Simvastatin attenuates silica-induced pulmonary inflammation and fibrosis in rats via the AMPK-NOX pathway.

BACKGROUND: Simvastatin (Sim), a hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has been widely used in prevention and treatment of cardiovascular diseases. Studies have suggested that Sim exerts anti-fibrotic effects by interfering fibroblast proliferation and collagen synthesis. This study was to determine whether Sim could alleviate silica-induced pulmonary fibrosis and explore the underlying mechanisms. METHODS: The rat model of silicosis was established by the tracheal perfusion method and treated with Sim (5 or 10 mg/kg), AICAR (an AMPK agonist), and apocynin (a NOX inhibitor) for 28 days. Lung tissues were collected for further analyses including pathological histology, inflammatory response, oxidative stress, epithelial mesenchymal transformation (EMT), and the AMPK-NOX pathway. RESULTS: Sim significantly reduced silica-induced pulmonary inflammation and fibrosis at 28 days after administration. Sim could reduce the levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α and transforming growth factor-ß1 in lung tissues. The expressions of hydroxyproline, α-SMA and vimentin were down-regulated, while E-cad was increased in Sim-treated rats. In addition, NOX4, p22pox, p40phox, p-p47phox/p47phox expressions and ROS levels were all increased, whereas p-AMPK/AMPK was decreased in silica-induced rats. Sim or AICAR treatment could notably reverse the decrease of AMPK activity and increase of NOX activity induced by silica. Apocynin treatment exhibited similar protective effects to Sim, including down-regulating of oxidative stress and inhibition of the EMT process and inflammatory reactions. CONCLUSIONS: Sim attenuates silica-induced pulmonary inflammation and fibrosis by downregulating EMT and oxidative stress through the AMPK-NOX pathway.

Plant-based zinc nanoflowers assisted molecularly imprinted polymer for the design of an electrochemical sensor for selective determination of abrocitinib.

The first electrochemical sensor application in the literature is described for the sensitive and selective determination of the selective Janus kinase (JAK)-1 inhibitor abrocitinib (ABR). ABR is approved by the U.S. Food and Drug Administration (FDA) for the treatment of atopic dermatitis. The molecularly imprinted polymer (MIP)-based sensor was designed to incorporate zinc nanoflower (ZnNFs)-graphene oxide (GO) conjugate (ZnNFs@GO), synthesized from the root methanolic extract (RME) of the species Alkanna cappadocica Boiss. et Bal. to improve the porosity and effective surface area of the glassy carbon electrode (GCE). Furthermore, the MIP structure was prepared using ABR as a template molecule, 4-aminobenzoic acid (4-ABA) as a functional monomer, and other additional components. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) were used to characterize the surface and structure of the synthesized nanomaterial and MIP-based surface. Among the electrochemical methods, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were preferred for detailed electrochemical characterization, and differential pulse voltammetry (DPV) was preferred for all other electrochemical measurements using 5.0 mM [Fe(CN)6]3-/4- solution as the redox probe. The MIP-based sensor, which was the result of a detailed optimization phase, gave a linear response in the 1.0 × 10-13 - 1.0 × 10-12 M range in standard solution and serum sample. The obtained limit of detection (LOD) and limit of quantification (LOQ) values and recovery studies demonstrated the sensitivity, accuracy, and applicability of the sensor. Selectivity, the most important feature of the MIP-based sensor, was verified by imprinting factor calculations using ibrutinib, ruxolitinib, tofacitinib, zonisamide, and acetazolamide.

The interaction between 20-hydroxyecdysone and AMPK through PI3K activation in Chinese mitten crab, Eriocheir sinensis.

In crustaceans, the steroid hormone 20-hydroxyecdysone (20E) initiates molting, and the molting process is also regulated by energy metabolism. AMPK is an energy sensor and plays a critical role in systemic energy balance. Here, the regulatory mechanism in the interaction between 20E and AMPK was investigated in Chinese mitten crab, Eriocheir sinensis. The results showed that the 20E concentration and the mRNA expression levels of 20E receptors in hepatopancreas were down-regulated post AMPK activator (AICAR) treatment, and were up-regulated after AMPK inhibitor (Compound C) injection in crabs. Besides, the molt-inhibiting hormone (MIH) gene expression in eyestalk showed the opposite patterns in response to the AICAR and Compound C treatment, respectively. Further investigation found that there was a significant reduction in 20E concentration post PI3K inhibitor (LY294002) treatment, and the phosphorylation level of PI3K was increased in hepatopancreas after AMPK inhibitor injection. On the other hand, the positive regulation of PI3K-mediated activation of AMPK was also observed, the phosphorylation levels of AMPKα, AMPKß and PI3K in hepatopancreas were significantly increased post 20E injection. In addition, the phosphorylation levels of AMPKα and AMPKß induced by 20E were decreased after the injection of PI3K inhibitor. Taken together, these results suggest that the regulatory cross-talk between 20E and AMPK is likely to act through PI3K pathway in E. sinensis, which appeared to be helpful for a better understanding in molting regulation.

Glucose uptake in trophoblasts of GDM mice is regulated by the AMPK-CLUT3 signaling pathway.

GDM, as a metabolic disease during pregnancy, regulates GLUT3 translocation by AMPK, thereby affecting glucose uptake in trophoblasts. It provides a new research idea and therapeutic target for alleviating intrauterine hyperglycemia in GDM. STZ was used to construct GDM mice, inject AICAR into pregnant mice, and observe fetal and placental weight; flow cytometry was employed for the detection of glucose uptake by primary trophoblast cells; immunofluorescence was applied to detect the localization of GLUT3 and AMPK in placental tissue; Cocofal microscope was used to detect the localization of GLUT3 in trophoblast cells;qRT-PCR and Western blot experiments were carried out to detect the expression levels of GLUT3 and AMPK in placental tissue; CO-IP was utilized to detect the interaction of GLUT3 and AMPK. Compared with the normal pregnancy group, the weight of the fetus and placenta of GDM mice increased (P < 0.001), and the ability of trophoblasts to take up glucose decreased (P < 0.001). In addition, AMPK activity in trophoblasts and membrane localization of GLUT3 in GDM mice were down-regulated compared with normal pregnant mice (P < 0.05). There is an interaction between GLUT3 and AMPK. Activating AMPK in trophoblasts can up-regulate the expression of GLUT3 membrane protein in trophoblasts of mice (P < 0.05) and increase the glucose uptake of trophoblasts (P < 0.05). We speculate that inhibition of AMPK activity in GDM mice results in aberrant localization of GLUT3, which in turn attenuates glucose uptake by placental trophoblast cells. AICAR activates AMPK to increase the membrane localization of GLUT3 and improve the glucose uptake capacity of trophoblasts.

Bronchial thermoplasty decreases airway remodeling by inhibiting autophagy via the AMPK/mTOR signaling pathway.

Bronchial thermoplasty (BT), an effective treatment for severe asthma, requires heat to reach the airway to reduce the mass of airway smooth muscle cells (ASMCs). Autophagy is involved in the pathological process of airway remodeling in patients with asthma. However, it remains unclear whether autophagy participates in controlling airway remodeling induced by BT. In this study, we aim to elucidate the autophagy-mediated molecular mechanisms in BT. Our study reveal that the number of autophagosomes and the level of alpha-smooth muscle actin (α-SMA) fluorescence are significantly decreased in airway biopsy tissues after BT. As the temperature increased, BT causes a decrease in cell proliferation and a concomitant increase in the apoptosis of human airway smooth muscle cells (HASMCs). Furthermore, increase in temperature significantly downregulates cellular autophagy, autophagosome accumulation, the LC3II/LC3I ratio, and Beclin-1 expression, upregulates p62 expression, and inhibits the AMPK/mTOR pathway. Furthermore, cotreatment with AICAR (an AMPK agonist) or RAPA (an mTOR antagonist) abolishes the inhibition of autophagy and attenuates the increase in the apoptosis rate of HASMCs induced by the thermal effect. Therefore, we conclude that BT decreases airway remodeling by blocking autophagy induced by the AMPK/mTOR signaling pathway in HASMCs.

AMPK restricts HHV-6A replication by inhibiting glycolysis and mTOR signaling.

AMP-activated protein kinase (AMPK) is a cellular energy sensor regulating metabolic homeostasis. In this study, we investigated the role of AMPK in response to human herpesvirus 6A (HHV-6A) infection. We show that HHV-6A infection significantly downregulates the active phosphorylated state of AMPK in infected T cells. Pharmacological activation of AMPK highly attenuated HHV-6A propagation. Mechanistically, we found that the activation of AMPK by AICAR blocked HHV-6-induced glycolysis by inhibiting glucose metabolism and lactate secretion, as well as decreasing expressions of key glucose transporters and glycolytic enzymes. In addition, mTOR signaling has been inactivated in HHV-6A infected T cells by AICAR treatment. We also showed that HHV-6A infection of human umbilical cord blood mononuclear cells (CBMCs) reduced AMPK activity whereas the activation of AMPK by metformin drastically reduced HHV-6A DNA replication and virions production. Taken together, this study demonstrates that AMPK is a promising antiviral therapeutic target against HHV-6A infection.

AICAR confers prophylactic cardioprotection in doxorubicin-induced heart failure in rats.

Doxorubicin (DOX) is a widely used chemotherapeutic agent that can cause serious cardiotoxic side effects, leading to heart failure (HF). Impaired mitochondrial function is thought to be key factor driving progression into HF. We have previously shown in a rat model of DOX-HF that heart failure with reduced ejection fraction correlates with mitochondrial loss and dysfunction. Adenosine monophosphate-dependent kinase (AMPK) is a cellular energy sensor, regulating mitochondrial biogenesis and energy metabolism, including fatty acid oxidation. We hypothesised that AMPK activation could restore mitochondrial function and therefore be a novel cardioprotective strategy for the prevention of DOX-HF. Consequently, we set out to assess whether 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR), an activator of AMPK, could prevent cardiac functional decline in this chronic intravenous rat model of DOX-HF. In line with our hypothesis, AICAR improved cardiac systolic function. AICAR furthermore improved cardiac mitochondrial fatty acid oxidation, independent of mitochondrial number, and in the absence of observable AMPK-activation. In addition, we found that AICAR prevented loss of myocardial mass. RNAseq analysis showed that this may be driven by normalisation of pathways associated with ribosome function and protein synthesis, which are impaired in DOX-treated rat hearts. AICAR furthermore prevented dyslipidemia and excessive body-weight loss in DOX-treated rats, which may contribute to preservation of myocardial mass. Though it is unclear whether AICAR exerted its cardioprotective effect through cardiac or extra-cardiac AMPK-activation or via an AMPK-independent effect, these results show promise for the use of AICAR as a cardioprotective agent in DOX-HF to both preserve cardiac function and mass.

Binding Mode and Molecular Mechanism of the Two-Component Histidine Kinase Bos1 of Botrytis cinerea to Fludioxonil and Iprodione.

Gray mold caused by Botrytis cinerea is among the 10 most serious fungal diseases worldwide. Fludioxonil is widely used to prevent and control gray mold due to its low toxicity and high efficiency; however, resistance caused by long-term use has become increasingly prominent. Therefore, exploring the resistance mechanism of fungicides provides a theoretical basis for delaying the occurrence of diseases and controlling gray mold. In this study, fludioxonil-resistant strains were obtained through indoor drug domestication, and the mutation sites were determined by sequencing. Strains obtained by site-directed mutagenesis were subjected to biological analysis, and the binding modes of fludioxonil and iprodione to Botrytis cinerea Bos1 BcBos1 were predicted by molecular docking. The results showed that F127S, I365S/N, F127S + I365N, and I376M mutations on the Bos1 protein led to a decrease in the binding energy between the drug and BcBos1. The A1259T mutation did not lead to a decrease in the binding energy, which was not the cause of drug resistance. The biological fitness of the fludioxonil- and point mutation-resistant strains decreased, and their growth rate, sporulation rate, and pathogenicity decreased significantly. The glycerol content of the sensitive strains was significantly lower than that of the resistant strains and increased significantly after treatment with 0.1 µg/ml of fludioxonil, whereas that of the resistant strains decreased. The osmotic sensitivity of the resistant strains was significantly lower than that of the sensitive strains. Positive cross-resistance was observed between fludioxonil and iprodione. These results will help to understand the resistance mechanism of fludioxonil in Botrytis cinerea more deeply.

Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) bifunctional enzyme from Candidatus Liberibacer asiaticus.

The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 µM and Vmax, 0.95 µmol/min/mg) and AICAR (Km, 34.81 µM and Vmax, 0.56 µmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 µM and Vmax, 2.87 µmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 µM and 34.2 µM, respectively) compared to AICAR (Kd, 83.4 µM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.

Genotoxic effects induced by iprodione and tebuconazole in meristematic cells of Allium cepa: responses dependent on concentration and exposure time.

The present work explores the genotoxicity of the fungicides iprodione (IP) and tebuconazole (TB) using the Allium cepa assay as an in vivo biological model. Both short-term and long-term exposures were studied, revealing concentration- and time-dependent cytological and genotoxic effects. IP exhibited genotoxicity over a wider concentration range (5-50 µg/ml) and required 30 h of exposure, while TB showed genotoxicity at higher concentrations (10 and 30 µg/ml) within a 4-h exposure period. The study highlights the importance of assessing potential risks associated with fungicide exposure, including handling, disposal practices, and concerns regarding food residue. Moreover, the research underscores the genotoxic effects of IP and TB on plant cells and provides valuable insights into their concentration and time-response patterns.

Kimchi attenuates endoplasmic reticulum stress-induced hepatic steatosis in HepG2 cells and C57BL/6N mice.

Kimchi is a traditional fermented food that contains abundant nutrients and functional ingredients with various health benefits. We previously reported that kimchi active components suppress hepatic steatosis caused by endoplasmic reticulum (ER) stress in vitro and in vivo. Therefore, we assessed the effect of kimchi on the inhibition of hepatic steatosis caused by ER stress in HepG2 cells and C57BL/6N mice to verify the hypothesis that kimchi may potentially inhibit nonalcoholic fatty liver disease. We investigated the effect of kimchi on cell viability and triglyceride concentrations in cells and on lipid profile, lipid accumulation, and expression of related genes in cells and mice with hepatic steatosis. A mechanistic study was also performed using the liver X receptor α agonist T0901317 and the AMP-activated protein kinase agonist AICAR. Kimchi was noncytotoxic and effectively reduced triglyceride concentrations and suppressed hepatic steatosis-related gene expression in cells and mice. Additionally, kimchi recovered weight loss, lowered the serum and liver tissue lipid profiles, suppressed lipid accumulation, and reduced the effects of T0901317 and AICAR on lipogenic gene expression in tunicamycin-treated mice. Our results highlight that kimchi could prevent hepatic steatosis caused by ER stress in cells and mice.

Central administration of AICAR attenuates hypertension via AMPK/Nrf2 pathway in the hypothalamic paraventricular nucleus of hypertensive rats.

BACKGROUND: Oxidative stress and inflammatory cytokines in the hypothalamus paraventricular nucleus (PVN) have been implicated in sympathetic nerve activity and the development of hypertension, but the specific mechanisms underlying their production in the PVN remains to be elucidated. Previous studies have demonstrated that activation of nuclear transcription related factor-2 (Nrf2) in the PVN reduced the production of reactive oxygen species (ROS) and inflammatory mediators. Moreover, AMP-activated protein kinase (AMPK), has been observed to decrease ROS and inflammatory cytokine production when activated in the periphery. 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) is an AMPK agonist. However, little research has been conducted on the role of AMPK in the PVN during hypertension. Therefore, we hypothesized that AICAR in the PVN is involved in regulating AMPK/Nrf2 pathway, affecting ROS and inflammatory cytokine expression, influencing sympathetic nerve activity. METHODS: Adult male Sprague-Dawley rats were utilized to induce two-kidney, one-clip (2K1C) hypertension via constriction of the right renal artery. Bilateral PVN was microinjected with either artificial cerebrospinal fluid or AICAR once a day for 4 weeks. RESULTS: Compared to the SHAM group, the PVN of 2K1C hypertensive rats decreased p-AMPK and p-Nrf2 expression, increased Fra-Like, NAD(P)H oxidase (NOX)2, NOX4, tumor necrosis factor-α and interleukin (IL)-1ß expression, elevated ROS levels, decreased superoxide dismutase 1 and IL-10 expression, and elevated plasma norepinephrine levels. Bilateral PVN microinjection of AICAR significantly ameliorated these changes. CONCLUSION: These findings suggest that repeated injection of AICAR in the PVN suppresses ROS and inflammatory cytokine production through the AMPK/Nrf2 pathway, reducing sympathetic nerve activity and improving hypertension.

Independent and combined effects of calorie restriction and AICAR on glucose uptake and insulin signaling in skeletal muscles from 24-month-old female and male rats.

We assessed the effects of two levels of calorie restriction (CR; eating either 15% or 35% less than ad libitum, AL, food intake for 8 weeks) by 24-month-old female and male rats on glucose uptake (GU) and phosphorylation of key signaling proteins (Akt; AMP-activated protein kinase, AMPK; Akt substrate of 160 kDa, AS160) measured in isolated skeletal muscles that underwent four incubation conditions (without either insulin or AICAR, an AMPK activator; with AICAR alone; with insulin alone; or with insulin and AICAR). Regardless of sex: (1) neither CR group versus the AL group had greater GU by insulin-stimulated muscles; (2) phosphorylation of Akt in insulin-stimulated muscles was increased in 35% CR versus AL rats; (3) prior AICAR treatment of muscle resulted in greater GU by insulin-stimulated muscles, regardless of diet; and (4) AICAR caused elevated phosphorylation of acetyl CoA carboxylase, an indicator of AMPK activation, in all diet groups. There was a sexually dimorphic diet effect on AS160 phosphorylation, with 35% CR exceeding AL for insulin-stimulated muscles in male rats, but not in female rats. Our working hypothesis is that the lack of a CR-effect on GU by insulin-stimulated muscles was related to the extended duration of the ex vivo incubation period (290 min compared to 40-50 min that was previously reported to be effective). The observed efficacy of prior treatment of muscles with AICAR to improve glucose uptake in insulin-stimulated muscles supports the strategy of targeting AMPK with the goal of improving insulin sensitivity in older females and males.

Iprodione induces hepatotoxicity in zebrafish by mediating ROS generation and upregulating p53 signalling pathway.

Iprodione is an effective and broad-spectrum fungicide commonly used for early disease control in fruit trees and vegetables. Due to rainfall, iprodione often finds its way into water bodies, posing toxicity risks to non-target organisms and potentially entering the human food chain. However, there is limited information available regarding the developmental toxicity of iprodione specifically on the liver in existing literature. In this study, we employed larval and adult zebrafish as models to investigate the toxicity of iprodione. Our findings revealed that iprodione exposure led to yolk sac edema and increased mortality in zebrafish. Notably, iprodione exhibited specific effects on zebrafish liver development. Additionally, zebrafish exposed to iprodione experienced an overload of reactive oxygen species, resulting in the upregulation of p53 gene expression. This, in turn, triggered hepatocyte apoptosis and disrupted carbohydrate/lipid metabolism as well as energy demand systems. These results demonstrated the substantial impact of iprodione on zebrafish liver development and function. Furthermore, the application of astaxanthin (an antioxidant) and p53 morpholino partially mitigated the liver toxicity caused by iprodione. To summarize, iprodione induces apoptosis through the upregulation of p53 mediated by oxidative stress signals, leading to liver toxicity in zebrafish. Our study highlights that exposure to iprodione can result in hepatotoxicity in zebrafish, and it may potentially pose toxicity risks to other aquatic organisms and even humans.

Regulation of the intestinal Na+/H+ exchanger NHE3 by AMP-activated kinase is dependent on phosphorylation of NHE3 at S555 and S563.

Electroneutral NaCl transport by Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and decreased NHE3 activity contributes to diarrhea. Patients with diabetes often experience gastrointestinal adverse effects and medications are often a culprit for chronic diarrhea in type 2 diabetes (T2D). We have shown previously that metformin, the most widely prescribed drug for the treatment of T2D, induces diarrhea by inhibition of Na+/H+ exchanger 3 (NHE3) in rodent models of T2D. Metformin was shown to activate AMP-activated protein kinase (AMPK), but AMPK-independent glycemic effects of metformin are also known. The current study is undertaken to determine whether metformin inhibits NHE3 by activation of AMPK and the mechanism by which NHE3 is inhibited by AMPK. Inhibition of NHE3 by metformin was abolished by knockdown of AMPK-α1 or AMPK-α2. AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) phosphorylated NHE3 at S555. S555 is the primary site of phosphorylation by protein kinase A (PKA), but AMPK phosphorylated S555 independently of PKA. Using Mass spectrometry, we found S563 as a newly recognized phosphorylation site in NHE3. Altering either S555 or S563 to Ala was sufficient to block the inhibition of NHE3 activity by AMPK. NHE3 inhibition is dependent on ubiquitination by the E3 ubiquitin ligase Nedd4-2 and metformin was shown to induce NHE3 internalization via Nedd4-2-mediated ubiquitination. AICAR did not increase NHE3 ubiquitination when S555 or S563 was mutated. We conclude that AMPK activation inhibits NHE3 activity and NHE3 inhibition is associated with phosphorylation of NHE3 at S555 and S563.NEW & NOTEWORTHY We show that AMP-activated protein kinase (AMPK) phosphorylates NHE3 at S555 and S563 to inhibit NHE3 activity in intestinal epithelial cells. Phosphorylation of NHE3 by AMPK is necessary for ubiquitination of NHE3.

Synthesis and Behavior of DNA Oligomers Containing the Ambiguous Z-Nucleobase 5-Aminoimidazole-4-carboxamide.

5-Amino-1-ß-D-ribofuranosylimidazole-4-carboxamide 5'-monophosphate (ZMP) is a central intermediate in de novo purine nucleotide biosynthesis. Its nucleobase moiety, 5-aminoimidazole-4-carboxamide (Z-base), is considered an ambiguous base that can pair with any canonical base owing to the rotatable nature of its 5-carboxamide group. This idea of ambiguous base pairing due to free rotation of the carboxamide has been applied to designing mutagenic antiviral nucleosides, such as ribavirin and T-705. However, the ambiguous base-pairing ability of Z-base has not been elucidated, because the synthesis of Z-base-containing oligomers is problematic. Herein, we propose a practical method for the synthesis of Z-base-containing DNA oligomers based on the ring-opening reaction of an N1-dinitrophenylhypoxanthine (HxaDNP) base. Thermal denaturation studies of the resulting oligomers revealed that the Z-base behaves physiologically as an A-like nucleobase, preferentially forming pairs with T. We tested the behavior of Z-base-containing DNA oligomers in enzyme-catalyzed reactions: in single nucleotide insertion, Klenow fragment DNA polymerase recognized Z-base as an A-like analog and incorporated dTTP as a complementary nucleotide to Z-base in the DNA template; in PCR amplification, Taq DNA polymerase similarly incorporated dTTP as a complementary nucleotide to Z-base. Our findings will contribute to the development of new mutagenic antiviral nucleoside analogs.

Design, synthesis, and evaluation of 1H-benzo[d]imidazole-4-carboxamide PARP-1 inhibitors using different saturated nitrogen-contained heterocycle as linker group.

Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors have been successfully applied in the clinical treatment of various cancer. Side effects and drug resistant cases were reported, and more effective PARP-1 inhibitors were required. However, studies on the AD site of PARP-1 inhibitors are currently incomplete. Therefore, to synthesize more potential candidate PARP-1 inhibitors and disclose some AD site SAR of the PARP-1 inhibitors, herein, a series of 2-phenyl-benzimidazole-4-carboxamide derivatives using different saturated nitrogen-contained heterocycles as linker group (6a-6t) have been designed, synthesized, and evaluated PARP-1 inhibitory activity and proliferation inhibitory against BRCA-1 mutant MDA-MB-436 cell line in vitro. The results showed 6b (IC50 = 8.65 nM) exhibited the most PARP-1 enzyme inhibitory activity comparable with Veliparib (IC50 = 15.54 nM) and Olaparib (IC50 = 2.77 nM); 6m exhibited the strongest MDA-MB-436 cell anti-proliferation activity (IC50 = 25.36 ± 6.06 µM) comparable with Olaparib (IC50 = 23.89 ± 3.81 µM). The compounds 6b, 6r, and 6m could be potential candidates for effective PARP-1 inhibitors and valuable for further optimization. The analysis of activity data also showed that the holistically anti-proliferation activity of the 1,4-diazepane group was about~twofold than that of the piperazine group. Meanwhile, the terminal 3-methyl-furanyl group exhibited the most robust PARP-1 inhibitory and anti-proliferation activity. It is hoped that the results could benefitable for further optimization of PARP-1 inhibitors. Furthermore, we note that some compounds (6d,6g,6n,6p,6s) showed poor PARP-1 inhibitory (>500 nM) but relatively good anti-proliferation activity, which indicates the proliferation inhibitory mechanism against MDA-MB-436 cell line was worth investigating in-depth.

AICAr to SAICAr ratio can serve as additional marker of AICAr use.

AICAr (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, commonly referred to as AICAR) is an adenosine monophosphate-activated protein kinase agonist previously investigated for its therapeutic potential which has been shown to improve exercise performance in laboratory animals. For this reason, the World Anti-Doping Agency prohibits the use of AICAr in sports. AICAr can easily be detected by means of liquid chromatography-mass spectrometry, but being an endogenous metabolite, it cannot be discriminated from AICAr of a non-natural origin. Population-based concentration thresholds have been suggested as a means to identify suspicious samples that would require further analysis by carbon isotope ratio mass spectrometry (CIR); however, it remains at the discretion of the laboratory how to apply them. Here, the urinary ratio of AICAr to SAICA-riboside (SAICAr) that is a closely related purine metabolite was investigated. In an athlete population of 5517 samples, this ratio was relatively narrowly distributed with median values and 99th percentiles of 3.3 and 9.3, and 4.2 and 14 in male and female athletes, respectively. Analysis of urine samples obtained from an AICAr administration study demonstrated that the AICAr/SAICAr ratio can serve in addition to AICAr concentration as a valuable diagnostic trigger for follow-up analysis by CIR. Conceivably, this combination can offer better retrospectivity than AICAr concentration alone by allowing to decrease the AICAr concentration threshold without significantly increasing the number of suspicious samples.

Myostatin Knockout Affects Mitochondrial Function by Inhibiting the AMPK/SIRT1/PGC1α Pathway in Skeletal Muscle.

Myostatin (Mstn) is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes. The deletion of the Mstn gene in mice leads to reduced mitochondrial functions. However, the underlying regulatory mechanisms remain unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. Mstn-KO mice exhibited significantly larger skeletal muscles. Meanwhile, Mstn knockout regulated the organ weights of mice. Moreover, we found that Mstn knockout reduced the basal metabolic rate, muscle adenosine triphosphate (ATP) synthesis, activities of mitochondrial respiration chain complexes, tricarboxylic acid cycle (TCA) cycle, and thermogenesis. Mechanistically, expressions of silent information regulator 1 (SIRT1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were down-regulated, while peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) acetylation modification increased in the Mstn-KO mice. Skeletal muscle cells from Mstn-KO and WT were treated with AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR), and the AMPK inhibitor Compound C, respectively. Compared with the wild-type (WT) group, Compound C treatment further down-regulated the expression or activity of pAMPK, SIRT1, citrate synthase (CS), isocitrate dehydrogenase (ICDHm), and α-ketoglutarate acid dehydrogenase (α-KGDH) in Mstn-KO mice, while Mstn knockout inhibited the AICAR activation effect. Therefore, Mstn knockout affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway. The present study reveals a new mechanism for Mstn knockout in regulating energy homeostasis.

AICAR attenuates postoperative abdominal adhesion formation by inhibiting oxidative stress and promoting mesothelial cell repair.

BACKGROUND: Postoperative abdominal adhesion is one of most common complications after abdominal operations. 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) is an adenosine 5'-monophosphate activated protein kinase (AMPK) pathway agonist that inhibits inflammation, reduces cell fibrosis and cellular reactive oxygen species (ROS) injury, promotes autophagy and mitochondrial function. This study aimed to explore the mechanism of AICAR in inhibiting adhesion formation. MATERIALS AND METHODS: Forty rats were randomly divided into five groups. All of the rats except the sham group received cecal abrasion to establish an adhesion model. The rats in the sodium hyaluronate group were treated with 2 mL sodium hyaluronate before closing the peritoneal cavity. The AICAR 1 and 2 groups were treated with 100 mg/kg and 200 mg/kg AICAR, respectively. Seven days after the operation, all of the rats were euthanized, and the adhesion condition was evaluated by Nair's system. Inflammation was assessed by Eosin-hematoxylin (HE) staining and transforming growth factor-ß (TGF-ß1) detection. Oxidative stress effect was determined by ROS, nitric oxide (NO) level, superoxide dismutase (SOD), catalase, glutathione peroxidase (Gpx) and malondialdehyde (MDA) levels in adhesion tissue. Then, Sirius red picric acid staining was used to detect the fiber thickness. Immunohistochemical staining of cytokeratin-19 (CK-19), alpha-smooth muscle actin (α-SMA) and nuclear factor erythroid 2-related factor 2 (Nrf2) was also performed. Finally, HMrSV5 cells were treated with TGF-ß1 and AICAR, the mRNA expression of E-cadherin, α-SMA and vimentin was assessed by q-PCR and cellular immunofluorescent staining. RESULTS: The rats in the AICAR-treated group had fewer adhesion formation incidences and a reduced Nair's score. The inflammation was determined by HE staining and TGF-ß1 concentration. The ROS, SOD, Catalase, Gpx, MDA levels and fiber thickness were decreased by AICAR treatments compared to the control. However, the NO production, Nrf2 levels and peritoneal mesothelial cell integrity were promoted after AICAR treatments. In vitro work, AICAR treatments reduced E-cadherin, α-SMA and vimentin mRNA level compared to that in the TGF-ß1 group. CONCLUSION: AICAR can inhibit postoperative adhesion formation by reducing inflammation, decreasing oxidative stress response and promoting peritoneal mesothelial cell repair.