Cryo-EM structure of the polyphosphate polymerase VTC reveals coupling of polymer synthesis to membrane transit.

The eukaryotic vacuolar transporter chaperone (VTC) complex acts as a polyphosphate (polyP) polymerase that synthesizes polyP from adenosine triphosphate (ATP) and translocates polyP across the vacuolar membrane to maintain an intracellular phosphate (Pi ) homeostasis. To discover how the VTC complex performs its function, we determined a cryo-electron microscopy structure of an endogenous VTC complex (Vtc4/Vtc3/Vtc1) purified from Saccharomyces cerevisiae at 3.1 Å resolution. The structure reveals a heteropentameric architecture of one Vtc4, one Vtc3, and three Vtc1 subunits. The transmembrane region forms a polyP-selective channel, likely adopting a resting state conformation, in which a latch-like, horizontal helix of Vtc4 limits the entrance. The catalytic Vtc4 central domain is located on top of the pseudo-symmetric polyP channel, creating a strongly electropositive pathway for nascent polyP that can couple synthesis to translocation. The SPX domain of the catalytic Vtc4 subunit positively regulates polyP synthesis by the VTC complex. The noncatalytic Vtc3 regulates VTC through a phosphorylatable loop. Our findings, along with the functional data, allow us to propose a mechanism of polyP channel gating and VTC complex activation.

Prognostic value of combined coronary angiography-derived IMR and myocardial perfusion imaging by CZT SPECT in INOCA.

BACKGROUND: A significant proportion of ischemia with non-obstructive coronary artery disease (INOCA) demonstrate coronary microvascular dysfunction (CMD), a condition associated with abnormal myocardial perfusion imaging (MPI) and adverse outcomes. Coronary angiography-derived index of microvascular resistance (caIMR) is a novel non-invasive technique to assess CMD. We aimed to investigate the prognostic value of combined caIMR and MPI by CZT SPECT in INOCA patients. METHODS: Consecutive 151 patients with chest pain and < 50% coronary stenosis who underwent coronary angiography and MPI within 3 months were enrolled. caIMR was calculated by computational pressure-flow dynamics. CMD was defined as caIMR ≥ 25. The endpoint was major adverse cardiac events (MACE: cardiovascular death, nonfatal myocardial infarction, revascularization, angina-related rehospitalization, heart failure, and stroke). RESULTS: Of all INOCA patients, CMD was present in 93 (61.6%) patients. The prevalence of abnormal MPI was significantly higher in CMD compared with non-CMD patients (40.9% vs 13.8%, P < .001). CMD showed a higher risk of MACE than non-CMD patients. Patients with both CMD and abnormal MPI had the worst prognosis, followed by patients with CMD and normal MPI (log-rank P < .001). Cox regression analysis identified CMD (HR 3.121, 95%CI 1.221-7.974, P = .017) and MPI (HR 2.704, 95%CI 1.030-7.099, P = .043) as predictive of MACE. The prognostic value of INOCA patients enhanced significantly by adding CMD and MPI to the model with clinical risk factors (AUC = 0.777 vs 0.686, P = .030). CONCLUSION: caIMR-derived CMD is associated with increased risk of MACE among INOCA patients. Patients with abnormalities on both caIMR and MPI had the worse outcomes.

PCT, IL-6, and IL-10 facilitate early diagnosis and pathogen classifications in bloodstream infection.

BACKGROUND: In the diagnosis of bloodstream infection (BSI), various inflammatory markers such as C-reactive protein (CRP), procalcitonin (PCT), interleukins (IL), white blood cell count (WBC), neutrophil percentage (NE%), platelet count (PLT), and erythrocyte sedimentation rate (ESR) have been extensively utilized. However, their specific roles in distinguishing BSI from local bacterial infection (LBI) and in classifying BSI pathogens remain uncertain. METHODS: A historical cohort study was conducted, involving the enrollment of 505 patients with BSI and 102 patients with LBI. To validate the reliability of the clinical data obtained from this cohort, mouse models of BSI were utilized. RESULTS: Our findings revealed that patients with BSI had significantly higher levels of inflammatory markers, including CRP, PCT, IL-6, IL-10, WBC, NE%, and ESR, compared to those with LBI (p < 0.05). The receiver operating characteristic (ROC) curve analysis demonstrated that CRP, PCT, IL-6, IL-10, ESR and NE% exhibited excellent diagnostic efficacy for BSI. Additionally, we observed significant differences in CRP, PCT, IL-6, and IL-10 levels between patients with BSI caused by Gram-positive bacteria (GP-BSI) and Gram-negative bacteria (GN-BSI), but no significant variations were found among specific bacterial species. Furthermore, our study also found that CRP, PCT, and IL-10 have good discriminatory ability for vancomycin-resistant Enterococcus (VRE), but they show no significant diagnostic efficacy for other multidrug-resistant organisms (MDROs) such as carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and methicillin-resistant Staphylococcus aureus (MRSA). In our mouse model experiments, we observed a remarkable increase in PCT, IL-6, and IL-10 levels in mice with GN-BSI compared to those with GP-BSI. CONCLUSION: Our study has confirmed that PCT, IL-6, and IL-10 are efficient biomarkers for distinguishing between BSI and LBI. Furthermore, they can be utilized to classify BSI pathogens and differentiate between VRE and vancomycin-susceptible Enterococcus. These findings are extremely valuable for clinicians as they enable timely initiation of empiric antibiotic therapies and ultimately lead to improved clinical outcomes for patients with BSI.

Integrated Metabolome and Transcriptome Analysis Provides New Insights into the Glossy Graft Cucumber Fruit (Cucumis sativus L.).

Cucumber is an important vegetable crop, and grafts often affect the quality and wax loss in cucumber fruit and affect its value. However, their metabolites and molecular mechanisms of action remain unclear. Metabolome and transcriptome analyses were conducted on the fruit peels of self-rooted plants (SR) grafted with white seed pumpkin (WG). The results showed that there were 352 differential metabolites in the fruit peels of the SR and WG. The transcriptome analysis showed 1371 differentially expressed genes (DEGs) between the WG and SR. These differentially expressed genes were significantly enriched in plant hormone signal transduction, cutin, suberin, wax biosynthesis, phenylpropanoid biosynthesis, and zeatin biosynthesis. By analyzing the correlation between differential metabolites and differentially expressed genes, six candidate genes related to the synthesis of glycitein, kaempferol, and homoeriodictyol were identified as being potentially important. Key transcription factors belonging to the TCP and WRKY families may be the main drivers of transcriptional changes in the peel between the SR and WG. The results of this study have provided a basis for the biosynthesis and regulation of wax loss and quality in grafted cucumbers and represents an important step toward identifying the molecular mechanisms of grafting onto cucumber fruit.

Integrated Physiological and Transcriptomic Analyses Revealed Improved Cold Tolerance in Cucumber (Cucumis sativus L.) by Exogenous Chitosan Oligosaccharide.

Cucumber (Cucumis sativus L.), sensitive to cold stress, is one of the most economically important vegetables. Here, we systematically investigated the roles of exogenous glycine betaine, chitosan, and chitosan oligosaccharide in alleviating cold stress in cucumber seedlings. The results showed that 50 mg·L-1 chitosan oligosaccharide had the best activity. It effectively increases plant growth, chlorophyll content, photosynthetic capacity, osmotic regulatory substance content, and antioxidant enzyme activities while reducing relative electrical conductivity and malondialdehyde levels in cucumber seedlings under cold stress. To reveal the protective effects of chitosan oligosaccharide in cold stress, cucumber seedlings pretreated with 50 mg·L-1 chitosan oligosaccharide were sampled after 0, 3, 12, and 24 h of cold stress for transcriptome analysis, with distilled water as a control. The numbers of differentially expressed genes in the four comparison groups were 656, 1274, 1122, and 957, respectively. GO functional annotation suggested that these genes were mainly involved in "voltage-gated calcium channel activity", "carbohydrate metabolic process", "jasmonic acid biosynthetic", and "auxin response" biological processes. KEGG enrichment analysis indicated that these genes performed important functions in "phenylpropanoid biosynthesis", "MAPK signaling pathway-plant", "phenylalanine metabolism", and "plant hormone signal transduction." These findings provide a theoretical basis for the use of COS to alleviate the damage caused by cold stress in plant growth and development.

Cefazolin and imipenem enhance AmpC expression and resistance in NagZ-dependent manner in Enterobacter cloacae complex.

BACKGROUND: Enterobacter cloacae complex (ECC) is a common opportunistic pathogen and is responsible for causing various infections in humans. Owing to its inducible chromosomal AmpC ß-lactamase (AmpC), ECC is inherently resistant to the 1st- and 2nd- generation cephalosporins. However, whether ß-lactams antibiotics enhance ECC resistance remains unclear. RESULTS: In this study, we found that subinhibitory concentrations (SICs) of cefazolin (CFZ) and imipenem (IMP) can advance the expression of AmpC and enhance its resistance towards ß-lactams through NagZ in Enterobacter cloacae (EC). Further, AmpC manifested a substantial upregulation in EC in response to SICs of CFZ and IMP. In nagZ knockout EC (ΔnagZ), the resistance to ß-lactam antibiotics was rather weakened and the effect of CFZ and IMP on AmpC induction was completely abrogated. NagZ ectopic expression can rescue the induction effects of CFZ and IMP on AmpC and increase ΔnagZ resistance. More importantly, CFZ and IMP have the potential to induce the expression of AmpR's target genes in a NagZ-dependent manner. CONCLUSIONS: Our findings suggest that NagZ is a critical determinant for CFZ and IMP to promote AmpC expression and resistance and that CFZ and IMP should be used with caution since they may aggravate ECC resistance. At the same time, this study further improves our understanding of resistance mechanisms in ECC.

Prognostic value of coronary microvascular dysfunction assessed by coronary angiography-derived index of microcirculatory resistance in diabetic patients with chronic coronary syndrome.

BACKGROUND: Coronary microvascular dysfunction (CMD) is common and is associated with unfavorable cardiovascular events in patients with diabetes mellitus (DM). Coronary angiography-derived index of microcirculatory resistance (caIMR) is a recently developed wire- and hyperemic agent-free method to assess CMD. We aimed to investigate the prognostic impact of CMD assessed by caIMR on clinical outcomes in patients with DM and chronic coronary syndrome (CCS). METHODS: CCS patients who underwent coronary angiography between June 2015 to May 2018 were included. Coronary microvascular function was measured by caIMR, and CMD was defined as caIMR ≥ 25U. The primary endpoint was major adverse cardiac events (MACE). Kaplan-Meier analysis and Cox proportional hazards models were used to assess the relationship between caIMR and the risk of MACE. RESULTS: Of 290 CCS patients, 102 patients had DM. Compared with non-diabetic patients, CMD (caIMR ≥ 25U) was higher among DM patients (57.8% vs. 38.3%; p = 0.001). During a mean 35 months follow-up, 40 MACE had occurred. Patients with caIMR ≥ 25 had a higher rate of MACE than patients with caIMR < 25 (20.6% vs. 8.2%, p = 0.002). Of these, the MACE rate was higher among DM patients with caIMR ≥ 25 than those with caIMR < 25 (33.9% vs. 14.0%; p = 0.022). In multivariable Cox analysis, caIMR ≥ 25 was independently associated with MACE in the DM patients but not in non-DM patients (HR, 2.760; 95% CI, 1.066-7.146; P = 0.036). CONCLUSION: CMD assessed by caIMR was common and is an independent predictor of MACE among diabetic patients with CCS. This finding potentially enables a triage of higher-risk patients to more intensive therapy.

Advanced Understanding of the Electron Transfer Pathway of Cytochrome P450s.

Cytochrome P450s are heme-thiolate enzymes that participate in carbon source assimilation, natural compound biosynthesis and xenobiotic metabolism in all kingdoms of life. P450s can catalyze various reactions by using a wide range of organic compounds, thus exhibiting great potential in biotechnological applications. The catalytic reactions of P450s are driven by electron equivalents that are sourced from pyridine nucleotides and delivered by cognate or matching redox partners (RPs). The electron transfer (ET) route from RPs to P450s involves one or more redox center-containing domains. As the rate of ET is one of the main determinants of P450 efficacy, an in-depth understanding of the P450 ET pathway should increase our knowledge of these important enzymes and benefit their further applications. Here, the various P450 RP systems along with current understanding of their ET routes will be reviewed. Notably, state-of-the-art structural studies of the two main types of self-sufficient P450 will also be summarized.

Degradation of epigallocatechin and epicatechin gallates by a novel tannase TanHcw from Herbaspirillum camelliae.

BACKGROUND: Herbaspirillum camelliae is a gram-negative endophyte isolated from the tea plant. Both strains WT00C and WT00F were found to hydrolyze epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG) to release gallic acid (GA) and display tannase activity. However, no tannase gene was annotated in the genome of H. camelliae WT00C. RESULTS: The 39 kDa protein, annotated as the prolyl oligopeptidase in the NCBI database, was finally identified as a novel tannase. Its gene was cloned, and the enzyme was expressed in E. coli and purified to homogeneity. Moreover, enzymatic characterizations of this novel tannase named TanHcw were studied. TanHcw was a secretary enzyme with a Sec/SPI signal peptide of 48 amino acids at the N-terminus, and it catalyzed the degradation of tannin, methyl gallate (MG), epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG). The optimal temperature and pH of TanHcw activities were 30 °C, pH 6.0 for MG and 40 °C, pH 7.0 for both EGCG and ECG. Na+, K+ Mn2+ and Triton-X100, Tween80 increased the enzyme activity of TanHcw, whereas Zn2+, Mg2+, Hg2+, EMSO, EDTA and ß-mercaptoethanol inhibited enzyme activity. Km, kcat and kcat /Km of TanHcw were 0.30 mM, 37.84 s-1, 130.67 mM-1 s-1 for EGCG, 0.33 mM, 34.59 s-1, 105.01 mM-1 s-1 for ECG and 0.82 mM, 14.64 s-1, 18.17 mM-1 s-1 for MG, respectively. CONCLUSION: A novel tannase TanHcw from H. camelliae has been identified and characterized. The biological properties of TanHcw suggest that it plays a crucial role in the specific colonization of H. camelliae in tea plants. Discovery of the tannase TanHcw in this study gives us a reasonable explanation for the host specificity of H. camelliae. In addition, studying the characteristics of this enzyme offers the possibility of further defining its potential in industrial application.

Multiple exposures to high concentrations of selenate significantly improve selenate tolerability, red elemental selenium (Se0) and selenoprotein biosynthesis in Herbaspirillum camelliae WT00C.

Herbaspirillum camelliae WT00C is a gram-negative endophyte isolated from the tea plant. It has an intact selenate metabolism pathway but poor selenate tolerability. In this study, microbiological properties of the strain WT00C were examined and compared with other three strains CT00C, NCT00C and NT00C, which were obtained respectively from four, six and eight rounds of 24-h exposures to 200 mM selenate. The selenate tolerability and the ability to generate red elemental selenium (Se0) and selenoproteins in H. camelliae WT00C has significantly improved by the forced evolution via 4-6 rounds of multiple exposures a high concentration of selenate. The original strain WT00C grew in 200 mM selenate with the lag phase of 12 h and 400 mM selenate with the lag phase of 60 h, whereas the strains CT00C and NCT00C grew in 800 mM selenate and showed a relatively short lag phase when they grew in 50-400 mM selenate. Besides selenate tolerance, the strains CT00C and NCT00C significantly improved the biosynthesis of red elemental selenium (Se0) and selenoproteins. Two strains exhibited more than 30% selenium conversion efficiency and 40% selenoprotein biosynthesis, compared to the original strain WT00C. These characteristics of the strains CT00C and NCT00C make them applicable in pharmaceuticals and feed industries. The strain NT00C obtained from eight rounds of 24-h exposures to 200 mM selenate was unable to grow in ≥ 400 mM selenate. Its selenium conversion efficiency and selenoprotein biosynthesis were similar to the strain WT00C, indicating that too many exposures may cause gene inactivation of some critical enzymes involving selenate metabolism and antioxidative stress. In addition, bacterial cells underwent obviously physiological and morphological changes, including gene activity, cell enlargement and surface-roughness alterations during the process of multiple exposures to high concentrations of selenate.

Structural insights into thebaine synthase 2 catalysis.

Thebaine synthase 2 (THS2) that can transform (7S)-salutaridinol 7-O-acetate to thebaine catalyzes the final step of thebaine biosynthesis in Papaver somniferum. Here, the crystal structures of THS2 and its complex with thebaine are reported, revealing the interaction network in the substrate-binding pocket. Subsequent docking and QM/MM studies was performed to further explore the catalytic mechanism of THS2. Our results suggest that T105 may abstract the proton of C4-OH from the substrate under the assistance of H89. The resulting C4-O- phenolate anion then attacks the nearby C5, and triggers intramolecular SN2' syn displacement with the elimination of O-acetyl group. Moreover, the latter SN2' reaction is the rate-determining step of the whole enzymatic reaction with an overall energy barrier of 18.8 kcal/mol. These findings are of pivotal importance to understand the mechanism of action of thebaine biosynthesis, and would guide enzyme engineering to enhance the production of opiate alkaloids via metabolic engineering.

Structural insights to heterodimeric cis-prenyltransferases through yeast dehydrodolichyl diphosphate synthase subunit Nus1.

The polyprenoid glycan carriers are produced by cis-prenyltransferases (cis-PTs), which function as heterodimers in metazoa and fungi or homodimers in bacteria, but both are found in plants, protista and archaea. Heterodimeric cis-PTs comprise catalytic and non-catalytic subunits while homodimeric enzymes contain two catalytic subunits. The non-catalytic subunits of cis-PT shows low sequence similarity to known cis-PTs and their structure information is of great interests. Here we report the crystal structure of Nus1, the non-catalytic subunit of cis-PT from Saccharomyces cerevisiae. We also investigate the heterodimer formation and active site conformation by constructing a homology model of Nus1 and its catalytic subunit. Nus1 does not contain an active site, but its C-terminus may participate in catalysis by interacting with the substrates bound to the catalytic subunit. These results provide important basis for further investigation of heterodimeric cis-PTs.

Crystal structure and proposed mechanism of an enantioselective hydroalkoxylation enzyme from Penicillium herquei.

Hydroalkoxylation is a useful and efficient reaction which generates C-O bond and produces cyclic ethers, the common structural elements of natural products. The dedicative enzyme which can catalyze enantioselective hydroalkoxylation named PhnH was recently identified in the herqueinone biosynthetic gene from Penicillium herquei. It catalyzes addition of a phenol to the terminal olefin on substrate to produce a dihydrobenzofuran. Here, the crystal structure of PhnH is reported and the putative substrate-binding pocket is illustrated. Through docking experiment, possible substrate-binding poses are displayed and the catalytic mechanism is therefore proposed. Our findings form the basis for further studies of enantioselective hydroalkoxylation enzymes.

Complex structures of MoeN5 with substrate analogues suggest sequential catalytic mechanism.

The antibiotic moenomycin A is a phosphoglycerate derivative with a C25-moenocinyl chain and a branched oligosaccharide. Formation of the C25-chain is catalyzed by the enzyme MoeN5 with geranyl pyrophosphate (GPP) and the sugar-linked 2-Z,E-farnesyl-3-phosphoglycerate (FPG) as its substrates. Previous complex crystal structures with GPP and long-chain alkyl glycosides suggested that GPP binds to the S1 site in a similar way as in most other α-helical prenyltransferases (PTs), and FPG is likely to assume a bent conformation in the S2 site. However, two FPG derivatives synthesized in the current study were found in the S1 site rather than S2 in their complex crystal structures with MoeN5. Apparently S1 is the preferred site for prenyl-containing ligand, and S2 binding may proceed only after S1 is occupied. Thus, like most trans-type PTs, MoeN5 may employ a sequential ionization-condensation-elimination mechanism that involves a carbocation intermediate.

Absence of phosphatidylcholine in bacterial membranes facilitates translocation of Sec-dependent ß-lactamase AmpC from cytoplasm to periplasm in two Pseudomonas strains.

Phosphatidylcholine (PC) is a rare membrane lipid in bacteria but crucial for virulence of various plant and animal pathogens. The pcs- mutant lacking PC in bacterial membranes of Pseudomonas syringae pv. syringae van Hall 1336 displayed more ampicillin resistance. Ampicillin susceptibility tests gave an IC50 (half maximal inhibitory concentration) of 52 mg/ml for Pseudomonas syringae pv. syringae van Hall 1336, 53 mg/ml for the complemented strain 1336 RM (pcs-/+) and 90 mg/ml for the 1336 pcs- mutant. Activity assay of ß-lactamase in periplasmic extracts gave 0.050 U/mg for the 1336 wild type, 0.052 U/mg for the 1336RM (pcs-/+), 0.086 U/mg for the 1336 pcs- mutant. Analysis by western blotting showed that the content of AmpC enzyme was markedly different in periplasmic extracts between the wild-type and pcs- mutant strains. Reverse transcriptase PCR also showed that the presence or absence of PC in bacterial membranes did not affect the transcription of ampC gene. The phenotype of the pcs- mutant was able to be recovered to the wild type by introducing a wild-type pcs gene into the pcs- mutant. Similar results were also obtained from the soil-dwelling bacterium Pseudomonas sp. 593. Our results demonstrate that the absence of PC in bacterial membranes facilitates the translocation of Sec-dependent ß-lactamase AmpC from cytoplasm to periplasm, and the enhanced ampicillin-resistance in the pcs- strains mainly comes from effective translocation of AmpC via Sec-pathway.

Dual specific phosphatase 12 ameliorates cardiac hypertrophy in response to pressure overload.

Pathological cardiac hypertrophy is an independent risk factor of heart failure. However, we still lack effective methods to reverse cardiac hypertrophy. DUSP12 is a member of the dual specific phosphatase (DUSP) family, which is characterized by its DUSP activity to dephosphorylate both tyrosine and serine/threonine residues on one substrate. Some DUSPs have been identified as being involved in the regulation of cardiac hypertrophy. However, the role of DUSP12 during pathological cardiac hypertrophy is still unclear. In the present study, we observed a significant decrease in DUSP12 expression in hypertrophic hearts and cardiomyocytes. Using a genetic loss-of-function murine model, we demonstrated that DUSP12 deficiency apparently aggravated pressure overload-induced cardiac hypertrophy and fibrosis as well as impaired cardiac function, whereas cardiac-specific overexpression of DUPS12 was capable of reversing this hypertrophic and fibrotic phenotype and improving contractile function. Furthermore, we demonstrated that JNK1/2 activity but neither ERK1/2 nor p38 activity was increased in the DUSP12 deficient group and decreased in the DUSP12 overexpression group both in vitro and in vivo under hypertrophic stress conditions. Pharmacological inhibition of JNK1/2 activity (SP600125) is capable of reversing the hypertrophic phenotype in DUSP12 knockout (KO) mice. DUSP12 protects against pathological cardiac hypertrophy and related pathologies. This regulatory role of DUSP12 is primarily through c-Jun N-terminal kinase (JNK) inhibition. DUSP12 could be a promising therapeutic target of pathological cardiac hypertrophy. DUSP12 is down-regulated in hypertrophic hearts. An absence of DUSP12 aggravated cardiac hypertrophy, whereas cardiomyocyte-specific DUSP12 overexpression can alleviate this hypertrophic phenotype with improved cardiac function. Further study demonstrated that DUSP12 inhibited JNK activity to attenuate pathological cardiac hypertrophy.

Deciphering transcriptome profile of the yellow catfish (Pelteobagrus fulvidraco) in response to Edwardsiella ictaluri.

Edwardsiella ictaluri is one of the most important pathogens posing a serious threat for yellow catfish (Pelteobagrus fulvidraco), a highly valuable fish species of increasing commercial interest in China. Here, a transcriptomic strategy was undertaken to investigate the yellow catfish gene expression profile against infection by the bacterial pathogen E. ictaluri. Comparison of the transcriptome profiles between the infected and uninfected samples showed that a massive gene expression change occurred in yellow catfish following bacterial exposure. A total of 5527 differentially expressed genes (DEGs) were detected, of which 2265 showed up-regulation and 3262 down-regulation. Gene set enrichment analysis revealed the presence of canonical pathways directly linked to innate and adaptive immune response, such as pattern recognition receptor (PRR) signaling pathways, complement and coagulation cascades, as well as T-cell receptor (TCR) and B-cell receptor (BCR) signaling pathways. Additionally, 47,526 putative EST-liked simple sequence repeats (SSRs) markers were retrieved for use in genetic studies. This study establishes the first molecular clues to understand the potential mechanisms of yellow catfish resistance to E. ictaluri, thus enabling future efforts on disease control programs in this valuable aquaculture species.

Plasma Long Noncoding RNA Urothelial Carcinoma Associated 1 Predicts Poor Prognosis in Chronic Heart Failure Patients.

BACKGROUND Chronic heart failure (CHF) is a leading cause of death worldwide. A long noncoding RNA (lncRNA) named urothelial carcinoma associated 1 (UCA1) is important in multiple diseases. However, the role of UCA1 in CHF is still unknown. Our study investigated whether UCA1 could be applied as an ideal marker to diagnose and evaluate prognosis in CHF. MATERIAL AND METHODS Total plasma RNA was extracted from 67 CHF patients and 67 controls. Quantitative real-time polymerase chain reaction was used to determine the plasma level of UCA1. Correlations between UCA1 and clinical parameters were analyzed by Pearson correlation. Receiver operating characteristic curves (ROC) were obtained to analyze the predictive power of UCA1 and BNP for CHF. Kaplan-Meier survival curves were used to evaluate prognosis of CHF within 1 year. RESULTS There was no significant difference in elementary data between CHF and controls. Plasma UCA1 was much higher in CHF patients compared with controls. Plasma UCA1 was positively and negatively correlated with brain natriuretic peptide (BNP) and left ventricle ejection fraction (LVEF), respectively. Plasma UCA1 diagnosed CHF with a diagnostic power of 0.89 and a sensitivity and specificity of 100% [95% CI (0.9464-1)] and 76.12% [95%CI (0.6414-0.8569)] (P<0.05), respectively. CHF patients with higher plasma UCA1 had a lower survival rate than those with a lower level, and survival rate predicted by UCA1 had a similar tendency with BNP. However, there was no significant difference between these 2 markers in predicting the prognosis of CHF (P>0.05). CONCLUSIONS Plasma UCA1 might be an excellent indicator to diagnose CHF and it might predict poor outcomes of CHF.

Expression of cocaine- and amphetamine-regulated transcript (CART) in hen ovary.

BACKGROUND: Cocaine- and amphetamine-regulated transcript (CART), discovered initially by via differential display RT-PCR analysis of brains of rats administered cocaine, is expressed mainly in central nervous system or neuronal origin cells, and is involved in a wide range of behaviors, such as regulation of food intake, energy homeostasis, and reproduction. The hens egg-laying rate mainly depends on the developmental status of follicles, expression of CART have not been identified from hen follicles, the regulatory mechanisms of CART biological activities are still unknown. The objective of this study was to characterize the mRNA expression of CART in hen follicular granulosa cells and determine CART peptide localization and regulatory role during follicular development. METHODS: Small white follicles (1-2 mm in diameter) were treated for RNA isolation; Small white follicles (1-2 mm in diameter) and large white follicles (4-6 mm in diameter) were treated for immunohistochemical localization and large white follicles (4-6 mm in diameter), small yellow follicles (6-8 mm in diameter), large yellow follicles (9-12 mm in diameter), mature follicles (F5, F4, F3, F2, F1, >12 mm in diameter) were treated for RNA isolation and Real time PCR. RESULTS: The results showed that full length of the CDS of hen CART was 336 bp encoding a 111 amino acid polypeptide. In the hen ovary, CART peptide was primarily localized to the theca layer, but not all, the oocyte and granulosa layer, with diffused, weaker staining than relative to the theca cell layer. Further, amount of CART mRNA was more (P < 0.05) in granulosa cells of 6-8 mm follicles compared with that in granulosa cells of other follicles. However, CART mRNA amount was greater in theca cells of 4-6 mm follicles relative to follicles of other sizes (P < 0.05). CONCLUSIONS: Results suggest that CART could play a potential role in developmental regulation of chicken follicles.

De novo annotation of the immune-enriched transcriptome provides insights into immune system genes of Chinese sturgeon (Acipenser sinensis).

Chinese sturgeon (Acipenser sinensis), one of the oldest extant actinopterygian fishes with very high evolutionary, economical and conservation interest, is considered to be one of the critically endangered aquatic animals in China. Up to date, the immune system of this species remains largely undetermined with little sequence information publicly available. Herein, the first comprehensive transcriptome of immune tissues for Chinese sturgeon was characterized using Illumina deep sequencing. Over 67 million high-quality reads were generated and de novo assembled into the final set of 91,739 unique sequences. The annotation pipeline revealed that 25,871 unigenes were successfully annotated in the public databases, of which only 2002 had significant match to the existing sequences for the genus Acipenser. Overall 22,827 unigenes were categorized into 52 GO terms, 12,742 were classified into 26 KOG categories, and 4968 were assigned to 339 KEGG pathways. A more detailed annotation search showed the presence of a notable representation of immune-related genes, which suggests that this non-teleost actinopterygian fish harbors the same intermediates as in the well known immune pathways from mammals and teleosts, such as pattern recognition receptor (PRR) signaling pathway, JAK-STAT signaling pathway, complement and coagulation pathway, T-cell receptor (TCR) and B-cell receptor (BCR) signaling pathways. Additional genetic marker discovery led to the retrieval of 20,056 simple sequence repeats (SSRs) and 327,140 single nucleotide polymorphisms (SNPs). This immune-enriched transcriptome of Chinese sturgeon represents a rich resource that adds to the currently nascent field of chondrostean fish immunogenetics and furthers the conservation and management of this valuable fish.