Cyclic di-GMP inhibits nitrate assimilation by impairing the antitermination function of NasT in Pseudomonas putida.

The ubiquitous bacterial second messenger cyclic diguanylate (c-di-GMP) coordinates diverse cellular processes through its downstream receptors. However, whether c-di-GMP participates in regulating nitrate assimilation is unclear. Here, we found that NasT, an antiterminator involved in nitrate assimilation in Pseudomonas putida, specifically bound c-di-GMP. NasT was essential for expressing the nirBD operon encoding nitrite reductase during nitrate assimilation. High-level c-di-GMP inhibited the binding of NasT to the leading RNA of nirBD operon (NalA), thus attenuating the antitermination function of NasT, resulting in decreased nirBD expression and nitrite reductase activity, which in turn led to increased nitrite accumulation in cells and its export. Molecular docking and point mutation assays revealed five residues in NasT (R70, Q72, D123, K127 and R140) involved in c-di-GMP-binding, of which R140 was essential for both c-di-GMP-binding and NalA-binding. Three diguanylate cyclases (c-di-GMP synthetases) were found to interact with NasT and inhibited nirBD expression, including WspR, PP_2557, and PP_4405. Besides, the c-di-GMP-binding ability of NasT was conserved in the other three representative Pseudomonas species, including P. aeruginosa, P. fluorescens and P. syringae. Our findings provide new insights into nitrate assimilation regulation by revealing the mechanism by which c-di-GMP inhibits nitrate assimilation via NasT.

The phosphodiesterase DibA interacts with the c-di-GMP receptor LapD and specifically regulates biofilm in Pseudomonas putida.

The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase and degraded by c-di-GMP-specific phosphodiesterase. The genome of Pseudomonas putida contains dozens of genes encoding diguanylate cyclase/phosphodiesterase, but the phenotypical-genotypical correlation and functional mechanism of these genes are largely unknown. Herein, we characterize the function and mechanism of a P. putida phosphodiesterase named DibA. DibA consists of a PAS domain, a GGDEF domain, and an EAL domain. The EAL domain is active and confers DibA phosphodiesterase activity. The GGDEF domain is inactive, but it promotes the phosphodiesterase activity of the EAL domain via binding GTP. Regarding phenotypic regulation, DibA modulates the cell surface adhesin LapA level in a c-di-GMP receptor LapD-dependent manner, thereby inhibiting biofilm formation. Moreover, DibA interacts and colocalizes with LapD in the cell membrane, and the interaction between DibA and LapD promotes the PDE activity of DibA. Besides, except for interacting with DibA and LapD itself, LapD is found to interact with 11 different potential diguanylate cyclases/phosphodiesterases in P. putida, including the conserved phosphodiesterase BifA. Overall, our findings demonstrate the functional mechanism by which DibA regulates biofilm formation and expand the understanding of the LapD-mediated c-di-GMP signaling network in P. putida.

Genome-wide identification and molecular evolution of Dof gene family in Camellia oleifera.

DNA binding with one finger(Dof) gene family is a class of transcription factors which play an important role on plant growth and development. Genome-wide identification results indicated that there were 45 Dof genes(ColDof) in C.oleifera genome. All 45 ColDof proteins were non-transmembrane and non-secretory proteins. Phosphorylation site analysis showed that biological function of ColDof proteins were mainly realized by phosphorylation at serine (Ser) site. The secondary structure of 44 ColDof proteins was dominated by random coil, and only one ColDof protein was dominated by α-helix. ColDof genes' promoter region contained a variety of cis-acting elements, including light responsive regulators, gibberellin responsive regulators, abscisic acid responsive regulators, auxin responsive regulators and drought induction responsive regulators. The SSR sites analysis showed that the proportion of single nucleotide repeats and the frequency of A/T in ColDof genes were the largest. Non-coding RNA analysis showed that 45 ColDof genes contained 232 miRNAs. Transcription factor binding sites of ColDof genes showed that ColDof genes had 5793 ERF binding sites, 4381 Dof binding sites, 2206 MYB binding sites, 3702 BCR-BPC binding sites. ColDof9, ColDof39 and ColDof44 were expected to have the most TFBSs. The collinearity analysis showed that there were 40 colinear locis between ColDof proteins and AtDof proteins. Phylogenetic analysis showed that ColDof gene family was most closely related to that of Camellia sinensis var. sinensis cv.Biyun and Camellia lanceoleosa. Protein-protein interaction analysis showed that ColDof34, ColDof20, ColDof28, ColDof35, ColDof42 and ColDof26 had the most protein interactions. The transcriptome analysis of C. oleifera seeds showed that 21 ColDof genes were involved in the growth and development process of C. oleifera seeds, and were expressed in 221 C. oleifera varieties. The results of qRT-PCR experiments treated with different concentrations NaCl and PEG6000 solutions indicated that ColDof1, ColDof2, ColDof14 and ColDof36 not only had significant molecular mechanisms for salt stress tolerance, but also significant molecular functions for drought stress tolerance in C. oleifera. The results of this study provide a reference for further understanding of the function of ColDof genes in C.oleifera.

Pressure Tuning and Sn Particle Size Optimization for Enhanced Performance in PbSnF4-Based All-Solid-State Fluoride Ion Batteries.

All-solid-state fluoride ion batteries (ASSFIBs) show remarkable potential as energy storage devices due to their low cost, superior safety, and high energy density. However, the poor ionic conductivity of F- conductor, large volume expansion, and the lack of a suitable anode inhibit their development. In this work, PbSnF4 solid electrolytes in different phases (ß- and γ-PbSnF4) are successfully synthesized and characterized. The ASSFIBs composed of ß-PbSnF4 electrolytes, a BiF3 cathode, and micrometer/nanometer size (µ-/n-) Sn anodes, exhibit substantial capacities. Compared to the µ-Sn anode, the n-Sn anode with nanostructure exhibits superior battery performance in the BiF3/ß-PbSnF4/Sn battery. The optimized battery delivers a high initial discharge capacity of 181.3 mAh g-1 at 8 mA g-1 and can be reversibly cycled at 40 mA g-1 with a high discharge capacity of over 100.0 mAh g-1 after 120 cycles at room temperature. Additionally, it displays high discharge capacities over 90.0 mAh g-1 with excellent cyclability over 100 cycles under -20 °C. Detailed characterization has confirmed that reducing Sn particle size and boosting external pressure are crucial for achieving good defluorination/fluorination behaviors in the Sn anode. These findings pave the way to designing ASSFIBs with high capacities and superior cyclability under different operating temperatures.

Simultaneous Rosiglitazone Release and Low-Density Lipoprotein Removal by Chondroitin Sodium Sulfate/Cyclodextrin/Poly(acrylic acid) Composite Adsorbents for Atherosclerosis Therapy.

Atherosclerosis (AS) is characterized by the accumulation of substantial low-density lipoprotein (LDL) and inflammatory response. Hemoperfusion is commonly employed for the selective removal of LDL from the body. However, conventional hemoperfusion merely focuses on LDL removal and does not address the symptom of plaque associated with AS. Based on the LDL binding properties of acrylated chondroitin sodium sulfate (CSA), acrylated beta-cyclodextrin (CD) and acrylic acid (AA), along with the anti-inflammatory property of rosiglitazone (R), the fabricated AA-CSA-CD-R microspheres could simultaneously release R and facilitate LDL removal for hemoperfusion. The AA and CSA offer electrostatic adsorption sites for LDL, while the CD provides hydrophobic adsorption sites for LDL and weak binding sites for R. According to the Sips model, the maximum static LDL adsorption capacity of AA-CSA-CD-R is determined to be 614.73 mg/g. In dynamic simulated perfusion experiments, AA-CSA-CD-R exhibits an initial cycle LDL adsorption capacity of 150.97 mg/g. The study suggests that the weakened inflammatory response favors plaque stabilization. The anti-inflammatory property of the microspheres is verified through an inflammation model, wherein the microsphere extracts are cocultured with mouse macrophages. Both qualitative analysis of iNOS\TNF-α and quantitative analysis of IL-6\TNF-α collectively demonstrate the remarkable anti-inflammatory effect of the microspheres. Therefore, the current study presents a novel blood purification treatment of eliminating pathogenic factors and introducing therapeutic factors to stabilize AS plaque.

Exosomes derived from adipose tissue-derived stem cells alleviated H2O2-induced oxidative stress and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells by inhibition of the mir-486-3p/Sirt6/Smad signaling pathway.

Hypertrophic scar (HS) is characterized by excessive collagen deposition and myofibroblasts activation. Endothelial-to-mesenchymal transition (EndoMT) and oxidative stress were pivotal in skin fibrosis process. Exosomes derived from adipose tissue-derived stem cells (ADSC-Exo) have the potential to attenuate EndoMT and inhibit fibrosis. The study revealed reactive oxygen species (ROS) levels were increased during EndoMT occurrence of dermal vasculature of HS. The morphology of endothelial cells exposure to H2O2, serving as an in vitro model of oxidative stress damage, transitioned from a cobblestone-like appearance to a spindle-like shape. Additionally, the levels of endothelial markers decreased in H2O2-treated endothelial cell, while the expression of fibrotic markers increased. Furthermore, H2O2 facilitated the accumulation of ROS, inhibited cell proliferation, retarded its migration and suppressed tube formation in endothelial cell. However, ADSC-Exo counteracted the biological effects induced by H2O2. Subsequently, miRNAs sequencing analysis revealed the significance of mir-486-3p in endothelial cell exposed to H2O2 and ADSC-Exo. Mir-486-3p overexpression enhanced the acceleration of EndoMT, its inhibitors represented the attenuation of EndoMT. Meanwhile, the target regulatory relationship was observed between mir-486-3p and Sirt6, whereby Sirt6 exerted its anti-EndoMT effect through Smad2/3 signaling pathway. Besides, our research had successfully demonstrated the impact of ADSC-Exo and mir-486-3p on animal models. These findings of our study collectively elucidated that ADSC-Exo effectively alleviated H2O2-induced ROS and EndoMT by inhibiting the mir-486-3p/Sirt6/Smad axis.

Nonthermal and reversible control of neuronal signaling and behavior by midinfrared stimulation.

Various neuromodulation approaches have been employed to alter neuronal spiking activity and thus regulate brain functions and alleviate neurological disorders. Infrared neural stimulation (INS) could be a potential approach for neuromodulation because it requires no tissue contact and possesses a high spatial resolution. However, the risk of overheating and an unclear mechanism hamper its application. Here we show that midinfrared stimulation (MIRS) with a specific wavelength exerts nonthermal, long-distance, and reversible modulatory effects on ion channel activity, neuronal signaling, and sensorimotor behavior. Patch-clamp recording from mouse neocortical pyramidal cells revealed that MIRS readily provides gain control over spiking activities, inhibiting spiking responses to weak inputs but enhancing those to strong inputs. MIRS also shortens action potential (AP) waveforms by accelerating its repolarization, through an increase in voltage-gated K+ (but not Na+) currents. Molecular dynamics simulations further revealed that MIRS-induced resonance vibration of -C=O bonds at the K+ channel ion selectivity filter contributes to the K+ current increase. Importantly, these effects are readily reversible and independent of temperature increase. At the behavioral level in larval zebrafish, MIRS modulates startle responses by sharply increasing the slope of the sensorimotor input-output curve. Therefore, MIRS represents a promising neuromodulation approach suitable for clinical application.

Wsp system oppositely modulates antibacterial activity and biofilm formation via FleQ-FleN complex in Pseudomonas putida.

Type VI secretion systems (T6SS) are specific antibacterial weapons employed by diverse bacteria to protect themselves from competitors. Pseudomonas putida KT2440 possesses a functional T6SS (K1-T6SS) and exhibits antibacterial activity towards a broad range of bacteria. Here we found that the Wsp signal transduction system regulated K1-T6SS expression via synthesizing the second messenger cyclic di-GMP (c-di-GMP), thus mediating antibacterial activity in P. putida. High-level c-di-GMP produced by Wsp system repressed the transcription of K1-T6SS genes in structural operon and vgrG1 operon. Transcriptional regulator FleQ and ATPase FleN functioned as repressors in the Wsp system-modulated K1-T6SS transcription. However, FleQ and FleN functioned as activators in biofilm formation, and Wsp system promoted biofilm formation largely in a FleQ/FleN-dependent manner. Furthermore, FleQ-FleN complex bound directly to the promoter of K1-T6SS structural operon in vitro, and c-di-GMP promoted the binding. Besides, P. putida biofilm cells showed higher c-di-GMP levels and lower antibacterial activity than planktonic cells. Overall, our findings reveal a mechanism by which Wsp system oppositely modulates antibacterial activity and biofilm formation via FleQ-FleN, and demonstrate the relationship between plankton/biofilm lifestyles and antibacterial activity in P. putida.

Second Messenger c-di-GMP Modulates Exopolysaccharide Pea-Dependent Phenotypes via Regulation of eppA Expression in Pseudomonas putida.

The exopolysaccharide (EPS) Pea is essential for wrinkly colony morphology, pellicle formation, and robust biofilm production in Pseudomonas putida. The second messenger cyclic diguanylate monophosphate (c-di-GMP) induces wrinkly colony morphology in P. putida through an unknown mechanism(s). Herein, we found that c-di-GMP modulates wrinkly colony morphology via the regulation of expression of eppA (PP_5586), a small individually transcribed gene of 177 bp, and this gene was adjacent to the upstream region of the pea cluster. Phenotype observation revealed that eppA was essential for Pea-dependent phenotypes. The deletion of eppA led to a smooth colony morphology and impaired biofilm, which was analogous to the phenotypes with loss of the entire pea operon. eppA expression was positively regulated by c-di-GMP via the transcriptional effector FleQ, and eppA was essential for the c-di-GMP-induced wrinkly colony morphology. Structure prediction results implied that EppA had two transmembrane regions, and Western blotting revealed that EppA was located on the cell membrane. Transcriptomic analysis indicated that EppA had no significant effect on the transcriptomic profile of P. putida. A bacterial two-hybrid (BTH) assay suggested that there was no direct interaction between EppA and the proteins in the pea cluster and adjacent operons. Overall, these findings reveal that EppA is essential for Pea-dependent phenotypes and that c-di-GMP modulates Pea-dependent phenotypes via regulation of eppA expression in P. putida. IMPORTANCE Microbe-secreted EPSs are high-molecular-weight polysaccharides that have the potential to be used as industrially important biomaterials. The EPS Pea in P. putida is essential for wrinkly colony morphology and pellicle formation. Here, we identified a function-unknown protein, EppA, which was also essential for Pea-dependent wrinkly colony morphology and pellicle formation, and EppA was probably involved in Pea secretion. Meanwhile, our results indicated that the second messenger c-di-GMP positively regulated the expression of EppA, resulting in Pea-dependent wrinkly colony morphology. Our results reveal the relationship of c-di-GMP, EppA, and Pea-dependent phenotypes and provide a possible pathway to construct genetically engineered strains for high Pea production.

Acute sleep deprivation increases inflammation and aggravates heart failure after myocardial infarction.

Sleep disorders have been observed among patients with heart failure. The aim of this study was to investigate whether acute sleep deprivation (SD) aggravates left heart function. Male C57B/L6 mice were assigned to four experimental groups. Ligation of the left anterior descending branch (LAD) caused myocardial infarction (MI) in mice in the LAD group and the LAD+SD group, while mice in the sham and sham+SD groups underwent the same surgery without ligation. Echocardiography was performed before and 8 weeks after ligation of the LAD to evaluate the left ventricular internal diameter at diastole (LVIDd), left ventricular internal diameter at systole (LVIDs), ejection fraction (EF), and fractional shortening (FS). Seven days of sleep deprivation induced using the modified single platform method resulted in a lower EF and FS and a higher LVIDd and LVIDs, as well as increased expression of the IL-1ß, IL-18, and IL-10 mRNAs in the left ventricular tissue of MI mice. ELISA also indicated higher levels of IL-1ß and IL-10 in the LAD+SD group. It was concluded that acute sleep deprivation induced cardiovascular alterations in cardiac structure and function in HF mice, accompanied by increased levels of inflammatory cytokines.

The two-component system TarR-TarS is regulated by c-di-GMP/FleQ and FliA and modulates antibiotic susceptibility in Pseudomonas putida.

Two-component systems (TCSs) are predominant means by which bacteria sense and respond to environment signals. Genome of Pseudomonas putida contains dozens of putative TCS-encoding genes, but phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterized function and transcriptional regulation of a conserved P. putida TCS, named TarR-TarS. TarS (PP_0769) encodes a potential histidine kinase, and tarR (PP_0768) encodes a potential response regulator. Protein-protein interaction assay and phosphorylation assay confirmed that TarR-TarS was a functional TCS. Growth assay under antibiotics revealed that TarR-TarS positively regulated bacterial resistance to multiple antibiotics. Pull-down assay revealed that TarR directly interacted with PP_0800 (a hypothetical protein) and GroEL (the chaperonin). GroEL played a positive role in antibiotic resistance, while PP_0800 seemed to have no effect on antibiotic resistance. The regulator FleQ indirectly activated tarR-tarS transcription. However, the second messenger c-di-GMP antagonized FleQ activation to inhibit tarR-tarS transcription. The sigma factor FliA directly activated tarR-tarS transcription via a consensus motif. These findings reveal function and transcriptional regulation of TarR-TarS, and enrich knowledge regarding the relationship between c-di-GMP and antibiotic susceptibility in P. putida.

Diagnostic Values of Intraoperative (1-84) Parathyroid Hormone Levels are Superior to Intact Parathyroid Hormone for Successful Parathyroidectomy in Patients With Chronic Kidney Disease.

OBJECTIVE: Persistent secondary hyperparathyroidism (SHPT) may occur because of residual cervicothoracic parathyroids in parathyroidectomy (PTX) patients with chronic kidney disease. We prospectively compared the predictive values of intraoperative plasma (1-84) parathyroid hormone (PTH) and intact PTH (iPTH) levels to improve the safety and efficacy of PTX. METHODS: We included 100 healthy controls, 162 stage 5 chronic kidney disease patients without SHPT, and 214 patients who underwent PTX because of SHPT. Plasma iPTH and (1-84) PTH levels were measured before incision (io-iPTH0 and io-[1-84]PTH0, respectively) and 10 minutes (io-iPTH10 and io-[1-84]PTH10, respectively) and 20 minutes (io-iPTH20 and io-[1-84]PTH20, respectively) after removing all parathyroids. The percentage reduction of iPTH and (1-84) PTH at 10 minutes (io-iPTH10% and io-[1-84]PTH10%, respectively) and 20 minutes (io-iPTH20%, and io-[1-84]PTH20%, respectively) was calculated. iPTH and (1-84) PTH were measured using second- and third-generation PTH assays, respectively. RESULTS: Compared with the controls and non-PTX patients, the PTX group had more obvious mineral metabolism disorders. There were 187 successful PTXs, 19 patients with persistent SHPT, and 8 patients lost to follow-up. The receiver operating characteristic curves revealed that io-(1-84)PTH10% >86.6% and io-(1-84)PTH20% >87.5% suggested successful PTX. The sensitivity of io-iPTH20% and io-(1-84)PTH20% were higher than those at the timepoint of 10 minutes. Moreover, the specificity and sensitivity of the (1-84) PTH reduction percentage were superior to that of iPTH. CONCLUSION: Intraoperative reduction percentages of plasma (1-84) PTH levels are superior to iPTH for accurately predicting successful PTX, especially at 20 minutes after all cervicothoracic parathyroids had been resected.

Novel in vivo and in vitro mechanisms of positive inotropic effect of atractylodin.

This study was to investigate the inotropic effect of atractylodin and its underlying mechanism. The cardiac pressure-volume loop (P-V loop), Langendroff-perfused isolated rat heart, patch-clamp, Ca2+ transient and western blot techniques were used. The results demonstrated that atractylodin (3 mg/kg, ip) remarkably increased the left ventricular stroke work, cardiac output, stroke volume, heart rate, ejection fraction, end-systolic pressure, peak rates of rise and fall of left ventricular pressures (+dP/dtmax , -dP/dtmax ), the slopes of end-systolic pressure-volume relationship (also named as end-systolic elastance, Ees) and reducing end-systolic volume and end-diastolic volume in the in vivo rat study. Also, atractylodin (3 mg/kg, ip) significantly decreased diastolic blood pressure and the arterial elastance (Ea) without significant systolic blood pressure change. In addition, atractylodin (0.1, 1, 10 µmol/L) also increased the isolated rat heart left ventricular developed pressure which is the difference between the systolic and diastolic pressure in non-pacing and pacing modes. Furthermore, JMV-2959 (1 µmol/L), a ghrelin receptor unbiased antagonist, blocked the increased left ventricular developed pressure of atractylodin in isolated rat hearts. Finally, atractylodin (5 µmol/L) increased the amplitude of Ca2+ transient by enhancing SERCA2a activity, the sarcoplasmic reticulum Ca2+ content and the phosphorylation of phospholamban at 16-serine. These results demonstrated that atractylodin had a positive inotropic effect by enhancing SERCA2a activity which might be mediated by acting ghrelin receptor in myocardium. In conclusion, atractylodin which had the positive inotropic effect and decreased diastolic blood pressure might serve as an agent for the treatment of heart failure in clinical settings.

Effects of parathyroidectomy on plasma PTH fragments and heart rate variability in stage 5 chronic kidney disease patients.

INTRODUCTION: Circulating intact parathyroid hormone (iPTH) levels include full-length (1-84) PTH and long C-PTH fragments, but primarily (7-84) PTH, which have been reported to have antagonistic effects on the bones and kidneys. However, their effects on the cardiovascular system remain unclear. In this study, the relationships between the plasma PTH fragments levels and heart rate variability (HRV) in stage 5 chronic kidney disease (CKD5) patients are explored. Furthermore, the effects of parathyroidectomy (PTX) on the above indices are investigated. METHODS: In this cross-sectional study, 164 healthy controls and 354 CKD5 patients, including 208 secondary hyperparathyroidism (SHPT) subgroup with PTX, were enrolled. Circulating (7-84) PTH levels were calculated by subtracting plasma (1-84) PTH levels from iPTH levels. The HRV parameters were measured using a 24-hour Holter. RESULTS: The baseline levels of plasma iPTH, (1-84) PTH, and (7-84) PTH in the CKD5 patients were 930.40 (160.65, 1792.50) pg/mL, 448.60 (99.62, 850.45) pg/mL, and 468.20 (54.22, 922.55) pg/mL, respectively. In the CKD5 patients, plasma (1-84) PTH levels were independently correlated with the standard deviation of the normal-to-normal R-R intervals (SDNN) and the standard deviation of the five-minute average of the normal R-R intervals (SDANN). With a median follow up time of 6.50 months after PTX in the SHPT patients (n = 30), improved SDNN and SDANN markers were related with decreased (1-84) PTH levels. Furthermore, an improved SDNN was related with decreased (7-84) PTH levels. CONCLUSIONS: The CKD5 patients' baseline (1-84) PTH levels were correlated with the SDNN and SDANN. After PTX, an improved SDNN was related with decreased (1-84) PTH and (7-84) PTH levels, while improved SDANN was related with decreased (1-84) PTH levels. No antagonistic effects of (1-84) PTH and (7-84) PTH on HRV were found in the CKD5 patients.

[Positive inotropic effect of phosphodiesterase type 9 inhibitor PF-04449613 in rats and its underlying mechanism].

This study aimed to explore the positive inotropic effect of phosphodiesterase type 9 (PDE9) inhibitor PF-04449613 in ratsand its cellular and molecular mechanisms. The heart pressure-volume loop (P-V loop) analysis was used to detect the effects of PF-04449613 on rat left ventricular pressure-volume relationship, aortic pressures and peripheral vessel resistance in healthy rats. The Langendorff perfusion of isolated rat heart was used to explore the effects of PF-04449613 on heart contractility. The cardiomyocyte sarcoplasmic reticulum (SR) Ca2+ transients induced by field stimulation and caffeine were used to analyze the mechanism underlying the effect of PF-04449613 using Fluo-4 AM as a Ca2+ indicator. The results indicated as follows: (1) PF-04449613 (5.5 mg/kg, ip) significantly increased the stroke work, cardiac output, stroke volume, end-systolic pressure and ejection fraction (P < 0.05), and decreased the end-systolic volume, end-diastolic volume and end-diastolic pressure (P < 0.05). Meanwhile, the systolic blood pressure was increased and diastolic blood pressure and arterial elastance were decreased after PF-04449613 treatment (P < 0.05). (2) PF-04449613 (0.001, 0.01, 0.1, 1 µmol/L) significantly increased the left ventricular developed pressure (LVDP) in a concentration-dependent manner in vitro (P < 0.05). (3) PF-04449613 (5 µmol/L) significantly increased the amplitude of SR Ca2+ transients mediated by facilitating sarcoplasmic reticulum Ca2+-ATPase-2a (SERCA2a) (P < 0.05). (4) PF-04449613 (5 µmol/L) decreased the SR Ca2+ leak rate via ryanodine receptor 2 (RyR2) (P < 0.05). In conclusion, PF-04449613 exerted positive inotropic effect both in vivo and in vitro by enhancing SERCA2a activity.

A crosstalk between c-di-GMP and cAMP in regulating transcription of GcsA, a diguanylate cyclase involved in swimming motility in Pseudomonas putida.

The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). Pseudomonas putida has dozens of DGC/PDE-encoding genes in its genome, but the phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterize function and transcriptional regulation of a P. putida c-di-GMP-metabolizing enzyme, GcsA. GcsA consists of two per-ARNT-sim (PAS) domains, followed by a canonical conserved central sequence pattern (GGDEF) domain and a truncated EAL domain. In vitro analysis confirmed the DGC activity of GcsA. The phenotypic observation revealed that GcsA inhibited swimming motility in an FlgZ-dependent manner. In terms of transcriptional regulation, gcsA was found to be cooperatively regulated by c-di-GMP and cAMP via their effectors, FleQ and Crp respectively. The transcription of gcsA was promoted by c-di-GMP and inhibited by cAMP. In vitro binding analysis revealed that FleQ indirectly regulated the transcription of gcsA, while Crp directly regulated the transcription of gcsA by binding to its promoter. Besides, an inverse relationship between the cellular c-di-GMP and cAMP levels in P. putida was confirmed. These findings provide basic knowledge regarding the function and transcriptional regulation of GcsA and demonstrate a crosstalk between c-di-GMP and cAMP in the regulation of the expression of GcsA in P. putida.

Expression and Clinical Significance of POLR1D in Colorectal Cancer.

PURPOSE: RNA polymerase I subunit D (POLR1D) is involved in the synthesis of ribosomal RNA precursors and small RNAs, but its mechanism in the development and progression of colorectal cancer (CRC) remains ambiguous. Thus, this research aimed to investigate POLR1D's expression and significance in human CRC patients and evaluate its association with clinicopathological characteristics. METHODS: Matched fresh-frozen cancerous and non-cancerous tissues were collected from 100 patients diagnosed with CRC. Immunohistochemical, Western blot, and quantitative real-time polymerase chain reaction analyses were adopted to validate the correlation between POLR1D expression and clinicopathological parameters in CRC tissues and adjacent normal tissues (ANTs). RESULTS: POLR1D expression in CRC tissues was significantly higher than in the ANTs. χ2 test and Spearman's correlative analysis showed that a high POLR1D expression is significantly associated with clinical stage, Dukes stage, tumor differentiation, depth of invasion, and metastasis (p < 0.05). It is not correlated with gender, age, and tumor location and size (p > 0.05). Kaplan-Meier survival curves show that the overall survival (OS) time for the low expression group is remarkably longer than for the high expression group (p < 0.0015). Univariate and multivariate analyses indicate that a high POLR1D expression is an independent prognostic factor for poor OS (p = 0.000). CONCLUSION: The findings of this study strongly indicate that POLR1D plays a pivotal role in the occurrence and progression of CRC. It might be an independent adverse prognostic factor for CRC patients and could serve as a potential therapeutic target for clinical diagnosis in CRC and anticancer drug development.

Using RIGHT (Reporting Items for Practice Guidelines in Healthcare) to evaluate the reporting quality of WHO guidelines.

BACKGROUND: Without adequate reporting of research, valuable time and resources are wasted. In the same vein, adequate reporting of practice guidelines to optimise patient care is equally important. Our study examines the quality of reporting of published WHO guidelines, over time, using the RIGHT (Reporting Items for Practice Guidelines in HealThcare) reporting checklist. METHODS: We examined English-language guidelines approved by the WHO Guidelines Review Committee from inception of the committee in 2007 until 31 December 2017. Pairs of independent, trained reviewers assessed the reporting quality of these guidelines. Descriptive data were summarised with frequencies and percentages. RESULTS: We included 182 eligible guidelines. Overall, 25 out of the 34 RIGHT items were reported in 75% or more of the WHO guidelines. The reporting rates improved over time. Further, 90% of the guidelines reported document type in the title. The identification of evidence, the rationale for recommendations and the review process were reported in more than 80% of guidelines. The certainty of the evidence using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system was assessed in 81% of the guidelines assessed. While 82% of guidelines reported funding sources, only 25% mentioned the role of funders. CONCLUSIONS: WHO guidelines provide adequate reporting of many of the RIGHT items and reporting has improved over time. WHO guidelines compare favourably to guidelines produced by other organisations. However, reporting can be further improved in a number of areas.

High c-di-GMP promotes expression of fpr-1 and katE involved in oxidative stress resistance in Pseudomonas putida KT2440.

Oxidative stress is an unavoidable consequence of interactions with various reactive oxygen species (ROS)-inducing agents that would damage cells or even cause cell death. Bacteria have developed defensive systems, including induction of stress-sensing proteins and detoxification enzymes, to handle oxidative stress. Cyclic diguanylate (c-di-GMP) is a ubiquitous intracellular bacterial second messenger that coordinates diverse aspects of bacterial growth and behavior. In this study, we revealed a mechanism by which c-di-GMP regulated bacterial oxidative stress resistance in Pseudomonas putida KT2440. High c-di-GMP level was found to enhance bacterial resistance towards hydrogen peroxide. Transcription assay showed that expression of two oxidative stress resistance genes, fpr-1 and katE, was promoted under high c-di-GMP level. Deletion of fpr-1 and katE both decreased bacterial tolerance to hydrogen peroxide and weakened the effect of c-di-GMP on oxidative stress resistance. The promoted expression of fpr-1 under high c-di-GMP level was caused by increased cellular ROS via a transcriptional regulator FinR. We further demonstrated that the influence of high c-di-GMP on cellular ROS depend on the existence of FleQ, a transcriptional regulatory c-di-GMP effector. Besides, the regulation of katE by c-di-GMP was also FleQ dependent in an indirect way. Our results proved a connection between c-di-GMP and oxidative stress resistance and revealed a mechanism by which c-di-GMP regulated expression of fpr-1 and katE in P. putida KT2440.

FinR Regulates Expression of nicC and nicX Operons, Involved in Nicotinic Acid Degradation in Pseudomonas putida KT2440.

Many proteobacteria harbor FinR homologues in their genomes as putative LysR-type proteins; however, the function of FinR is poorly studied except in the induction of fpr-1 under superoxide stress conditions in Pseudomonas putida and Pseudomonas aeruginosa Here, by analyzing the influence of finR deletion on the transcriptomic profile of P. putida KT2440 through RNA sequencing and real-time quantitative PCR (RT-qPCR), we found 11 operons that are potentially regulated by FinR. Among them, the expression of nicC and nicX operons, which were reported to be responsible for the aerobic degradation of nicotinic acid (NA), was significantly decreased in the finR mutant, and complementation with intact finR restored the expression of the two operons. The results of bacterial NA utilization demonstrated that the deletion of finR impaired bacterial growth in minimal medium supplemented with NA/6HNA (6-hydroxynicotinic acid) as the sole carbon source and that complementation with intact finR restored the growth of the mutant strain. The expression of nicC and nicX operons was previously revealed to be repressed by the NicR repressor and induced by NA/6HNA. Our transcriptional assay revealed that the deletion of finR weakened the induction of nicC and nicX by NA/6HNA. Meanwhile, the deletion of finR largely decreased the effect of nicR deletion on the expression of nicC and nicX operons. These results suggest that finR plays a positive role and cooperates with NicR in the regulation of nicC and nicX operons. In vitro experiments showed that both FinR and NicR bound to nicX and nicC promoter regions directly. The results of this study deepened our knowledge of FinR function and nicotinic acid degradation in P. putidaIMPORTANCE This study analyzed the influence of finR deletion on the transcriptomic profile of Pseudomonas putida KT2440. The FinR regulator is widely distributed but poorly studied in diverse proteobacteria. Here, we found 11 operons that potentially are regulated by FinR in KT2440. We further demonstrated that FinR played a positive role and cooperated with the NicR repressor in bacterial nicotinic acid (NA) degradation via regulating the expression of nicC and nicX operons. Furthermore, a transcriptomic analysis also indicated a potentially negative role of FinR in the expression of the hut cluster involved in bacterial histidine utilization. The work deepened our knowledge of FinR function and nicotinic acid degradation in P. putida.