Monophosphate Content Research Articles

Application of hydrogen-rich water maintains red pitaya fruit quality through regulation of ROS and energy metabolism

Hydrogen-rich water (HRW) treatment has been shown to enhance the shelf life of fruit and vegetables. This study aimed to investigate the effects of HRW on improving the quality of red pitaya and its underlying regulatory mechanisms. The results revealed that applying HRW at a 70% concentration reduced decay and weight loss rates, slowed the respiratory rate, and delayed the decline in titratable acidity and total soluble solids. Additionally, HRW treatment mitigated the increase in superoxide anion production and hydrogen peroxide levels, while enhancing antioxidant enzyme activities and gene expression, including those of peroxidase, superoxide dismutase, glutathione peroxidase, catalase, ascorbate peroxidase, monodehydroascorbate reductase, and glutathione reductase. Non-enzymatic antioxidant compounds such as ascorbate and glutathione also increased. Moreover, RT-qPCR analysis showed that HRW treatment upregulated HcMYB6 and downregulated HcR2R3MYB. Dual-luciferase reporter assays indicated that HcMYB6 activated the expression of HcSOD and HcCAT, whereas HcR2R3MYB repressed their expression. These findings suggest that HcMYB6 and HcR2R3MYB may be involved in reactive oxygen species metabolism, with HcMYB6 being induced and HcR2R3MYB inhibited by HRW treatment. Furthermore, HRW treatment led to increased adenosine triphosphate and adenosine diphosphate levels, a decrease in adenosine monophosphate content, and elevated gene expression and activity of Ca2⁺-ATPase, succinate hydrogenase, H⁺-ATPase, and cytochrome C. Collectively, these results demonstrate that HRW treatment effectively reduces oxidative damage and improves energy status, thereby preserving the quality of red pitaya fruit.

Effects of the phosphodiesterase 2 inhibitor BI 474121 on central nervous system cyclic guanosine monophosphate concentrations: Translational studies.

Phosphodiesterase 2 (PDE2) regulates intracellular cyclic adenosine monophosphate and guanosine monophosphate (cAMP/cGMP) levels, which contribute to processes crucial for learning and memory. BI 474121, a potent and selective PDE2 inhibitor, is in development for treating cognitive impairment associated with schizophrenia. The effects of BI 474121 on cGMP concentrations were first assessed in rat cerebrospinal fluid (CSF) to demonstrate central nervous system (CNS) and functional target engagement. Next, a Phase I study in healthy participants assessed the pharmacokinetics of BI 474121 in CSF vs. plasma, the pharmacodynamics of BI 474121 by measuring cGMP concentrations in the CSF, and the safety of BI 474121. In rats, BI 474121 was associated with a dose-dependent increase (71% at the highest dose tested [3.0mg kg-1]) in cGMP levels in the CSF relative to vehicle (P < 0.001). In healthy participants, the maximum-measured concentration CSF-to-plasma ratio for BI 474121 exposure was similar following single oral doses of BI 474121 2.5, 10, 20 and 40 mg (dose-adjusted geometric mean: 8.96% overall). BI 474121 2.5-40 mg administration in healthy participants also increased cGMP levels in CSF (maximum exposure-related change from baseline ratio, BI 474121: 1.44-2.20 vs. placebo: 1.26). The most common treatment-emergent adverse event (AE) was mild-to-moderate post-lumbar puncture syndrome, which resolved with standard treatment. No AEs of special interest were observed. BI 474121 crosses the blood-brain barrier to inhibit PDE2, supporting cGMP as a translational marker to monitor CNS target engagement. These findings promote further clinical development of BI 474121. gov number (NCT04672954).

The regulatory effects of arginine on ovary development in the Chinese mitten crab Eriocheir sinensis

This study aimed to investigate the effects of arginine (Arg) on the vitellogenesis and ovarian maturation of Chinese mitten crab. Six isonitrogenous and isolipidic diets (1.83, 2.52, 3.26, 3.96, 4.81 and 5.52 % Arg) were fed to adult female Chinese mitten crab (60.05 ± 4.33 g). The feeding period lasted for eleven weeks. The results showed that gonadosomatic index of crabs increased with increasing dietary Arg concentrations from 1.83 % to 2.52 % and significantly decreased from 2.52 % to 5.52 %. The quadratic regression analysis based on gonadosomatic index estimated that the optimal Arg requirement for adult female crabs was 2.60 %. Dietary supplementation with 2.52 % Arg significantly increased the crude protein concentration in the ovary. A dose of 2.52 % Arg significantly decreased the vitellogenesis-inhibiting hormone (VIH) content in the hemolymph. Arg (2.52 %) significantly upregulated the Vg and protein kinase A (PKA) gene expressions and increased the cyclic adenosine monophosphate (cAMP) concentration in the hepatopancreas. Moreover, dietary supplementation with 2.52 % Arg significantly decreased the cyclic guanosine monophosphate (cGMP) concentration and downregulated protein kinase G (PKG) and p38 mitogen-activated protein kinase (p38 MAPK) gene expression. Arg at 2.52 % significantly increased hemolymph Vg content and the gene expression of the vitellogenin receptor (VgR). In conclusion, Arg at 2.52 % improved vitellogenesis and ovarian maturation by reducing the VIH content and regulating Vg expression via the cGMP/cAMP pathway.

Effect of different processing steps in the production of beer fish on volatile flavor profile and their precursors determined by HS-GC-IMS, HPLC, E-nose, and E-tongue

Beer fish is characterized by its distinctive spicy flavor and strong beer aroma. Currently, there is a lack of comprehensive research analyzing the changes in taste and volatile compounds that occur during the processing of beer fish. Thus, this study used HS-GC-IMS, electronic tongue, and electronic nose to investigate the changes in flavor components during various processing stages of beer fish. The obtained results were subsequently analyzed using multivariate statistical analysis. The results showed that the final beer fish product (SF) had the greatest amount of free amino acids (888.28 mg/100 g), with alanine, glutamic acid, and glycine contributing to the taste of SF. The inosine monophosphate (IMP) content of beer fish meat varied noticeably depending on processing stages, with deep-fried fish (FF) having the greatest IMP content (61.93 mg/100 g), followed by the final product (SF) and ultrasonic-cured fish (UF). A total of 67 volatiles were detected by GC-IMS, mainly consisting of aldehydes, ketones, and alcohols, of which aldehydes accounted for >37%, which had a great influence on the volatile flavor of beer fish. The flavor components' composition varied noticeably depending on the stage of processing. PLS-DA model screened 35 volatile flavor components (VIP > 1) as markers; the most significant differences were 1-propanethiol, isoamyl alcohol, ethanol, and eucalyptol. Ultrasonic processing, frying, and soaking sauce can significantly improve the formation of flavor compounds, resulting in a notable enhancement of the final beer fish's umami taste and overall flavor quality.

RNA-Seq Reveals Pathways Responsible for Meat Quality Characteristic Differences between Two Yunnan Indigenous Chicken Breeds and Commercial Broilers.

Poultry is a source of meat that is in great demand in the world. The quality of meat is an imperative point for shoppers. To explore the genes controlling meat quality characteristics, the growth and meat quality traits and muscle transcriptome of two indigenous Yunnan chicken breeds, Wuding chickens (WDs) and Daweishan mini chickens (MCs), were compared with Cobb broilers (CBs). The growth and meat quality characteristics of these two indigenous breeds were found to differ from CB. In particular, the crude fat (CF), inosine monophosphate content, amino acid (AA), and total fatty acid (TFA) content of WDs were significantly higher than those of CBs and MCs. In addition, it was found that MC pectoralis had 420 differentially expressed genes (DEGs) relative to CBs, and WDs had 217 DEGs relative to CBs. Among them, 105 DEGs were shared. The results of 10 selected genes were also confirmed by qPCR. The differentially expressed genes were six enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways including lysosomes, phagosomes, PPAR signaling pathways, cell adhesion molecules, cytokine-cytokine receptor interaction, and phagosome sphingolipid metabolism. Interestingly, four genes (LPL, GK, SCD, and FABP7) in the PPAR signal pathway related to fatty acid (FA) metabolism were elevated in WD muscles, which may account for higher CF, inosine monophosphate content, and AA and FA contents, key factors affecting meat quality. This work laid the foundation for improving the meat quality of Yunnan indigenous chickens, especially WD. In future molecular breeding, the genes in this study can be used as molecular screening markers and applied to the molecular breeding of chicken quality characteristics.

Effects of chlorogenic acid-grafted-chitosan on biofilms, oxidative stress, quorum sensing and c-di-GMP in Pseudomonas fluorescens

This study determined the inhibitory mechanism as well as anti-biofilm activity of chlorogenic acid-grafted-chitosan (CS-g-CA) against Pseudomonas fluorescens (P. fluorescens) in terms of biofilm content, oxidative stress, quorum sensing and cyclic diguanosine monophosphate (c-di-GMP) concentration, and detected the changes in the expression levels of related genes by quantitative real-time PCR (qRT-PCR). Results indicated that treatment with sub-concentrations of CS-g-CA for P. fluorescens led to reduce the biofilm size of large colonies, decrease the content of biofilm and extracellular polymers, weaken the motility and adhesion of P. fluorescens. Moreover, CS-g-CA resulted in higher ROS levels, diminished catalase activity (CAT), and increased superoxide dismutase (SOD) in P. fluorescens. CS-g-CA reduced the production of quorum-sensing signaling molecules (AHLs) and the concentration of c-di-GMP in bacteria. Genes for flagellar synthesis (flgA), the resistance to stress (rpoS and hfq), and pde (phosphodiesterases that degrade c-di-GMP) were significantly down-regulated as determined by RT-PCR. Overall, CS-g-CA leads to the accumulation of ROS in bacteria via P. fluorescens environmental resistance genes and decreases the activity of enzymes in the bacterial antioxidant system, and interferes with the production and reception of quorum-sensing signaling molecules and the synthesis of c-di-GMP in P. fluorescens, which regulates the generation of biofilms.

Safety, Tolerability, and Pharmacodynamics of AZD3366 (Optimized Human CD39L3 Apyrase) Alone and in Combination With Ticagrelor and Acetylsalicylic Acid: A Phase 1, Randomized, Placebo-Controlled Study.

ADP and ATP are importantly involved in vascular and thrombotic homeostasis, via multiple receptor pathways. Blockade of ADP P2Y12 receptors inhibits platelet aggregation and represents an effective cardiovascular disease prevention strategy. AZD3366 (APT102), a long-acting recombinant form of an optimized CD39L3 human apyrase, has effectively reduced ATP, ADP, and platelet aggregation and provided tissue protection in preclinical models, features that could be very beneficial in treating patients with cardiovascular disease. We conducted this phase 1, first-in-human study of single ascending doses of intravenous AZD3366 or placebo, including doses added to dual antiplatelet therapy with ticagrelor and acetylsalicylic acid. The primary objective was safety and tolerability; secondary and exploratory objectives included pharmacokinetics, pharmacodynamics (measured as inhibition of platelet aggregation), adenosine diphosphatase (ADPase) activity, and ATP/ADP metabolism. In total, 104 participants were randomized. AZD3366 was generally well tolerated, with no major safety concerns observed. ADPase activity increased in a dose-dependent manner with a strong correlation to AZD3366 exposure. Inhibition of ADP-stimulated platelet aggregation was immediate, substantial, and durable. In addition, there was a prompt decrease in systemic ATP concentration and an increase in adenosine monophosphate concentrations, whereas ADP concentration appeared generally unaltered. At higher doses, there was a prolongation of capillary bleeding time without detectable changes in the exvivo thromboelastometric parameters. AZD3366 was well tolerated in healthy participants and demonstrated substantial and durable inhibition of platelet aggregation after single dosing. Higher doses prolonged capillary bleeding time without detectable changes in exvivo thromboelastometric parameters. URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04588727.

A cAMP-biosensor-based assay for measuring plasma arginine–vasopressin levels

Arginine–vasopressin (AVP), a cyclic peptide hormone composed of nine amino acids, regulates water reabsorption by increasing intracellular cyclic adenosine monophosphate (cAMP) concentrations via the vasopressin V2 receptor (V2R). Plasma AVP is a valuable biomarker for the diagnosis of central diabetes insipidus (CDI) and is commonly measured using radioimmunoassay (RIA). However, RIA has several drawbacks, including a long hands-on time, complex procedures, and handling of radioisotopes with special equipment and facilities. In this study, we developed a bioassay to measure plasma AVP levels using HEK293 cells expressing an engineered V2R and a cAMP biosensor. To achieve high sensitivity, we screened V2R orthologs from 11 various mammalian species and found that the platypus V2R (pV2R) responded to AVP with approximately six-fold higher sensitivity than that observed by the human V2R. Furthermore, to reduce cross-reactivity with desmopressin (DDAVP), a V2R agonist used for CDI treatment, we introduced a previously described point mutation into pV2R, yielding an approximately 20-fold reduction of responsiveness to DDAVP while maintaining responsiveness to AVP. Finally, a comparison of plasma samples from 12 healthy individuals demonstrated a strong correlation (Pearson's correlation value: 0.90) between our bioassay and RIA. Overall, our assay offers a more rapid and convenient method for quantifying plasma AVP concentrations than existing techniques.

Decoding the influence of low temperature on biofilm development: The hidden roles of c-di-GMP

Biofilms are widely used and play important roles in biological processes. Low temperature of wastewater inhibits the development of biofilms derived from wastewater activated sludge. However, the specific mechanism of temperature on biofilm development is still unclear. This study explored the mechanism of temperature on biofilm development and found a feasible method to enhance biofilm development at low temperature. The amount of biofilm development decreased by approximately 66 % and 55 % at 4 °C and 15 °C, respectively, as compared to 28 °C. The cyclic dimeric guanosine monophosphate (c-di-GMP) concentration also decreased at low temperature and was positively correlated with extracellular polymeric substance (EPS) content, formation, and adhesion strength. Microbial community results showed that low temperature inhibited the normal survival of most microorganisms, but promoted the growth of some psychrophile bacteria like Sporosarcina, Caldilineaceae, Gemmataceae, Anaerolineaceae and Acidobacteriota. Further analysis of functional genes demonstrated that the abundance of functional genes related to the synthesis of c-di-GMP (K18968, K18967 and K13590) decreased at low temperature. Subsequently, the addition of exogenous spermidine increased the level of intracellular c-di-GMP and alleviated the inhibition effect of low temperature on biofilm development. Therefore, the possible mechanism of low temperature on biofilm development could be the inhibition of the microorganism activity and reduction of the communication level between cells, which is the closely related to the EPS content, formation, and adhesion strength. The enhancement of c-di-GMP level through the exogenous addition of spermidine provides an alternative strategy to enhance biofilm development at low temperatures. The results of this study enhance the understanding of the influence of temperature on biofilm development and provide possible strategies for enhancing biofilm development at low temperatures.

Molecular characterization of adenosine monophosphate deaminase 1 and the correlation analysis between its mRNA expression levels and inosine monophosphate content in large yellow croaker (Larimichthys crocea)

Declining flesh quality has drawn considerable attention in the farmed large yellow croaker (LYC; Larimichthys crocea) industry. Inosine monophosphate (IMP) is the primary flavor substance in aquatic animals. Adenosine monophosphate deaminase 1 (AMPD1) plays a critical role in IMP formation by catalyzing the deamination of AMP to IMP in the purine nucleotide cycle. To further evaluate the correlation between ampd1 mRNA expression levels and IMP content in the LYC muscle tissue, the relevant open reading frame (ORF) of L. crocea (Lcampd1) was cloned, and the IMP content and Lcampd1 mRNA expression in the muscles of LYCs of different sizes were examined. The ORF cDNA of Lcampd1 was 2211 bp in length and encoded a polypeptide of 736 amino acids (AAs). The deduced protein, LcAMPD1, possesses conserved AMPD active regions (SLSTDDP) and shows high homology with AMPD proteins of other teleost fishes. The genomic DNA sequence of Lcampd1 exhibits a high degree of evolutionary conservation in terms of structural organization among species. Phylogenetic analysis of the deduced AA sequence revealed that teleost fish and mammalian AMPD1 were separate from each other and formed a cluster with AMPD3, suggesting that AMPD1 and AMPD3 arose by duplication of a common primordial gene. In healthy LYC, Lcampd1 mRNA was expressed only in the muscle tissue. The IMP content in the muscle of LYCs with different average body weights was measured by high-performance liquid chromatography; the results showed that the IMP content in the muscle of LYCs with greater body weight was significantly higher than that in LYC with lower body weight. Moreover, a similar trend in Lcampd1 expression was observed in these muscle tissues. The Pearson correlation analysis further showed that the Lcampd1 mRNA expression was positively correlated with IMP content in the muscles of different-sized LYCs. These results suggest the potential function of Lcampd1 in determining the IMP content in LYC and provide a theoretical basis for flesh quality improvement, as well as a scientific basis for the development of the molecular breeding of LYC.

The Effect of Bacterial AHL on the Cyclic Adenosine Monophosphate Content in Plants According to High-Performance Liquid Chromatography.

Cyclic adenosine monophosphate (cAMP) is an important second messenger in cells, mediating various stimulation signals such as the growth and development of organisms and stress and participating in regulating various biological processes of cells. This article explores the quantitative determination of cAMP in plants using High-Performance Liquid Chromatography (HPLC) and applies this method to analyzing the changes in cAMP content during the process of plant response to the bacterial quorum sensing signal N-acyl homoserine lactone (AHL). Research has shown that the optimal detection conditions for HPLC are as follows: the chromatographic column is Venusil MP C18 (2), the mobile phase is methanol-water (0.1% trifluoroacetic acid) (v:v, 10:90), the detection wavelength is 259 nm, the column temperature is 35 °C, and the flow rate is 0.8 mL/min. The precision of the standard sample of this method is 98.21%, the precision of the sample is 98.87%, and the recovery rate is 101.067%. The optimal extraction conditions for cAMP in Arabidopsis are to use 15% methanol ultrasonic extraction for 10 min, followed by a 40 °C water bath for 4 h. Bacterial AHL signal processing can significantly stimulate an increase in cAMP levels in Arabidopsis leaves and roots. The establishment of HPLC detection methods for the cAMP content in plants is of great significance for in-depth research on the signal transduction mechanisms of plant-bacterial interactions.

High-pressure processing enhances saltiness perception and sensory acceptability of raw but not of cooked cured pork loins-leveraging salty and umami taste.

The salt (NaCl) content in processed meats must be reduced because of its adverse effects on cardiovascular health. However, reducing salt in meat products typically leads to a lower taste intensity and, thus, consumer acceptability. Industry interventions must reduce salt content while maintaining taste, quality, and consumer acceptability. In this context, high-pressure processing (HPP) has been proposed to enhance saltiness perception, though there are contradictory reports to date. The present work aimed to conduct a targeted experiment to ascertain the influence of HPP (300/600 MPa) and cooking (71°C) on saltiness perception and sensory acceptability of meat products. HPP treatment (300/600 MPa) did enhance those two sensory attributes (approx. +1 on a 9-point hedonic scale) in raw (uncooked) cured pork loins but did not in their cooked counterparts. Further, the partition coefficient of sodium (PNa+), as an estimate of Na+ binding strength to the meat matrix, and the content of umami-taste nucleotides were investigated as potential causes. No effect of cooking (71°C) and HPP (300/600 MPa) could be observed on the PNa+ at equilibrium. However, HPP treatment at 300 MPa increased the inosine-5'-monophosphate (IMP) content in raw cured pork loins. Finally, hypothetical HPP effects on taste-mediating molecular mechanisms are outlined and discussed in light of boosting the sensory perception of raw meat products as a strategy to achieve effective salt reductions while keeping consumer acceptability.

Effect of Lupin Supplementation on the Growth, Carcass, and Meat Characteristics of Late-Fattening Hanwoo Steers.

This study aimed to investigate the effects of lupin flake supplementation on the growth, plasma parameters, carcass characteristics, and meat composition of late-fattening Hanwoo steers. The steers (n = 40) were randomly divided into the four groups with 10 steers each: LP0 (lupin flake 0%), LP3 (lupin flake 3%), LP6 (lupin flake 6%), and LP9 (lupin flake 9%). The total digestible nutriant intake increased as the concentration of lupin increased (linear and quadratic effects; p < 0.05). The thiobarbituric acid-reactive substance content in the strip loins decreased as lupin flake supplementation levels increased (linear and quadratic effects; p < 0.05), while carnosine levels increased linearly (p < 0.05). As the lupin flake supplementation level increased, anserine and creatinine contents increased linearly and quadratically (p < 0.05). Similarly, adenosine triphosphate (ATP) and adenosine monophosphate (AMP) content increased with increasing lupin flake supplementation levels in linear and quadratic effects (p < 0.001). Palmitoleic acid content increased significantly with increasing lupin flake supplementation level (linear and quadratic effects; p < 0.05). The content of oleic acid in the strip loin was not significant, but the unsaturated fatty acid (UFA) (p < 0.05) and n-6/n-3 ratio (p < 0.05) increased. The results of this study indicated that although lupin flake supplementation did not markedly affect the growth, carcass characteristics, or meat composition of late-fattening Hanwoo steers, it exerted a positive effect on the flavor, taste profiles (anserine, creatinine, ATP, and AMP), hypotonicity (TBARS), and healthy meat production (UFA and n-6/n-3 ratio) related to beef.

Role of alpha-7 nicotinic acetylcholine receptor down-regulation in cyclic nucleotide reduction in memory-related brain regions of Alzheimer’s rats

In this study, we investigated the effect of the downregulation of α7 nicotinic acetylcholine receptors (α7nAChRs) by Streptozotocin (STZ) on the level of cyclic nucleotides. Three groups of six rats each—Control, Sham (received an Intracerebroventricular injection of artificial cerebrospinal fluid, and STZ were formed. Except for the control group, all rats were anesthetized with 45 mg/kg of pentobarbitone. The STZ was administered intracerebroventricularly at a dose of 3 mg/kg to STZ group rats on Day 1 as well as Day 3 of an experimental period of 18 days. By using the Morris Water Maze (MWM) along with the Y-maze test, behavioral parameters were assessed, and biochemical analysis was assessed within the hippocampus, prefrontal cortex as well as amygdala of the rat brain. Statistical analysis was performed using repeated measures with one-way and two-way analysis of variance. Bonferroni and Student Newman–Keuls post hoc test was used to determine significance between experimental groups, with a significance threshold of p &lt; 0.05. STZ administration resulted in a notable decline in the cognitive function of rats, as evidenced by longer transfer latency, reduced duration spent, and decreased overall distance covered within the designated quadrant (in percentage) in the MWM assessment. Moreover, STZ lowered the spontaneous change in behavior observed in the rodents during the Y-maze evaluation and decreased Cyclic Adenosine Monophosphate and Cyclic Guanosine Monophosphate (cyclic nucleotides) concentrations within the HIP, PFC, and Amygdale of rat brain regions. The α7nAChR downregulation may form a basis that could serve as a foundation for cognitive impairments alongside reduced cyclic nucleotides’ concentration in specific areas of the rat brain. Thus, cyclic nucleotides could be a probable target to stimulate the α7nAChR mediated mechanism in STZ-mediated conditions.

Improved umami taste of the enzymatic hydrolysate of soybean protein isolate by Corynebacterium glutamicum P-45 fermentation

Improving the umami taste of seasoning is an important topic of discussion in the current scenario based on the conventional technology of using soybean protein isolates (SPI) as proposed as the base material for seasoning. In this study, a novel SPI seasoning was created by fermenting the SPI hydrolysate of Corynebacterium glutamicum to increase the umami taste of its fermented products. The differences between traditional SPI enzymatic hydrolysate fermentation (Sample S), traditional C. glutamicum fermentation (Sample F), and the aforementioned novel fermentation (Sample SF) were investigated. The results showed that Sample SF had the best umami taste, saltiness, and richness, which was compared with sample S and F showing significant differences, respectively (P < 0.05). Succinic acid, lactic acid, and acetic acid were the main organic acids in the seasonings, accounting for 92.24% of the total organic acids. The total fresh-sweet free amino acids content of sample SF was 7.22 and 2.15 times that of samples S and F, respectively, and 5′-nucleotide content was 2.85 and 2.35 times that of Samples S and F, respectively. Glu, 5′-inosine monophosphate, and 5′-guanosine monophosphate contents contributed remarkably to the umami taste of the seasoning (P < 0.05). Furthermore, among all the detected umami compounds, 18 conformed the variable important projection (VIP) values > 1.0. Therefore, SPI hydrolysate fermentation by C. glutamicum may improve the umami flavor of fermented products. This study presents a novel method for producing new SPI-containing seasonings on a commercial scale.

CP-25 inhibits the hyperactivation of rheumatic synoviocytes by suppressing the switch in Gαs-Gαi coupling to the β2-adrenergic receptor

In essence, the β2 adrenergic receptor (β2AR) plays an antiproliferative role by increasing the intracellular cyclic 3’,5’-adenosine monophosphate (cAMP) concentration through Gαs coupling, but interestingly, β2AR antagonists are able to effectively inhibit fibroblast-like synoviocytes (FLSs) proliferation, thus ameliorating experimental RA, indicating that the β2AR signalling pathway is impaired in RA FLSs via unknown mechanisms. The local epinephrine (Epi) level was found to be much higher in inflammatory joints than in normal joints, and high-level stimulation with Epi or isoproterenol (ISO) directly promoted FLSs proliferation and migration due to impaired β2AR signalling and cAMP production. By applying inhibitor of receptor internalization, and small interfering RNA (siRNA) of Gαs and Gαi, and by using fluorescence resonance energy transfer and coimmunoprecipitation assays, a switch in Gαs-Gαi coupling to β2AR was observed in inflammatory FLSs as well as in FLSs with chronic ISO stimulation. This Gαi coupling was then revealed to be initiated by G protein coupled receptor kinase 2 (GRK2) but not β-arrestin2 or protein kinase A-mediated phosphorylation of β2AR. Inhibiting the activity of GRK2 with the novel GRK2 inhibitor paeoniflorin-6′-O-benzene sulfonate (CP-25), a derivative of paeoniflorin, or the accepted GRK2 inhibitor paroxetine effectively reversed the switch in Gαs-Gαi coupling to β2AR during inflammation and restored the intracellular cAMP level in ISO-stimulated FLSs. As expected, CP-25 significantly inhibited the hyperplasia of FLSs in a collagen-induced arthritis (CIA) model (CIA FLSs) and normal FLSs stimulated with ISO and finally ameliorated CIA in rats. Together, our findings revealed the pathological changes in β2AR signalling in CIA FLSs, determined the underlying mechanisms and identified the pharmacological target of the GRK2 inhibitor CP-25 in treating CIA.7HWufKwpH5ZfY_hx8fyY6aVideo

Expression of the umami taste receptor T1R1/T1R3 in porcine testis of: Function in regulating testosterone synthesis and autophagy in Leydig cells

Testosterone is a vital male hormone responsible for male sexual characteristics. The taste receptor family 1 subunit 3 (T1R3) regulates testosterone synthesis and autophagy in non-taste cells, and the links with the taste receptor family 1 subunit 1 (T1R1) for umami perception. However, little is known about these mechanisms. Thus, we aimed to determine the relationship between the umami taste receptor (T1R1/T1R3) and testosterone synthesis or autophagy in testicular Leydig cells of the Xiang pig. There was a certain proportion of spermatogenic tubular dysplasia in the Xiang pig at puberty, in which autophagy was enhanced, and the testosterone level was increased with a weak expression of T1R3. Silenced T1R3 decreased testosterone level and intracellular cyclic adenosine monophosphate (cAMP) content and inhibited the messenger RNA (mRNA) expression levels of testosterone synthesis enzyme genes [steroidogenic acute regulatory protein (StAR), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (3β-HSD1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and hydroxysteroid 17-beta dehydrogenase 3 (17β-HSD3)]. In addition, T1R3 increased the number of acidic autophagy bubbles and upregulated the expression levels of autophagy markers [Microtubule-associated protein 1 A/1B-light chain 3 (LC3) and Beclin-1] in testicular Leydig cells of the Xiang pig. Using an umami tasting agonist (10 mM L-glutamate for 6 h), the activation of T1R1/T1R3 enhanced the testosterone synthesis ability by increasing the intracellular cAMP level and upregulated the expression levels of StAR, 3β-HSD1, CYP17A1 and 17β-HSD3 in Leydig cells. Furthermore, the number of acidic autophagy bubbles decreased in the T1R1/T1R3-activated group with the downregulation of the expression levels of the autophagy markers, including LC3 and Beclin-1. These data suggest that the function of T1R1/T1R3 expressed in testicular Leydig cells of the Xiang pig is related to testosterone synthesis and autophagy.

Novel Melanocortin-3 and -4 Receptor Functional Variants in Asian Children With Severe Obesity.

Genetic variants in melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) genes are strongly associated with childhood obesity. This study aims to identify and functionally characterize MC3R and MC4R variants in an Asian cohort of children with severe early-onset obesity. Whole-exome sequencing was performed to screen for MC3R and MC4R coding variants in 488 Asian children with severe early-onset obesity (body mass index for age ≥97th percentile). Functionality of the identified variants were determined via measurement of intracellular cyclic adenosine monophosphate (cAMP) concentrations and luciferase activity. Four MC3R and 2 MC4R heterozygous nonsynonymous rare variants were detected. There were 3 novel variants: MC3R c.151G > C (p.Val51Leu), MC4R c.127C > A (p.Gln43Lys), and MC4R c.272T > G (p.Met91Arg), and 3 previously reported variants: MC3R c.127G > A (p.Glu43Lys), MC3R c.97G > A (p.Ala33Thr), and MC3R c.437T > A (p.Ile146Asn). Both MC3R c.127G > A (p.Glu43Lys) and MC4R c.272T > G (p.Met91Arg) variants demonstrated defective downstream cAMP signaling activity. The MC4R c.127C > A (p.Gln43Lys) variant showed reduced cAMP signaling activity at low substrate concentration but the signaling activity was restored at high substrate concentration. The MC3R c.151G > C (p.Val51Leu) variant did not show a significant reduction in cAMP signaling activity compared to wild-type (WT) MC3R. Coexpression studies of the WT and variant MC3R/MC4R showed that the heterozygous variants did not exhibit dominant negative effect. Our functional assays demonstrated that MC3R c.127G > A (p.Glu43Lys) and MC4R c.272T > G (p.Met91Arg) variants might predispose individuals to early-onset obesity, and further studies are needed to establish the causative effect of these variants in the pathogenesis of obesity.

Metabolic and microbial analyses of the surface and inner part of wet-aged and dry-aged beef.

The effects of aging and microbial growth on the metabolome of aged beef were investigated in this study. The metabolome of beef is influenced by the aging method applied. This includes the aging-related changes in metabolism and the presence of microorganisms on the beef during aging that may affect the beef and its quality. The inner part and the trimmed surface of dry-aged (the surface of dry-aged beef is also called the "crust" due to its drying during aging) and wet-aged beef were analyzed by 1 H nuclear magnetic resonance (NMR) spectroscopy over aging periods up to 28 days at intervals of 7 days, and the former also by microbiological analysis. The metabolome detected by 1 H NMR spectroscopy demonstrated changes over the aging time of beef and differed depending on the sampling location (surface or inner part of beef). The influence of the microbiota on changes in the metabolome can be negligible due to the low microbial growth on the surface of dry-aged beef (<3logCFU/g). Therefore, the aging-related metabolism postmortem of the analyzed dry-aged beef might be the main factor for metabolic changes. The significantly (p<0.05) higher amino acids and inosine concentrations and lower inosine 5'-monophosphate concentrations suggested enhanced protein degradation and energy metabolism in the wet-aged beef compared to the dry-aged beef, probably due to the combined influence of the aging and the microbiota on the wet-aged beef and, thus, its metabolic changes.

Inhibitory Activity and Mechanism of Action with Thymol against the Blueberry Pathogenic Fungi Caused by Neopestalotiopsis clavispora

Decay caused by Neopestalotiopsis clavispora is an important postharvest disease of blueberries that seriously affects the commercial value of blueberry fruit. In this paper, we studied the inhibitory activity and mode of action of thymol against the pathogenic fungus of blueberries caused by Neopestalotiopsis clavispora. The results demonstrated that thymol administration could limit mycelial growth in vitro; the inhibitory effect was positively connected with thymol mass concentrations, and the minimal inhibitory concentration (MIC) was 100 mg/L. Further investigations revealed that MIC thymol treatment dramatically reduced the germination of pathogenic spores and led to an increase in the conductivity of the pathogen, leakage of contents, and a decrease in pH. Propidium iodide (PI) staining experiments demonstrated that MIC thymol caused damage to mycelial cell membranes. Additionally, MIC thymol treatment promoted mycelium malondialdehyde content accumulation, inhibited superoxide dismutase (SOD) and catalase (CAT) enzyme activities, decreased adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) content and energy charge levels, and the fluorescence intensity of mycelium caused by MIC thymol treatment was significantly increased by the 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) assay. The results of this study indicate that thymol suppresses the proliferation of Neopestalotiopsis clavispora by compromising the integrity of their cell membranes, promoting the accumulation of cellular reactive oxygen species (ROS), and interfering with energy metabolism.