Development of the Low-Temperature Inducible System for Recombinant Protein Production in Escherichia coli Nissle 1917.

The pET system is commonly used for producing foreign proteins in Escherichia coli, but its reliance on the costly and metabolically demanding inducer IPTG limits its industrial use. This study engineered a low-temperature inducible system (LTIS) in E. coli Nissle 1917 (EcN) by combining the T7 expression system with the thermal inducible mechanism CI857-λPRPL to generate the new LTIS strain, ENL7P. The strain ENL7P-sfGFP-Km underwent overnight culture at 37 °C for 14-16 h, followed by subculturing at 30 °C for 24 h. This resulted in a notable 5.53-fold increase in the sfGFP induction rate when the strain was cultivated under 37-30 °C conditions. Moreover, gene expression was induced using a two-stage strategy. Initially, the strain was cultured overnight at 39 °C for 14-16 h, followed by a subculture at 30 °C for 6 h, and finally, another subculture at 30 °C for 24 h. This cultivation strategy led to an impressive 158.37-fold induction rate for sfGFP. Similar effects could be achieved through utilization of the LTIS system for inducing the production of thermophilic trehalose synthase from Thermus antranikianii (TaTS). The results of this study proved that the LTIS system has the potential for industrial applications.

Inflammatory myofibroblastic tumor of the heart in an older woman with paroxysmal atrial fibrillation: a case report and review of the literature.

Inflammatory myofibroblastic tumors (IMTs) of the heart are rarely observed in the eldly. We report a case involving an elderly woman with an IMT situated on the right atrial wall. The tumor was fully excised. The patient had a smooth recovery post-surgery and remained free of recurrence for three years.

GP73 enhances the ox-LDL-induced inflammatory response in THP-1 derived macrophages via affecting NLRP3 inflammasome signaling.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease with its molecular basis incompletely understood. Here, we determined whether the Golgi phosphoprotein 73 (GP73), a novel protein highly related to inflammation and disrupted lipid metabolism, was involved in the development of atherosclerosis. METHODS: Public microarray databases of human vascular samples were analyzed for expression patterns. Apolipoprotein-E-gene-deficient (ApoE-/-) mice (8-week-old) were randomly assigned to either a chow diet group or a high-fat diet group. The levels of serum GP73, lipid profiles and key inflammatory cytokines were determined by ELISA. The aortic root plaque was isolated and used for by Oil Red O staining. PMA-differentiated THP-1 macrophages were transfected with GP73 small interfering RNA (siRNA) or infected with adenovirus expressing GP73, and then stimulated with oxidized low density lipoprotein (ox-LDL). The expressions of pro-inflammatory cytokines and signal pathway key targets were determined by ELISA kit and Western blot respectively. In addition, ichloro-dihydro-fluorescein diacetate (DCFH-DA) was used to measure the intracellular ROS levels. RESULTS: The expressions of GP73 and NLRP3 were substantially upregulated in human atherosclerotic lesions. There were significant linear correlations between GP73 and inflammatory cytokines expressions. High-fat diet-induced atherosclerosis and increased levels of plasma inflammatory mediators (IL-1ß, IL-18, and TNF-α) were observed in ApoE-/- mice. Besides, the expressions of GP73 in the aorta and serum were significantly upregulated and positively correlated with the NLRP3 expression. In the THP-1 derived macrophages, ox-LDL treatment upregulated the expressions of GP73 and NLRP3 proteins and activated the inflammatory responses in a concentration-dependent and time-dependent manner. Silencing of GP73 attenuated the inflammatory response and rescued the decreased migration induced by ox-LDL, inhibiting the NLRP3 inflammasome signaling and the ROS and p-NF-κB activation. CONCLUSIONS: We demonstrated that GP73 promoted the ox-LDL-induced inflammation in macrophages by affecting the NF-κB/NLRP3 inflammasome signaling, and may play a role in atherosclerosis.

Targeting efficacy of spironolactone in patients with heart failure with preserved ejection fraction: the TOPCAT study.

AIMS: We aimed to explore the heterogeneous treatment effects (HTEs) for spironolactone treatment in patients with Heart failure with preserved ejection fraction (HFpEF) and examine the efficacy and safety of spironolactone medication, ensuring a better individualized therapy. METHODS AND RESULTS: We used the causal forest algorithm to discover the heterogeneous treatment effects (HTEs) from patients in the Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist (TOPCAT) trial. Cox regressions were performed to assess the hazard ratios (HRs) of spironolactone medication for cardiovascular death and drug discontinuation in each group. The causal forest model revealed three representative covariates and participants were partitioned into four subgroups which were Group 1 (baseline BMI ≤ 31.71 kg/m2 and baseline ALP ≤ 80 U/L, n = 759); Group 2 (BMI ≤ 31.71 kg/m2 and ALP > 80 U/L, n = 1088); Group 3 (BMI > 31.71 kg/m2 , and WBC ≤ 6.6 cells/µL, n = 633); Group 4 (BMI > 31.71 kg/m2 and WBC > 6.6 cells/µL, n = 832), respectively. In the four subgroups, spironolactone therapy reduced the risk of cardiovascular death in high-risk group (Group 4) with both high BMI and WBC count (HR: 0.76; 95% CI 0.58 to 0.99; P = 0.045) but increased the risk in low-risk group (Group 1) with both low BMI and ALP (HR: 1.45; 95% CI 1.02 to 2.07; P = 0.041; P for interaction = 0.020) but showed similar risk of drug discontinuation (P for interaction = 0.498). CONCLUSION: Our study manifested the HTEs of spironolactone in patients with HFpEF. Spironolactone treatment in HFpEF patients is feasible and effective in patients with high BMI and WBC while harmful in patients with low BMI and ALP. Machine learning model could be meaningful for improved categorization of patients with HFpEF, ensuring a better individualized therapy in the clinical setting.

The efficacy of Lactobacillus acidophilus and rhamnosus in the reduction of bacterial load of Helicobacter pylori and modification of gut microbiota-a double-blind, placebo-controlled, randomized trial.

BACKGROUND: Probiotics may alter the gut microbiota and may reduce antibiotic-related dysbiosis after H. pylori eradication. However, whether probiotics are effective in reducing the bacterial load of H. pylori and modifying the gut microbiota remains unknown. We aimed to assess the efficacy of Lactobacillus acidophilus and Lactobacillus rhamnosus in reducing the bacterial load of H. pylori and modifying the gut microbiota. MATERIALS AND METHODS: In this double-blind, randomized, placebo-controlled trial, we recruited 40 adult subjects with moderate to high bacterial loads of H. pylori, defined as a mean delta over baseline (DOB) value of the 13 C-urea breath test (13 C-UBT) of 10 or greater every 4 days 6 times. Eligible subjects were randomized in a 1:1 ratio to receive either probiotics containing Lactobacillus acidophilus and Lactobacillus rhamnosus or placebo twice daily for 4 weeks. 13 C-UBT was measured weekly from the beginning of treatment to 2 weeks after treatment. Amplification of the V3 and V4 hypervariable regions of the 16S rRNA was performed for fecal microbiota. RESULTS: A total of 40 subjects were randomized to receive probiotics or placebo. The DOB value was significantly lower in the probiotic group than in the placebo group after 4 weeks of treatment (26.0 vs. 18.5, p = .045). The DOB value was significantly reduced compared to that at baseline in the probiotic group (18.5 vs. 26.7, p = .001) but not in the placebo group (26.0 vs. 25.0, p = .648). However, the eradication rate for H. pylori was 0% in both groups. There was no significant difference in the DOB values between the two groups 1 and 2 weeks after discontinuation of the probiotics. There were also no significant changes observed in the α-diversity and ß-diversity at week 4 compared to baseline in the probiotic group (p = .77 and 0.91) and the placebo group (p = .26 and 0.67). CONCLUSIONS: Although the use of Lactobacillus acidophilus and Lactobacillus rhamnosus may reduce the bacterial load of H. pylori, there were no significant changes in the composition of gut microbiota. This trial is registered with ClinicalTrials.gov, NCT02725138.

Association between serum cystatin C level and hemodynamically significant aortic stenosis: a prospective cohort study.

BACKGROUND: Cystatin C (CysC) is a cysteine protease inhibitor involved in proteins catabolism and plays an essential role in human vascular pathophysiology. CysC may also increase the risk of aortic stenosis (AS), but limited studies have reported on this association. This study aimed to investigate if elevated serum CysC levels are associated with hemodynamically significant AS. METHODS: Serum CysC levels were estimated in 4,791 participants, samples were collected in 1990-1992. The study population was divided into quintile groups. Follow-up continued in 2011-2013 when participants returned for echocardiography examination. Incidence of aortic valve disease (AVD) was ascertained by Doppler echocardiography through the end of 2013. AVD defined in hemodynamic progression was assessed and classified as aortic sclerosis, mild stenosis, and moderate-to-severe stenosis. RESULTS: Overall, a total of 4,791 participants (mean age: 54.8 ± 5.0 years, females: 57.6%, blacks: 8.2%) were included in this study. During a follow-up of 21 years, we identified 736 cases (15.4%) of aortic sclerosis, 194 cases (4.0%) of mild stenosis, and 42 cases (0.7%) of moderate-to-severe stenosis. Compared with serum CysC levels within individual quintile groups, the odds ratio (OR) was per standard deviation associated with an increased incidence of AVD (OR = 1.15, 95% CI: 1.05-1.26,P = 0.002). CONCLUSIONS: In this large population-based study, an increased serum CysC levels is independently associated with the incidence of hemodynamically significant AS. However, this association appears not to extend to patients with extremely high serum CysC levels and necessitate further investigation.

Production and characterization of a recombinant thermophilic trehalose synthase from Thermus antranikianii.

Trehalose synthase converts maltose into trehalose in a single conversion step via intramolecular transformation and is thus useful for industrial production. In this study, we synthesized a thermophilic trehalose synthase from Thermus antranikianii (TaTS), which was recombinantly expressed in Escherichia coli BL21(DE3). The recombinant TaTS showed the highest activity at pH 7.0 and 60°C, with the maximum trehalose yield (76.8%) obtained at pH 7.0 and 30°C. TaTS activity was stable over a wide pH and temperature range of 6-10 and 4-70°C, respectively, over 6 h of incubation. The enzyme activity was strongly inhibited by Co2+, Cu2+, Zn2+, sodium dodecyl sulfate, and Tris. TaTS showed a 1.48-fold higher catalytic efficiency (kcat/Km) for maltose than for trehalose. Overall, these results demonstrate the good application potential of the recombinant enzyme TaTS in the efficient conversion of trehalose from maltose, with superior environmental tolerance to other trehalose synthases reported.

How moles walk; it's all thumbs.

A recurring theme in the evolution of tetrapods is the shift from sprawling posture with laterally orientated limbs to erect posture with the limbs extending below the body. However, in order to invade particular locomotor niches, some tetrapods secondarily evolved a sprawled posture. This includes moles, some of the most specialized digging tetrapods. Although their forelimb anatomy and posture facilitates burrowing, moles also walk long distances to forage for and transport food. Here, we use X-ray Reconstruction Of Moving Morphology (XROMM) to determine if the mole humerus rotates around its long axis during walking, as it does when moles burrow and echidnas walk, or alternatively protracts and retracts at the shoulder in the horizontal plane as seen in sprawling reptiles. Our results reject both hypotheses and demonstrate that forelimb kinematics during mole walking are unusual among those described for tetrapods. The humerus is retracted and protracted in the parasagittal plane above, rather than below the shoulder joint and the 'false thumb', a sesamoid bone (os falciforme), supports body weight during the stance phase, which is relatively short. Our findings broaden our understanding of the diversity of tetrapod limb posture and locomotor evolution, demonstrate the importance of X-ray-based techniques for revealing hidden kinematics and highlight the importance of examining locomotor function at the level of individual joint mobility.

How moles destroy your lawn: the forelimb kinematics of eastern moles in loose and compact substrates.

The interplay between morphological specialization and kinematic flexibility is important for organisms that move between habitats within different substrates. Burrowing is energetically expensive and requires substantial interaction with soil to dislodge and transport it. True moles (Talpidae) have extraordinary forelimb morphologies and a unique ability to dig in loose as well as compact soils, yet we know little of how moles coordinate their forelimb joint kinematics when digging in soils of different compactness. Using marker-based X-ray Reconstruction of Moving Morphology (XROMM), we tested the hypothesis that moles burrow using different forelimb kinematics in loose and compact substrates. We predicted that moles raise mounds of loose soil by performing powerful compacting strokes mainly with long-axis rotation of the humerus (i.e. pronation/supination), but shear compact soil away by performing scratching strokes involving amplified elbow extension, similar to most scratching diggers. We also predicted that in both types of substrate, moles displace soil rearward like other mammalian diggers. Our results support our hypothesis but not the predictions. Eastern moles (Scalopus aquaticus) move substrates upward using compacting strokes in loose substrates and outward from the body midline using scratching strokes in compact substrates; unlike the digging strokes of most mammalian forelimb diggers, the power-stroke of moles itself does not displace substrates directly rearward. Compacting and scratching strokes involve similar ranges of humeral pronation and retraction at the scapulohumeral (shoulder) joint, yet the movements at the elbow and carpal joints differ. Our results demonstrate that the combination of stereotypic movements of the shoulder joint, where the largest digging muscles are located, and flexibility in the elbow and carpal joints makes moles extremely effective diggers in both loose and compact substrates.

The Changes in Metabolisms of Membrane Lipids and Phenolics Induced by Phomopsis longanae Chi Infection in Association with Pericarp Browning and Disease Occurrence of Postharvest Longan Fruit.

This study investigated the changes in metabolisms of membrane lipids and phenolics caused by Phomopsis longanae Chi infection in association with pericarp browning and fruit disease occurrence of postharvest longans. Compared with the uninoculated-longans, the longans inoculated by P. longanae exhibited higher cellular membrane permeability; higher PLD, lipase, and LOX activities; and higher levels of saturated fatty acids (SFAs) and phosphatidic acid but lower levels of phosphatidylinositol, phosphatidylcholine, and unsaturated fatty acids (USFAs). Additionally, the longans inoculated by P. longanae showed higher activities of POD and PPO but a lower amount of total phenolics. These findings suggested that infection of P. longanae enhanced activities of PLD-, lipase-, and LOX- stimulated degradations of membrane lipids and USFAs, which destroyed the integrity of the cell membrane structure, resulting in enzymatic browning by contact of phenolics with POD and PPO, and resulting in reduction of resistance to pathogen infection and accordingly accelerated disease occurrence of postharvest longan fruit.

Hydrogen Peroxide Induced Changes in Energy Status and Respiration Metabolism of Harvested Longan Fruit in Relation to Pericarp Browning.

Energy status and respiration metabolism of "Fuyan" longan fruit treated by hydrogen peroxide (H2O2) and their relationship to pericarp browning were studied. The results displayed that H2O2 significantly increased the respiration rate, increased activities of respiratory terminal oxidases like cytochrome C oxidase (CCO) and ascorbic acid oxidase (AAO), decreased NAD kinase activity, maintained lower contents of NADP and NADPH as well as higher amounts of NAD and NADH, and accelerated the decrease of energy charge. These results gave convincing evidence that the treatment of H2O2 for accelerating longan pericarp browning was due to an increase of energy deficiency, an increase of respiratory metabolic pathways of Embden-Meyerhof pathway (EMP) and tricarboxylic acid (TCA) cycle, a decrease of pentose phosphate pathway (PPP) of respiratory pathway, and an increase of activities of respiratory terminal oxidases like CCO and AAO.

Simultaneous determination of caffeine and some selected polyphenols in Wuyi Rock tea by high-performance liquid chromatography.

The primary taste and healthy benefits of tea are mainly attributed to tea polyphenols and caffeine. Due to very many kinds of flavonoid glycosides in tea and the lack of commercial standards of flavonoid glycosides, it is critical to develop a rapid and cheap method for determining flavonoid glycosides of tea. Contents of myricetin glycosides and quercetin glycosides in Wuyi Rock tea were determined by detecting contents of corresponding myricetin and quercetin. Optimizing hydrolysis conditions for hydrolyzing flavonoid glycosides to their corresponding flavonols including quercetin and myricetin in Wuyi Rock tea was a key technology for detecting contents of corresponding myricetin and quercetin. The results showed that hydrolysis at 2 mol/L HCl solution and at 90 °C for 1 h was an optimizing condition for hydrolyzing flavonoid glycosides to myricetin and quercetin in Wuyi Rock tea. Caffeine and seven kinds of polyphenols (GA, EGC, C, EGCG, EC, ECG, and CGA) in 20 samples of Wuyi Rock tea were simultaneously determined using a simple and fast reverse-phase high-performance liquid chromatography procedure coupled with photodiode array detector (RP-HPLC-PDAD). The results indicated that there were significant (P < 0.05) differences of ECG, CGA, ECG, and myricetin glycosides in 'Wuyi Rougui' and 'Wuyi Shuixian', which were credited with causing the difference in taste between these two cultivar of Wuyi Rock tea. The study may be useful for clarifying the cause of "cultivated varieties flavor" of Wuyi Rock tea.

Inhibitory effects of propyl gallate on tyrosinase and its application in controlling pericarp browning of harvested longan fruits.

Tyrosinase (EC 1.14.18.1), also known as polyphenol oxidase (PPO), is a key enzyme in pigment biosynthesis of organisms. The inhibitory effects of propyl gallate on the activity of mushroom tyrosinase and effects of propyl gallate on pericarp browning of harvested longan fruits in relation to phenolic metabolism were investigated. The results showed that propyl gallate could potently inhibit diphenolase activity of tyrosinase. The inhibitor concentration leading to 50% activity lost (IC50) was determined to be 0.685 mM. Kinetic analyses showed that propyl gallate was a reversible and mixed type inhibitor on this enzyme. The inhibition constants (K(IS) and K(I)) were determined to be 2.135 and 0.661 mM, respectively. Furthermore, the results also showed that propyl gallate treatment inhibited activities of PPO and POD in pericarp of harvested longan fruits, and maintained higher contents of total phenol and flavonoid of longan pericarp. Moreover, propyl gallate treatment also delayed the increases of browning index and browning degree in pericarp of harvested longan fruits. Therefore, application of propyl gallate may be a promising method for inhibiting tyrosinase activity, controlling pericarp browning, and extending shelf life of harvested longan fruits.

Inhibitory effects of naphthols on the activity of mushroom tyrosinase.

Tyrosinase (EC 1.14.18.1), a copper-containing multifunctional oxidase, was known to be a key enzyme for biosynthesis in fungi, plants and animals. In this work, the inhibition properties α-naphthol and ß-naphthol toward the activity of tyrosinase have been evaluated, and the effects of α-naphthol and ß-naphthol on monophenolase and diphenolase activity of tyrosinase have been investigated. The results showed that both α-naphthol and ß-naphthol could potently inhibit both monophenolase activity and diphenolase activity of mushroom tyrosinase, and that ß-naphthol exhibited stronger inhibitory effect against tyrosinase than α-naphthol. For monophenolase activity, ß-naphthol could not only lengthen the lag time but also decrease the steady-state activity, while α-naphthol just only decreased the steady-state activity. For diphenolase activity, both α-naphthol and ß-naphthol displayed revisible inhibition. Kinetic analyses showed that both α-naphthol and ß-naphthol were competetive inhibitors.