicated that long non-coding RNA is involved in the formation of atherosclerosis, which is the pathological basis of coronary heart disease. Here, we reported the function and regulatory mechanism of RMRP in coronary atherosclerosis. MATERIALS AND METHODS: qPCR was used to investigate the expression of IL-6, IL-8, RMRP, and miR-128-1-5P in coronary atherosclerosis and human vascular smooth muscle cells. Luciferase reporter assay confirmed the direct target effect of RMRP with miR-128-1-5P and miR-128-1-5P with Gadd45g on HEK293T. Western blot was used to detect protein expression in coronary atherosclerosis and human vascular smooth muscle cells. RESULTS: RMRP expression and Gadd45g protein level were up-regulated in coronary atherosclerosis and human vascular smooth muscle cells, while miR-128-1-5P was down-regulated. RMRP downregulation remarkably inhibited the expression of IL-6, IL-8, and apoptosis related protein in human vascular smooth muscle cells after ox-LDL treatment. In addition, bioinformatics analysis and Luciferase report experiments confirmed that RMRP was the direct target of miR-128-1-5P. Moreover, miR-128-1-5P inhibitor reserved evidently the effect of IL-6, IL-8, and apoptosis related protein induced RMRP-si after treatment of human vascular smooth muscle cells with ox-LDL, implying RMRP negatively and directly regulated miR-128-1-5P in coronary atherosclerosis. More importantly, RMRP silencing increased Gadd45g protein level in human vascular smooth muscle cells. The same results were found when miR-128 was upregulated. Meanwhile, Gadd45g-si extremely reversed the result of IL-6, IL-8, and apoptosis related protein induced miR-128-1-5P inhibitor after treatment of human vascular smooth muscle cells with ox-LDL and Luciferase report experiments showed that Gadd45g was a direct target of miR-128-1-5P, implying Gadd45g negatively and directly regulated miR-128-1-5P in coronary atherosclerosis. Furthermore, liraglutide restrained evidently the expression of IL-6, IL-8, and apoptosis related protein in coronary atherosclerosis. After all, these results showed that liraglutide could regulate RMRP/miR-128-1-5P/Gadd45g signal pathway to improve coronary atherosclerosis. CONCLUSIONS: Liraglutide could curb the expression of inflammatory cytokines and apoptosis related protein in coronary atherosclerosis by regulating RMRP/miR-128-1-5P/Gadd45g signaling pathway, providing a new potential strategy for the treatment of coronary atherosclerosis.

LncRNA SNHG16 promoted proliferation and inflammatory response of macrophages through miR-17-5p/NF-κB signaling pathway in patients with atherosclerosis.

OBJECTIVE: Atherosclerosis (AS) is a leading cause of cardiovascular disease (CVD), which is also the leading reason for mortality and morbidity worldwide. Growing evidence has shown that long non-coding RNAs (lncRNAs) play some roles in the development of AS; however, their roles remain unclear. In this study, we aimed to explore the function of lncRNA SNHG16 in AS. PATIENTS AND METHODS: qRT-PCR was used to detect the expressions of SNHG16 and miR-17-5p in AS serum samples and THP-1 macrophage-derived foam cells; the correlations were also analyzed. THP-1 macrophages were respectively treated with ox-LDL and several inflammatory factors to explore the affecting factors. What's more, SNHG16 overexpression lentivirus (LV-SNHG16) and downregulation lentivirus (LV-sh SNHG16) were purchased and infected into THP-1 macrophages. CCK8 assay was used to measure cell proliferation; the levels of IKKß, p-IkBα and p-p65 were detected by western blot (WB), and the levels of TNF-α, IL-1ß and IL-6 were detected by ELISA kit. Moreover, the luciferase assay was performed to explore the binding site of SNHG16 and miR-17-5p. Furthermore, we transfected miR-17-5p mimic and inhibitor into THP-1 macrophages; the proliferation, NF-κB signaling pathway factors and inflammatory factors were detected. Finally, JSH, a NF-κB signaling inhibitor, was added into LV-SNHG16 THP-1 macrophages and miR-17-5p inhibitor was transfected into LV-sh SNHG16 THP-1 macrophages to confirm that SNHG16 functions via miR-17-5p/ NF-κB signaling pathway. RESULTS: We found that SNHG16 was increased in AS patients and THP-1 macrophage-derived foam cells. Additionally, SNHG16 was increased in THP-1 macrophages by ox-LDL with time-dependence and dose-dependence. Furthermore, SNHG16 overexpression promoted proliferation, inflammatory response and increased levels of IKKß, p-IkBα, p-p65 in THP-1 macrophages, while SNHG16 downregulation led to the opposite results. Most importantly, we found that miR-17-5p expressions were significantly decreased in AS patients, which were negatively correlated with SNHG16. Luciferase gene reporter assay confirmed that SNHG16 could directly bind with miR-17-5p. Moreover, the proliferation, inflammatory factors and NF-κB signaling factors were significantly repressed after transfecting miR-17-5p mimic into THP-1 macrophages, while it led to the opposite results after transfecting miR-17-5p inhibitor. Then, we added JSH, a NF-κB signaling inhibitor, into LV-SNHG16 THP-1 macrophages; as a result, the increased cell proliferation rate and inflammatory response were both decreased. Finally, we found that the repressed cell proliferation, inflammatory factors and expressions of NF-κB signaling factors in LV-sh SNHG16 group were increased after co-transfected with miR-17-5p inhibitor. CONCLUSIONS: According to the results, we found that SNHG16 was upregulated in AS patients. Furthermore, we firstly found that SNHG16 was increased by ox-LDL in THP-1 macrophages. Most importantly, we uncovered a previously unappreciated SNHG16/miR-17-5p/ NF-κB signaling axis in promoting proliferation and inflammatory response in AS patients and THP-1 macrophages, which might provide a potential target for treating AS.

Single layer centrifugation improves the quality of frozen-thawed sperm of giant panda (Ailuropoda melanoleuca).

The reproductive capacity of captive giant pandas is poor and sperm cryopreservation is necessary for the reproduction and conservation of this species. Cryopreservation, however, leads to a significant decrease in sperm quality, including sperm motility, acrosome integrity and DNA integrity. In the present study, a method was developed based on colloid single layer centrifugation that could significantly improve frozen-thawed sperm quality. Two colloids were compared for post-thaw giant panda sperm preparation; the sperm samples had greater total motility (Colloid 1: 44.5 ±â€¯16.0%, Colloid 2: 42.4 ±â€¯10.1% compared with Control: 25.4 ±â€¯8.4%, P < 0.05), linear velocity (Colloid 1: 17.2 ±â€¯8.3 µm/s; Colloid 2: 19.0 ±â€¯9.0 µm/s compared with Control: 6.6 ±â€¯1.7 µm/s, P < 0.05) and membrane integrity (Colloid: 46.9 ±â€¯13.2%; Colloid 2: 54.3 ±â€¯5.7% compared with Control: 36.0 ±â€¯9.1%; P < 0.05). This method could be a useful tool to enable the use of poor quality sperm samples and benefit this population by using available genetic material.

Paternal age as an independent factor does not affect embryo quality and pregnancy outcomes of testicular sperm extraction-intracytoplasmic sperm injection in azoospermia.

This study was performed to evaluate the independent influence of paternal age affecting embryo development and pregnancy using testicular sperm extraction (TESE)-intracytoplasmic sperm injection (ICSI) in obstructive azoospermia (OA) and nonobstructive azoospermia (NOA). Paternal patients were divided into the following groups: ≤30 years, 31-35 years, 36-40 years, 41-45 years and ≥46 years. There were no differences in the rates of fertilisation or embryo quality according to paternal and maternal age. However, clinical pregnancy and implantation rates were significantly lower between those ≥46 years of paternal age compared with other age groups. Fertilisation rate was higher in the OA than the NOA, while embryo quality, pregnancy and delivery results were similar. Clinical pregnancy and implantation rates were significantly lower for patients ≥46 years of paternal age compared with younger age groups. In conclusion, fertilisation using TESE in azoospermia was not affected by the independent influence of paternal age; however, as maternal age increased concomitantly with paternal age, rates of pregnancy and delivery differed between those with paternal age <41 years and ≥46 years. Therefore, paternal age ≥46 years old should be considered when applying TESE-ICSI in cases of azoospermia, and patients should be advised of the associated low pregnancy rates.

Endpoints for screening thyroid cancer in the Republic of Korea: thyroid specialists' perspectives.

Cancer screening is aimed primarily at reducing deaths from the specific cancer. Thyroid-specific cancer mortality may be the most ambitious endpoint for obtaining estimates of screening effect. Numerous observations have accumulated over the years, indicating that thyroid cancer mortality endpoint has been difficult to study and is confounded by population heterogeneity, provision of randomization, and requirement of large cohorts with sufficiently long follow-up due to the excellent prognosis of the cancer. Accordingly, it may be important to reconsider how to best measure thyroid cancer screening efficacy. Recommendations against thyroid cancer screening should be based upon trials designed to evaluate its effectiveness not only in significant reduction in cancer mortality, but also of other distinct endpoints. It is desirable to evaluate derivative endpoints that can reliably predict reductions in mortality. The term "derivative" means a variable that is related to the true endpoint and is likely to be observable before the primary endpoint. Derivative endpoints may include thyroid cancer incidence, the proportion of early-stage tumors detected, more treatable stage, the identification of small tumors (to maintain in observation), decrease in the number of people who develop metastatic disease, the increased chance of lesser extent surgery, and the application of minimally invasive approaches, as well as no need for lifelong thyroid replacement therapy, a consistent follow-up, low-dose or no RAI administration and risk factor assessments where case findings should be continuous. The Korean guidelines for thyroid cancer national-level screening were published by a relevant group of multidisciplinary thyroid experts. It was concluded that the evidence is insufficient to balance the benefits and harms of thyroid cancer screening. However, the paper seems to raise the necessary investments in future research and demand a complete analysis for derivative endpoints, and offer screening participants with complete information necessary to make decisions that will provide them with the most value when a small thyroid cancer is screen-identified.

Serum troponin I concentrations assessed 18-24 hours after coronary artery bypass grafting are significant predictors of early patient prognosis.

OBJECTIVE: Coronary heart disease (CHD) is a frequent medical condition in developed countries and is one of the most serious diseases threatening patients' lives. Perioperative myocardial infarction is the major cause of perioperative cardiac death and cardiac arrest, but is difficult to be precisely identified by observing clinical symptoms or assessing cardiac enzyme levels or by ECG examination. Therefore, assessment of patient prognosis requires reliable predictors. In this regard, we tested the prognostic value of serum troponin I (TnI) concentrations. PATIENTS AND METHODS: 98 patients undergoing elective simple off-pump coronary artery bypass grafting were recruited. Venous blood samples were collected within 3-5 hours, 18-24 hours, and 36-48 hours post-operation, and associations of TnI concentrations with early outcomes measures (duration of assisted ventilation, length of stay in the ICU, length of postoperative stay, administration of antihypotensive medications, use of intra-aortic balloon pump, and ECG abnormalities) were evaluated. Correlations of postoperative TnI concentrations with the outcomes measures were analyzed by using median TnI concentrations as the cut-off value. RESULTS: TnI concentrations assessed within 18-24 hours post-operation showed significant associations with most tested outcome measures (p < 0.05 for four out of five comparisons). Furthermore, after building ROC curves, the highest AUC values (> 0.9) were also observed for TnI1 concentrations assessed within this time frame. The optimal cutoff value for TnI concentration was 1.78 ng/ml. CONCLUSIONS: TnI concentrations assessed within 18-24 hours after elective off-pump coronary artery bypass grafting can effectively predict early patient prognosis.

KAT5-mediated SOX4 acetylation orchestrates chromatin remodeling during myoblast differentiation.

Transcription factor SOX4 has been implicated in skeletal myoblast differentiation through the regulation of Cald1 gene expression; however, the detailed molecular mechanism underlying this process is largely unknown. Here, we demonstrate that SOX4 acetylation at lysine 95 by KAT5 (also known as Tip60) is essential for Cald1 promoter activity at the onset of C2C12 myoblast differentiation. KAT5 chromodomain was found to facilitate SOX4 recruitment to the Cald1 promoter, which is involved in chromatin remodeling at the promoter. Chromatin occupancy analysis of SOX4, KAT5, and HDAC1 indicated that the expression of putative SOX4 target genes during C2C12 myoblast differentiation is specifically regulated by the molecular switching of the co-activator KAT5 and the co-repressor HDAC1 on SOX4 transcriptional activation.

Outcome of percutaneous transluminal angioplasty in diabetic patients with critical limb ischaemia.

OBJECTIVE: We investigated the clinical outcome of percutaneous transluminal angioplasty (PTA) which has not been fully established in diabetic patients with critical limb Ischaemia (CLI) compared with non-diabetics. DESIGN AND PATIENTS: A total of 73 limbs of 52 patients (50 limbs of 34 diabetic patients and 23 limbs of 18 non-diabetics) who underwent PTA for CLI (Rutherford-Becker category 4, 5 or 6) were enrolled. Rates of amputation and restenosis, and ankle brachial index (ABI), were assessed before and after PTA during a 36-month follow-up period. RESULTS: Diabetic patients had a higher rate of major amputations after PTA (10 vs. 0%, P<0.05); however, total amputation (12.0 vs. 8.7%, P=0.62) and restenosis rates (4.0 vs. 8.7%, P=0.38) were not significantly different compared with non-diabetic patients. ABI at 3 months after PTA was significantly improved in both diabetic and non-diabetic patients (0.70±0.20 vs. 0.93±0.19, P<0.01 in diabetic patients; 0.69±0.25 vs. 0.92±0.17, P<0.01 in non-diabetics). Improved ABI was maintained for 36 months in both groups and did not show a significant difference (0.88±0.21 vs. 0.89±0.20, P=0.89). CONCLUSION: Our results, showing that the outcome of PTA in diabetic patients is not inferior to that in non-diabetics, suggest the potential benefit of primary PTA, instead of bypass surgery, for CLI in diabetic patients who are at high risk of perioperative complications.

Cross-species amplification of Bovidae microsatellites and low diversity of the endangered Korean goral.

The Korean goral (Nemorhaedus caudatus) is an endangered species of wild goat. The conservation and management of this species could benefit from a better understanding of its genetic diversity and structure. Cross-species amplification of 34 Bovidae microsatellite loci was tested on a panel of 6 Korean gorals and 10 domestic goats. After polymerase chain reaction (PCR) optimization, 29 (85.3%) microsatellite loci amplified successfully for the Korean gorals and 27 (79.4%) for the domestic goats. Of the amplified products, 16 (55.2%) were polymorphic in the Korean goral and 22 (81.5%) in domestic goats. Nei's unbiased mean heterozygosity and mean allele number per locus were, respectively, 0.356 and 2.6 in the Korean goral and 0.636 and 4.8 in domestic goats. Low genetic diversity in the Korean gorals observed in this preliminary microsatellite survey suggests an urgent need for further detailed study of genetic diversity in Korean goral populations and a population management strategy based on these studies. Current results of cross-species amplification of domestic Bovidae microsatellites could be employed for the necessary population genetic studies on the Korean goral and other endangered Caprinae species.

Identification and characterization of a novel transcriptional regulator, MatR, for malonate metabolism in Rhizobium leguminosarum bv. trifolii.

A novel gene, matR, located upstream of matABC, transcribed in the opposite direction, and encoding a putative regulatory protein by sequence analysis was discovered from Rhizobium leguminosarum bv. trifolii. The matA, matB, and matC genes encode malonyl-CoA decarboxylase, malonyl-CoA synthetase, and a presumed malonate transporter, respectively. Together, these enzymes catalyze the uptake and conversion of malonate to acetyl-CoA. The deduced amino-acid sequence of matR showed sequence similarity with GntR from Bacillus subtilis in the N-terminal region encoding a helix-turn-helix domain. Electrophoretic mobility shift assay indicated that MatR bound to a fragment of DNA corresponding to the mat promoter region. The addition of malonate or methylmalonate increased the association of MatR and DNA fragment. DNase I footprinting assays identified a MatR binding site encompassing 66 nucleotides near the mat promoter. The mat operator region included an inverted repeat (TCTTGTA/TACACGA) centered -46.5 relative to the transcription start site. Transcriptional assays, using the luciferase gene, revealed that MatR represses transcription from the mat promoter and malonate alleviates MatR-mediated repression effect on the expression of Pmat-luc+ reporter fusion.

The active site and substrates binding mode of malonyl-CoA synthetase determined by transferred nuclear Overhauser effect spectroscopy, site-directed mutagenesis, and comparative modeling studies.

The active sites and substrate bindings of Rhizobium trifolii molonyl-CoA synthetase (MCS) catalyzing the malonyl-CoA formation from malonate and CoA have been determined based on NMR spectroscopy, site-directed mutagenesis, and comparative modeling methods. The MCS-bound conformation of malonyl-CoA was determined from two-dimensional-transferred nuclear Overhauser effect spectroscopy data. MCS protein folds into two structural domains and consists of 16 alpha-helices, 24 beta-strands, and several long loops. The core active site was determined as a wide cleft close to the end of the small C-terminal domain. The catalytic substrate malonate is placed between ATP and His206 in the MCS enzyme, supporting His206 in its catalytic role as it generates reaction intermediate, malonyl-AMP. These findings are strongly supported by previous biochemical data, as well as by the site-directed mutagenesis data reported here. This structure reveals the biochemical role as well as the substrate specificity that conservative residues of adenylate-forming enzymes have.

Identification of residues essential for a two-step reaction by malonyl-CoA synthetase from Rhizobium trifolii.

Malonyl-CoA synthetase (MCS) catalyses the formation of malonyl-CoA in a two-step reaction consisting of the adenylation of malonate with ATP followed by malonyl transfer from malonyl-AMP to CoA. In order to identify amino acid residues essential for each step of the enzyme, catalysis based on chemical modification and database analysis, Arg-168, Lys-170, and His-206 were selected for site-directed mutagenesis. Glutathione-S-transferase-fused enzyme (GST-MCS) was constructed and mutagenized to make R168G, K170M, R168G/K170M and H206L mutants, respectively. The MCS activity of soluble form GST-MCS was the same as that of wild-type MCS. Circular dichroism spectra for the four mutant enzymes were nearly identical to that for the GST-MCS, indicating that Arg-168, Lys-170 and His-206 are not important for conformation but presumably for substrate binding and/or catalysis. HPLC analysis of products revealed that the intermediate malonyl-AMP is not accumulated during MCS catalysis and that none of the mutant enzymes accumulated it either. Kinetic analysis of the mutants revealed that Lys-170 and His-206 play a critical role for ATP binding and the formation of malonyl-AMP, whereas Arg-168 is critical for formation of malonyl-CoA and specificity for malonyl-AMP. Molecular modelling based on the crystal structures of luciferase and gramicidin S synthetase 1 provided MCS structure which could fully explain all these biochemical data even though the MCS model was generated by comparative modelling.

A gene cluster encoding malonyl-CoA decarboxylase (MatA), malonyl-CoA synthetase (MatB) and a putative dicarboxylate carrier protein (MatC) in Rhizobium trifolii--cloning, sequencing, and expression of the enzymes in Escherichia coli.

A gene cluster consisting of three consecutive genes, matABC, was isolated using a probe prepared from amino acid sequence information of Rhizobium trifolii malonyl-CoA synthetase, and was subsequently sequenced. The sequences of matA and matB were overlapped by four base pairs, whereas the intergenic region between matB and matC had 95 base pairs. The upstream region contained DNA sequences which are typical for an Escherichia coli sigma70 promoter, and no other open reading frame was found within 400 bp downstream of matC. The ribosome-binding sites were found 7 to 12 base pairs upstream of each gene. MatA gene encoded a polypeptide of 462 amino acid residues, with deduced molecular mass of 51414 Da. A glutathione-S-transferase-MatA fusion protein has been purified and MatA was shown to have an intrinsic malonyl-CoA decarboxylase activity (Km = 0.47 mM; Vmax = 52 micromol x min(-1) x mg(-1)). MatB encoded a polypeptide of 504 amino acid residues with deduced molecular mass of 54612 Da. MatB was also purified from E. coli transformant carrying the gene cluster. The enzyme was essentially indistinguishable from the wild-type malonyl-CoA synthetase of R. trifolii by the criteria of polyacrylamide gel electrophoresis and biochemical properties. MatC encoded a 46453-Da protein with a high content of hydrophobic residues. The deduced amino acid sequences of matC showed identity to some extent with anaerobic C4-dicarboxylate carrier proteins from E. coli (25%) and Haemophilus influenzae (17%). MatC protein appears to be an integral membrane protein that could function as a malonate carrier. The formation of acetyl-CoA and malonyl-CoA from malonate was confirmed by thin-layer chromatographic analysis. These results strongly suggest that the gene cluster encodes proteins involved in the malonate-metabolizing system, malonate-->malonyl-CoA-->acetyl-CoA, in R. trifolii and that the metabolic pathway in the malonate-rich clover nodule might play an important role in symbiosis.

High-performance reversed-phase chromatographic mapping of 2-pyridylamino derivatives of xyloglucan oligosaccharides.

Xyloglucan oligosaccharides from cotton cell walls and tamarind seeds were derivatized with 2-aminopyridine and subsequently separated by reversed-phase chromatography (r.p.c.) using an octadecylsilyl silica stationary phase and aqueous-organic eluents with 0.01% (v/v) trifluoroacetic acid. The chromatographic behavior of the 2-pyridylamino derivatives of xyloglucan oligosaccharides was examined under a wide range of elution conditions, including gradient steepness and shape, initial acetonitrile concentration in the eluent, and pore size of the r.p.c. packings. Relatively steep acetonitrile gradients resulted in poor resolution of the different xyloglucan fragments, which is believed to be the result of acetonitrile-induced conformational changes. Under these circumstances the elution order of the derivatized xyloglucan oligosaccharides was such that the smaller fragments eluted from the column before the larger ones. R.p.c. packing with a 70-A pore size necessitated relatively high acetonitrile concentration in the eluent when compared with 300-A stationary phase. The r.p.c. mapping of 2-pyridylamino derivatives of xyloglucan oligosaccharides was best achieved when both a wide-pore octadecyl-silyl silica stationary phase and a shallow gradient with consecutive linear segments of increasing acetonitrile concentration in the eluent were employed. This combination yielded rapid r.p.c. maps of the xyloglucan fragments from different sources with high separation efficiencies and concomitantly high resolution. The effects of the nature of the sugar residues in the xyloglucan oligomers and their degree of branching on r.p.c. retention and selectivity are also highlighted.