The Na+ /Ca2+ antiporter slr0681 affects carotenoid production in Synechocystis sp. PCC 6803 under high-light stress.

BACKGROUND: Carotenoids play key roles in photosynthesis and are widely used in foods as natural pigments, antioxidants, and health-promoting compounds. Enhancing carotenoid production in microalgae via biotechnology has become an important area of research. RESULTS: We knocked out the Na+ /Ca2+ antiporter gene slr0681 in Synechocystis sp. PCC 6803 via homologous recombination and evaluated the effects on carotenoid production under normal (NL) and high-light (HL) conditions. On day 7 of NL treatment in calcium ion (Ca2+ )-free medium, the cell density of Δslr0681 decreased by 29% compared to the wild type (WT). After 8 days of HL treatment, the total carotenoid contents decreased by 35% in Δslr0681, and the contents of individual carotenoids were altered: myxoxanthophyll, echinenone, and ß-carotene contents increased by 10%, 50%, and 40%, respectively, while zeaxanthin contents decreased by ~40% in Δslr0681 versus the WT. The expression patterns of carotenoid metabolic pathway genes also differed: ipi expression increased by 1.2- to 8.5-fold, whereas crtO and crtR expression decreased by ~90% and 60%, respectively, in ∆slr0681 versus the WT. In addition, in ∆slr0681, the expression level of psaB (encoding a photosystem I structural protein) doubled, whereas the expression levels of the photosystem II genes psbA2 and psbD decreased by ~53% and 84%, respectively, compared to the WT. CONCLUSION: These findings suggest that slr0681 plays important roles in regulating carotenoid biosynthesis and structuring of the photosystems in Synechocystis sp. This study provides a theoretical basis for the genetic engineering of microalgae photosystems to increase their economic benefits and lays the foundation for developing microalgae germplasm resources with high carotenoid contents. © 2023 Society of Chemical Industry.

Master regulator NtrC controls the utilization of alternative nitrogen sources in Pseudomonas stutzeri A1501.

Pseudomonas stutzeri A1501 is a model strain used to study associative nitrogen fixation, and it possesses the nitrogen regulatory NtrC protein in the core genome. Nitrogen sources represent one of the important factors affecting the efficiency of biological nitrogen fixation in the natural environment. However, the regulation of NtrC during nitrogen metabolism in P. stutzeri A1501 has not been clarified. In this work, a phenotypic analysis of the ntrC mutant characterized the roles of NtrC in nitrogen metabolism and the oxidative stress response of P. stutzeri A1501. To systematically identify NtrC-controlled gene expression, RNA-seq was performed to further analyse the gene expression differences between the wild-type strain and the ∆ntrC mutant under nitrogen fixation conditions. A total of 1431 genes were found to be significantly altered by ntrC deletion, among which 147 associative genes had NtrC-binding sites, and the pathways for nitrogen fixation regulation, nitrogenous compound acquisition and catabolism and nitrate assimilation were discussed. Furthermore, the oxidative stress-related gene (katB), which was upregulated by ntrC deletion, was suggested to be a potential target gene of NtrC, thus highlighting the importance of NtrC in nitrogenase protection against oxygen damage. Based on these findings, we propose that NtrC is a high-ranking element in the regulatory network of P. stutzeri A1501 that controls a variety of nitrogen metabolic and oxidative stress responsive traits required for adaptation to complex rhizosphere environments.

Role of N,N-Dimethylglycine and Its Catabolism to Sarcosine in Chromohalobacter salexigens DSM 3043.

Chromohalobacter salexigens DSM 3043 can grow on N,N-dimethylglycine (DMG) as the sole C, N, and energy source and utilize sarcosine as the sole N source under aerobic conditions. However, little is known about the genes and enzymes involved in the conversion of DMG to sarcosine in this strain. In the present study, gene disruption and complementation assays indicated that the csal_0990, csal_0991, csal_0992, and csal_0993 genes are responsible for DMG degradation to sarcosine. The csal_0990 gene heterologously expressed in Escherichia coli was proven to encode an unusual DMG dehydrogenase (DMGDH). The enzyme, existing as a monomer of 79 kDa with a noncovalently bound flavin adenine dinucleotide, utilized both DMG and sarcosine as substrates and exhibited dual coenzyme specificity, preferring NAD+ to NADP+ The optimum pH and temperature of enzyme activity were determined to be 7.0 and 60°C, respectively. Kinetic parameters of the enzyme toward its substrates were determined accordingly. Under high-salinity conditions, the presence of DMG inhibited growth of the wild type and induced the production and accumulation of trehalose and glucosylglycerate intracellularly. Moreover, exogenous addition of DMG significantly improved the growth rates of the four DMG- mutants (Δcsal_0990, Δcsal_0991, Δcsal_0992, and Δcsal_0993) incubated at 37°C in S-M63 synthetic medium with sarcosine as the sole N source. 13C nuclear magnetic resonance (13C-NMR) experiments revealed that not only ectoine, glutamate, and N-acetyl-2,4-diaminobutyrate but also glycine betaine (GB), DMG, sarcosine, trehalose, and glucosylglycerate are accumulated intracellularly in the four mutants.IMPORTANCE Although N,N-dimethylglycine (DMG) dehydrogenase (DMGDH) activity was detected in cell extracts of microorganisms, the genes encoding microbial DMGDHs have not been determined until now. In addition, to our knowledge, the physiological role of DMG in moderate halophiles has never been investigated. In this study, we identified the genes involved in DMG degradation to sarcosine, characterized an unusual DMGDH, and investigated the role of DMG in Chromohalobacter salexigens DSM 3043 and its mutants. Our results suggested that the conversion of DMG to sarcosine is accompanied by intramolecular delivery of electrons in DMGDH and intermolecular electron transfer between DMGDH and other electron acceptors. Moreover, an unidentified methyltransferase catalyzing the production of glycine betaine (GB) from DMG but sharing no homology with the reported sarcosine DMG methyltransferases was predicted to be present in the cells. The results of this study expand our understanding of the physiological role of DMG and its catabolism to sarcosine in C. salexigens.

Glycine Betaine Monooxygenase, an Unusual Rieske-Type Oxygenase System, Catalyzes the Oxidative N-Demethylation of Glycine Betaine in Chromohalobacter salexigens DSM 3043.

Although some bacteria, including Chromohalobacter salexigens DSM 3043, can use glycine betaine (GB) as a sole source of carbon and energy, little information is available about the genes and their encoded proteins involved in the initial step of the GB degradation pathway. In the present study, the results of conserved domain analysis, construction of in-frame deletion mutants, and an in vivo functional complementation assay suggested that the open reading frames Csal_1004 and Csal_1005, designated bmoA and bmoB, respectively, may act as the terminal oxygenase and the ferredoxin reductase genes in a novel Rieske-type oxygenase system to convert GB to dimethylglycine in C. salexigens DSM 3043. To further verify their function, BmoA and BmoB were heterologously overexpressed in Escherichia coli, and 13C nuclear magnetic resonance analysis revealed that dimethylglycine was accumulated in E. coli BL21(DE3) expressing BmoAB or BmoA. In addition, His-tagged BmoA and BmoB were individually purified to electrophoretic homogeneity and estimated to be a homotrimer and a monomer, respectively. In vitro biochemical analysis indicated that BmoB is an NADH-dependent flavin reductase with one noncovalently bound flavin adenine dinucleotide (FAD) as its prosthetic group. In the presence of BmoB, NADH, and flavin, BmoA could aerobically degrade GB to dimethylglycine with the concomitant production of formaldehyde. BmoA exhibited strict substrate specificity for GB, and its demethylation activity was stimulated by Fe2+ Phylogenetic analysis showed that BmoA belongs to group V of the Rieske nonheme iron oxygenase (RO) family, and all the members in this group were able to use quaternary ammonium compounds as substrates.IMPORTANCE GB is widely distributed in nature. In addition to being accumulated intracellularly as a compatible solute to deal with osmotic stress, it can be utilized by many bacteria as a source of carbon and energy. However, very limited knowledge is presently available about the molecular and biochemical mechanisms for the initial step of the aerobic GB degradation pathway in bacteria. Here, we report the molecular and biochemical characterization of a novel two-component Rieske-type monooxygenase system, GB monooxygenase (BMO), which is responsible for oxidative demethylation of GB to dimethylglycine in C. salexigens DSM 3043. The results gained in this study extend our knowledge on the catalytic reaction of microbial GB degradation to dimethylglycine.

Establishment of a markerless gene deletion system in Chromohalobacter salexigens DSM 3043.

Chromohalobacter salexigens DSM 3043 can grow over a wide range of salinity, which makes it as an excellent model organism for understanding the mechanism of prokaryotic osmoregulation. Functional analysis of C. salexigens genes is an essential way to reveal their roles in cellular osmoregulation. However, the lack of an effective markerless gene deletion system has prevented construction of multiple gene deletion mutants for the members in the genus. Here, we report the development of a markerless gene deletion system in C. salexigens using allelic exchange method. In this system, the in vitro mutant allele of target gene was inserted into a pK18mobsacB-based integrative vector pMDC21, which contained a chloramphenicol resistance cassette as the positive selection marker and a sacB gene from Bacillus subtilis as the counterselectable marker. To validate this system, two single-gene deletion mutants and a double-gene deletion mutant were constructed. In addition, our results showed that growth of the merodiploids and sucrose screening at 25 °C were more effective to decrease the occurrence of spontaneous sucrose resistance colonies than at higher temperature (30 or 37 °C), and growth of the merodiploids in mineral salt medium instead of the complex medium was critical to increase the recovery rate of deletion mutants.

Evaluation of (188)Re-labeled NGR-VEGI protein for radioimaging and radiotherapy in mice bearing human fibrosarcoma HT-1080 xenografts.

Vascular endothelial growth inhibitor (VEGI) is an anti-angiogenic protein, which includes three isoforms: VEGI-174, VEGI-192, and VEGI-251. The NGR (asparagine-glycine-arginine)-containing peptides can specifically bind to CD13 (Aminopeptidase N) receptor which is overexpressed in angiogenic blood vessels and tumor cells. In this study, a novel NGR-VEGI fusion protein was prepared and labeled with (188)Re for radioimaging and radiotherapy in mice bearing human fibrosarcoma HT-1080 xenografts. Single photon emission computerized tomography (SPECT) imaging results revealed that (188)Re-NGR-VEGI exhibits good tumor-to-background contrast in CD13-positive HT-1080 tumor xenografts. The CD13 specificity of (188)Re-NGR-VEGI was further verified by significant reduction of tumor uptake in HT-1080 tumor xenografts with co-injection of the non-radiolabeled NGR-VEGI protein. The biodistribution results demonstrated good tumor-to-muscle ratio (4.98 ± 0.25) of (188)Re-NGR-VEGI at 24 h, which is consistent with the results from SPECT imaging. For radiotherapy, 18.5 MBq of (188)Re-NGR-VEGI showed excellent tumor inhibition effect in HT-1080 tumor xenografts with no observable toxicity, which was confirmed by the tumor size change and hematoxylin and eosin (H&E) staining of major mouse organs. In conclusion, these data demonstrated that (188)Re-NGR-VEGI has the potential as a theranostic agent for CD13-targeted tumor imaging and therapy.

A direct comparison of tumor angiogenesis with 68Ga-labeled NGR and RGD peptides in HT-1080 tumor xenografts using microPET imaging.

Peptides containing asparagine-glycine-arginine (NGR) and arginine-glycine-aspartic acid (RGD) sequence are being developed for tumor angiogenesis-targeted imaging and therapy. The aim of this study was to compare the efficacy of NGR- and RGD-based probes for imaging tumor angiogenesis in HT-1080 tumor xenografts. Two PET probes, (68)Ga-NOTA-G3-NGR2 and 68Ga-NOTA-G3-RGD2, were successfully prepared. In vitro stability, partition coefficient, tumor cell binding, as well as in vivo biodistribution properties were also analyzed for both PET probes. The results revealed that the two probes were both hydrophilic and stable in vitro and in vivo, and they were excreted predominately and rapidly through the kidneys. For both probes, the higher tumor uptake and lower accumulation in vital organs were determined. No significant difference between two probes was observed in terms of tumor uptake and the in vivo biodistribution properties. We concluded that these two probes are promising in tumor angiogenesis imaging. 68Ga-NOTA-G3-NGR2 has the potential as an alternative for PET imaging in patients with fibrosarcoma, and it may offer an opportunity to noninvasively monitor CD13-targeted therapy.

68Ga-labeled cyclic NGR peptide for microPET imaging of CD13 receptor expression.

Peptides containing the asparagines-glycine-arginine (NGR) motif have been identified as specific ligands binding to CD13/aminopeptidase N (APN) receptor, a tumor neovascular biomarker. In this study, we synthesized a novel NGR-containing peptide (NOTA-G3-NGR), and labeled NOTA-G3-NGR with (68)Ga (t1/2 = 67.7 min). The resulting (68)Ga-NOTA-G3-NGR peptide was subject to in vitro and in vivo characterization. The microPET imaging results revealed that the (68)Ga-NOTA-G3-NGR peptide exhibits rapid and specific tumor uptake, and high tumor-to-background contrast in a subcutaneous HT-1080 fibrosarcoma mouse model. We concluded that the (68)Ga-NOTA-G3-NGR peptide has potential in the diagnosis of CD13-targeted tumor angiogenesis.

Integrated Hfq-interacting RNAome and transcriptomic analysis reveals complex regulatory networks of nitrogen fixation in root-associated Pseudomonas stutzeri A1501.

The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated diazotrophs; however, its target RNAs and the mechanisms underlying nitrogen fixation remain largely unknown. Here, we used enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing to identify hundreds of Hfq-binding RNAs probably involved in nitrogen fixation, carbon substrate utilization, biofilm formation, and other functions. Collectively, these processes endow strain A1501 with the requisite capabilities to thrive in the highly competitive rhizosphere. Our findings revealed a previously uncharted landscape of Hfq target genes. Notable among these is nifM, encoding an isomerase necessary for nitrogenase reductase solubility; amtB, encoding an ammonium transporter; oprB, encoding a carbohydrate porin; and cheZ, encoding a chemotaxis protein. Furthermore, we identified more than 100 genes of unknown function, which expands the potential direct regulatory targets of Hfq in diazotrophs. Our data showed that Hfq directly interacts with the mRNA of regulatory proteins (RsmA, AlgU, and NifA), regulatory ncRNA RsmY, and other potential targets, thus revealing the mechanistic links in nitrogen fixation and other metabolic pathways. IMPORTANCE: Numerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing was employed to identify the Hfq-binding sites and potential targets in the root-associated Pseudomonas stutzeri A1501 and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown Hfq interactions with various regulatory proteins' mRNA and their potential targets at the posttranscriptional level. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.

Advanced treatment of secondary effluent from wastewater treatment plant by a newly isolated microalga Desmodesmus sp. SNN1.

Secondary effluents contain considerable amounts of nitrogen and phosphorous, which if dumped untreated can cause eutrophication of the receiving water bodies. Microalgae can remove these nutrients and other pollutants from the wastewater effluents and play an effective role in the secondary effluent treatment. In this study, six microalgae strains (SNN1, SNN2, SNN3, SNN4, SNS1, and SNS2) were isolated and screened from the water and mud of Yingxue Lake of Shandong Jianzhu University, and their efficiencies for the removal of COD, NH4 +-N, TN, and TP in the secondary effluent were assessed. By comparing the growth performances and nutrient removal ability of algal strains in domestic sewage, we found that SNN1 (identified and named as Desmodesmus sp. SNN1) has the highest efficiency for biomass accumulation and sewage purification. Hence, the algal strain SNN1 was selected for further screening and optimization experiments. The strain showed higher biomass yield and better nutrient removal rate when the pH of secondary effluent was 9.0 and the initial inoculum concentration (optical density at 680 nm) of algal strain was 0.4. After 12 days of treatment, the concentrations of COD, NH4 +-N, TN, and TP in the secondary effluent were 31.79, 0.008, 8.631, and 0.069 mg/L, respectively. Therefore, SNN1 with the removal rates of 52.69% (COD), 99.99% (NH4 +-N), 89.09% (TN), and 94.64% (TP) displayed its high potential in nutrient removal. In addition, it also yielded 5.30 mg/L of chlorophyll a and 168.33 mg/L of lipids. These results demonstrated that this strain exhibited an effective treatment capacity for secondary effluent and microalgal oil production. This study is helpful to provide a strategy for the resource utilization of secondary effluent and the conservation of freshwater resources required by microalgae culture.

Arbuscular mycorrhizal fungi increased peanut (Arachis hypogaea L.) yield by changing the rhizosphere microbial community structure in saline-alkali soil.

Arbuscular mycorrhizal fungi (AMF) have demonstrated the potential to enhance the saline-alkali tolerance in plants. Nevertheless, the extent to which AMF can ameliorate the tolerance of salt-sensitive plants to alkaline conditions necessitates further investigation. The current study is primarily centered on elucidating the impact of AMF on the growth of the Huayu22 (H22) when cultivated in saline-alkaline soil. We leveraged DNA of rhizosphere microorganisms extracted from saline-alkali soil subjected to AMF treatment and conducted high-throughput sequencing encompassing 16S rRNA gene and ITS sequencing. Our findings from high-throughput sequencing unveiled Proteobacteria and Bacillus as the prevailing phylum and genus within the bacterial population, respectively. Likewise, the predominant fungal phylum and genus were identified as Ascomycota and Haematonectria. It is noteworthy that the relative abundance of Proteobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, and Ascomycota exhibited significant increments subsequent to AMF inoculation. Our investigation into soil enzyme activity revealed a remarkable surge post-AMF inoculation. Notably, the amounts of pathogen growth inhibitory enzymes and organic carbon degrading enzymes rise, as predicted by the putative roles of microbial communities. In saline-alkali soil, inoculation of AMF did boost the yield of H22. Notable improvements were observed in the weight of both 100 fruits and 100 grains, which increased by 20.02% and 22.30%, respectively. Conclusively, this study not only provides a theoretical framework but also furnishes empirical evidence supporting the utilization of AMF as a viable strategy for augmenting the yield of salt-sensitive plants grown in alkaline conditions.

New normal values not related to age and sex, of glomerular filtration rate by (99m)Tc-DTPA renal dynamic imaging, for the evaluation of living kidney graft donors.

The aim of this study was to investigate the normal values of glomerular filtration rate (GFR) by technetium-99m diaethylene-triamine-pentaacetic acid ((99m)Tc-DTPA) renal dynamic imaging for living kidney graft donors. In a total of 212 candidate donors, GFR was examined using (99m)Tc-DTPA renal dynamic imaging. Donors with GFR≥80mL/(min×1.73m(2)) and as low as with GFR≥70mL/(min×1.73m(2)) but a normal endogenous creatinine clearance rate (CCr) were quantified for living kidney donation. Differences in GFR levels based on sex and age were analyzed using rank correlation coefficient. Out of the 212 candidates, 161 were finally selected as kidney graft donors. The double kidney total GFR between the male and female donor groups, the GFR levels among differently-aged donor groups, and the GFR levels between the elderly (>55 years) and young- and middle-aged (≤55 years) donor groups did not show any significant difference (P>0.05). After kidney donation, renal function measured by blood urea nitrogen (BUN) and serum creatinine of all donors returned to normal within one week, and no serious complications were noticed. In conclusion, renal dynamic imaging by (99m)Tc-DTPA had a good accuracy and repeatability in GFR evaluation for living kidney donors. Candidate donors with GFR between 70mL/(min×1.73m(2)) and 80mL/(min×1.73m(2)) can be selected as kidney donors after strict screening. In living kidney donors GFR is not significantly correlated with age or sex.

Regulation of hierarchical carbon substrate utilization, nitrogen fixation, and root colonization by the Hfq/Crc/CrcZY genes in Pseudomonas stutzeri.

Bacteria of the genus Pseudomonas consume preferred carbon substrates in nearly reverse order to that of enterobacteria, and this process is controlled by RNA-binding translational repressors and regulatory ncRNA antagonists. However, their roles in microbe-plant interactions and the underlying mechanisms remain uncertain. Here we show that root-associated diazotrophic Pseudomonas stutzeri A1501 preferentially catabolizes succinate, followed by the less favorable substrate citrate, and ultimately glucose. Furthermore, the Hfq/Crc/CrcZY regulatory system orchestrates this preference and contributes to optimal nitrogenase activity and efficient root colonization. Hfq has a central role in this regulatory network through different mechanisms of action, including repressing the translation of substrate-specific catabolic genes, activating the nitrogenase gene nifH posttranscriptionally, and exerting a positive effect on the transcription of an exopolysaccharide gene cluster. Our results illustrate an Hfq-mediated mechanism linking carbon metabolism to nitrogen fixation and root colonization, which may confer rhizobacteria competitive advantages in rhizosphere environments.

The Sigma Factor AlgU Regulates Exopolysaccharide Production and Nitrogen-Fixing Biofilm Formation by Directly Activating the Transcription of pslA in Pseudomonas stutzeri A1501.

Pseudomonas stutzeri A1501, a plant-associated diazotrophic bacterium, prefers to conform to a nitrogen-fixing biofilm state under nitrogen-deficient conditions. The extracytoplasmic function (ECF) sigma factor AlgU is reported to play key roles in exopolysaccharide (EPS) production and biofilm formation in the Pseudomonas genus; however, the function of AlgU in P. stutzeri A1501 is still unclear. In this work, we mainly investigated the role of algU in EPS production, biofilm formation and nitrogenase activity in A1501. The algU mutant ΔalgU showed a dramatic decrease both in the EPS production and the biofilm formation capabilities. In addition, the biofilm-based nitrogenase activity was reduced by 81.4% in the ΔalgU mutant. The transcriptional level of pslA, a key Psl-like (a major EPS in A1501) synthesis-related gene, was almost completely inhibited in the algU mutant and was upregulated by 2.8-fold in the algU-overexpressing strain. A predicted AlgU-binding site was identified in the promoter region of pslA. The DNase I footprinting assays indicated that AlgU could directly bind to the pslA promoter, and ß-galactosidase activity analysis further revealed mutations of the AlgU-binding boxes drastically reduced the transcriptional activity of the pslA promoter; moreover, we also demonstrated that AlgU was positively regulated by RpoN at the transcriptional level and negatively regulated by the RNA-binding protein RsmA at the posttranscriptional level. Taken together, these data suggest that AlgU promotes EPS production and nitrogen-fixing biofilm formation by directly activating the transcription of pslA, and the expression of AlgU is controlled by RpoN and RsmA at different regulatory levels.

A regulatory network involving Rpo, Gac and Rsm for nitrogen-fixing biofilm formation by Pseudomonas stutzeri.

Biofilm and nitrogen fixation are two competitive strategies used by many plant-associated bacteria; however, the mechanisms underlying the formation of nitrogen-fixing biofilms remain largely unknown. Here, we examined the roles of multiple signalling systems in the regulation of biofilm formation by root-associated diazotrophic P. stutzeri A1501. Physiological analysis, construction of mutant strains and microscale thermophoresis experiments showed that RpoN is a regulatory hub coupling nitrogen fixation and biofilm formation by directly activating the transcription of pslA, a major gene involved in the synthesis of the Psl exopolysaccharide component of the biofilm matrix and nifA, the transcriptional activator of nif gene expression. Genetic complementation studies and determination of the copy number of transcripts by droplet digital PCR confirmed that the regulatory ncRNA RsmZ serves as a signal amplifier to trigger biofilm formation by sequestering the translational repressor protein RsmA away from pslA and sadC mRNAs, the latter of which encodes a diguanylate cyclase that synthesises c-di-GMP. Moreover, RpoS exerts a braking effect on biofilm formation by transcriptionally downregulating RsmZ expression, while RpoS expression is repressed posttranscriptionally by RsmA. These findings provide mechanistic insights into how the Rpo/Gac/Rsm regulatory networks fine-tune nitrogen-fixing biofilm formation in response to the availability of nutrients.

Association of polymorphisms in the RAGE gene with serum CRP levels and coronary artery disease in the Chinese Han population.

The role of an advanced glycation end product/receptor for advanced glycation end product (AGE/RAGE) system in the pathogenesis of coronary artery disease (CAD) is not fully understood. To clarify whether polymorphisms of the RAGE gene were related to CAD, we performed a case-control study in Chinese Han patients. The allele frequencies and genotype distribution combinations of the -429T/C, 1704G/T and G82S polymorphisms of the RAGE gene were compared in 200 cases of hypertension (HT), 155 cases of CAD combined with HT (CAD&HT), 175 cases of CAD and 170 control subjects. Polymerase chain reaction-restriction fragment length polymorphism was used for detection of genotypic variants. The S allele frequency of the G82S polymorphism was higher in the CAD (odds ratio (OR), 2.303, 95% confidence interval (CI) 1.553-3.416; P<0.001, P(corr)<0.003) and CAD&HT (OR, 1.842; 95% CI 1.219-2.785; P<0.003, P(corr)<0.009) groups when compared with the control group. However, the S allele frequency was not significantly different between the CAD and the CAD&HT patient groups (P=0.223), and no statistically significant difference of genotype or allele frequency distributions was observed in the HT group (P>0.05). Meanwhile, serum CRP was significantly associated with the G82S variant. Haplotype-based logistic regression analysis revealed that haplotype G-Ser-T (OR, 1.670; 95% CI, 1.017-2.740; P=0.043), compared with the reference haplotype T-Gly-T, was associated with an increased risk of CAD after adjusting for other risk factors. Further analysis limited to non-diabetic participants exhibited similar significant findings. The haplotype carrying the G82S variant of the RAGE gene was significantly associated with an increased risk of CAD, but not with HT patients. Moreover, a remarkable association of the G82S variant with serum CRP levels implied that the prevalence of RAGE 82S allelic variation might influence susceptibility to CAD by affecting vascular inflammation.

Multimodality Imaging of Angiogenesis in a Rabbit Atherosclerotic Model by GEBP11 Peptide Targeted Nanoparticles.

Background and Aims: Angiogenesis is an important pathological process during progression of plaque formation, which can result in plaque hemorrhage and vulnerability. This study aims to explore non-invasive imaging of angiogenesis in atherosclerotic plaque through magnetic resonance imaging (MRI) and positron emission tomography (PET) by using GEBP11 peptide targeted magnetic iron oxide nanoparticles in a rabbit model of atherosclerosis. Methods: The dual-modality imaging probe was constructed by coupling 2, 3-dimercaptosuccinnic acid-coated paramagnetic nanoparticles (DMSA-MNPs) and the PET 68Ga chelator 1,4,7-triazacyclononane-N, N', N''-triacetic acid (NOTA) to GEBP11 peptide. The atherosclerosis model was induced in New Zealand white rabbits by abdominal aorta balloon de-endothelialization and atherogenic diet for 12 weeks. The plaque areas in abdominal artery were detected by ultrasound imaging and Oil Red O staining. Immunofluorescence staining and Prussian blue staining were applied respectively to investigate the affinity of GEBP11 peptide. MTT and flow cytometric analysis were performed to detect the effects of NGD-MNPs on cell proliferation, cell cycle and apoptosis in Human umbilical vein endothelial cells (HUVECs). In vivo MRI and PET imaging of atherosclerotic plaque were carried out at different time points after intravenous injection of nanoparticles. Results: The NGD-MNPs with hydrodynamic diameter of 130.8 nm ± 7.1 nm exhibited good imaging properties, high stability, low immunogenicity and little cytotoxicity. In vivo PET/MR imaging revealed that 68Ga-NGD-MNPs were successfully applied to visualize atherosclerotic plaque angiogenesis in the rabbit abdominal aorta. Prussian blue and CD31 immunohistochemical staining confirmed that NGD-MNPs were well co-localized within the blood vessels' plaques. Conclusion:68Ga-NGD-MNPs might be a promising MR and PET dual imaging probe for visualizing the vulnerable plaques.

[Transient cytosolic free calcium changes in cultured neonatal rat cardiac myocytes in response to advanced glycation end-product treatment].

OBJECTIVE: To investigate the effects of advanced glycation end-products (AGEs) on transient cytosolic free calcium in neonatal rat cardiac myocytes (CMs) cultured in vitro. METHODS: CMs cultured for 3 to 5 days in vitro were incubated with Ca(2+)-sensitive fluorescent indicator Fluo-3AM with light screening at 37 degrees celsius; with 5% CO(2) for 60 min. Changes of the fluorescence signal of free calcium caused by AGEs were measured under laser scanning confocal microscope (LSCM). RESULTS: Compared with the control cells, AGEs caused an increase in the concentration of cytosolic free calcium in a dose-dependent manner. CONCLUSION: AGEs may impair neonatal rat CMs by altering cytosolic free calcium concentration.

[Effects of captopril and losartan on expression of kidney aquaporin-2 mRNA and urine aquaporin-2 excretion in rats].

OBJECTIVE: To investigate effects of captopril and losartan on the expression of kidney aquaporin-2 (AQP2) mRNA and the excretion of urine AQP2 in rats. METHODS: Thirty healthy rats were randomized into 3 groups, namely the control group, captopril group and losartan group, respectively. Blood and urine samples were collected from the rats for detecting serum Na(+), urine volume and urine osmolality in the course of medication. Urine AQP2 concentration was measured by enzyme-linked immunosorbent assay (ELISA). Semi-quantitative RT-PCR was performed for measurement of kidney inner medullary AQP2 and vasopressin V(2) receptor mRNA. RESULTS: Urine volume was increased in rats of captopril and losartan groups as compared with that of the control group. However, urine osmolality was lower in captopril group than in the other two groups (P<0.05). RT-PCR revealed decreased quantity of the inner medullary AQP2 mRNA of the captopril group than that of the other two groups, but the quantity of V(2) receptor mRNA did not differ significantly between the 3 groups. Urine AQP2 concentration was significantly higher in captopril group than in the control (P<0.05) and losartan groups (P0<0.01). CONCLUSION: Captopril can reduce the expression of the kidney inner medullary AQP2 mRNA and accelerate the excretion of the urine AQP2 in normal rats.

[Calcification and biomechanics in vivo of ultra-microporous expanded polytetrafluoroethylene in rabbits].

OBJECTIVE: To study in vivo calcification and biomechanics of the ultra-microporous expanded polytetrafluo- roethylene (UePTFE) in rabbits in comparison with glutaral-treated bovine pericardium (BP), so as to assess the potential of UePTFE as a material for cardiac valve prosthesis. METHODS: Factorial analysis was adopted in the experiment. UePTFE and glutaral-treated BP of appropriate sizes were embedded beneath skin of young New Zealand rabbits, and at 0, 1, 3 and 6 months following the implantation, the materials were measured for the content of calcium and biomechanics properties. RESULTS: Lower level of calcification of the UePTFE occurred after the implantation, as compared with BP, and the biomechanics indices of the former UePTFE were obviously suprior to those of the latter. CONCLUSION: UePTFE is a better material than BP for cardiac valve prosthesis.