USP36-mediated PARP1 deubiquitination in doxorubicin-induced cardiomyopathy.

Doxorubicin (Dox) is a potent antineoplastic agent, but its use is curtailed by severe cardiotoxicity, known as Dox-induced cardiomyopathy (DIC). The molecular mechanism underlying this cardiotoxicity remains unclear. Our current study investigates the role of Ubiquitin-Specific Protease 36 (USP36), a nucleolar deubiquitinating enzyme (DUB), in the progression of DIC and its mechanism. We found increased USP36 expression in neonatal rat cardiomyocytes and H9C2 cells exposed to Dox. Silencing USP36 significantly mitigated Dox-induced oxidative stress injury and apoptosis in vitro. Mechanistically, USP36 upregulation positively correlated with Poly (ADP-ribose) polymerase 1 (PARP1) expression, and its knockdown led to a reduction in PARP1 levels. Further investigation revealed that USP36 could bind to and mediate the deubiquitination of PARP1, thereby increasing its protein stability in cardiomyocytes upon Dox exposure. Moreover, overexpression of wild-type (WT) USP36 plasmid, but not its catalytically inactive mutant (C131A), stabilized PARP1 in HEK293T cells. We also established a DIC model in mice and observed significant upregulation of USP36 in the heart. Cardiac knockdown of USP36 in mice using a type 9 recombinant adeno-associated virus (rAAV9)-shUSP36 significantly preserved cardiac function after Dox treatment and protected against Dox-induced structural changes within the myocardium. In conclusion, these findings suggest that Dox promotes DIC progression by activating USP36-mediated PARP1 deubiquitination. This novel USP36/PARP1 axis may play a significant regulatory role in the pathogenesis of DIC.

Characterization of two keystone taxa, sulfur-oxidizing, and nitrate-reducing bacteria, by tracking their role transitions in the benzo[a]pyrene degradative microbiome.

BACKGROUND: Keystone taxa are drivers of microbiome structure and functioning, which may play critical roles in microbiome-level responses to recalcitrant pollution and are a key to bioremediation. However, the characterization and manipulation of such taxa is a major challenge due to the complexity of microbial communities and rapid turnover in both time and space. Here, microcosms were set up with benzo[a]-pyrene (BaP) and/or nitrate based on C-rich, S-rich, and N-limited mangrove sediments as reductive experimental models to trigger and track the turnover of keystone taxa to address this challenge. RESULTS: Based on microbial co-occurrence network analysis, two keystone taxa, Sulfurovum and Sulfurimonas, were found to exhibit significant role transitions in different microcosms, where these two taxa played nonkeystone roles with neutral relationships in in situ mangrove sediments. However, Sulfurimonas transitioned to be keystone taxa in nitrate-replenished microcosms and formed a keystone guild with Thioalkalispira. Sulfurovum stood out in BaP-added microcosms and mutualized in a densely polycyclic aromatic hydrocarbon (PAH)-degrader-centric keystone guild with Novosphingobium and Robiginitalea, where 63.25% of added BaP was removed. Under the occurrence of nitrate and BaP, they simultaneously played roles as keystone taxa in their respective guilds but exhibited significant competition. Comparative genomics and metagenome-assembled genome (MAG) analysis was then performed to reveal the metabolic potential of those keystone taxa and to empirically deduce their functional role in keystone guilds. Sulfurimonas possesses a better sense system and motility, indicative of its aggressive role in nitrate acquisition and conversion; Sulfurovum exhibited a better ability for oxidation resistance and transporting nutrients and electrons. High-efficiency thermal asymmetric interlaced polymerase reaction (hiTAIL-PCR) and enhanced green fluorescent protein (eGFP)-labeling approaches were employed to capture and label the BaP key degrader to further experimentally verify the roles of keystone taxa Sulfurovum in the keystone guilds. Observations of the enhancement in reactive oxygen species (ROS) removal, cell growth, and degradation efficiency by co-culture of isolated keystone taxa strains experimentally demonstrated that Sulfurovum contributes to the BaP degradative microbiome against BaP toxicity. CONCLUSIONS: Our findings suggest that the combined use of co-occurrence network analysis, comparative genomics, and co-culture of captured keystone taxa (3C-strategy) in microbial communities whose structure is strongly shaped by changing environmental factors can characterize keystone taxa roles in keystone guilds and may provide targets for manipulation to improve the function of the microbiome. Video Abstract.

Al and Ta co-doped LLZO as active filler with enhanced Li+conductivity for PVDF-HFP composite solid-state electrolyte.

Battery safety calls for solid state batteries and how to prepare solid electrolytes with excellent performance are of significant importance. In this study, hybrid solid electrolytes combined with organic PVDF-HFP and inorganic active fillers are studied. The modified active fillers of Li7-x-3yAlyLa3Zr2-xTaxO12are obtained by co-element doping with Al and Ta when LLZO is synthesized by calcination. And an high room temperature ionic conductivity of 5.357 × 10-4S cm-1is exhibited by ATLLZO ceramic sheet. The composite solid electrolyte PVDF-HFP/LiTFSI/ATLLZO (PHL-ATLLZO) is prepared by solution casting method, and its electrochemical properties are investigated. The results show that when the contents of lithium salt LiTFSI and active filler ATLLZO are controlled at 40 wt% and 10%, respectively, the ionic conductivity of the resulting composite solid electrolyte is as high as 2.686 × 10-4S cm-1at room temperature, and a wide electrochemical window of 4.75 V is exhibited. The LiFePO4/PHL-ATLLZO/Li all-solid-state battery assembled based on the composite solid-state electrolyte exhibits excellent cycling stability at room temperature. The cell assembled by casting the composite solid-state electrolyte on the cathode surface shows a discharge specific capacity of 134.3 mAh g-1and 96.2% capacity retention after 100 cycles at 0.2 C. The prepared composite solid-state electrolyte demonstrates excellent electrochemical performance.

Simultaneous determination of ZL-01, a novel nucleotide prodrug, and its metabolites in rat plasma by LC-MS/MS: Application to pharmacokinetic study.

ZL-01 is a novel dual-prodrug which shows promise to be an antiviral candidate for hepatitis C virus. Here we have established a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of ZL-01 and its four metabolites (M1, M7, M8, and M9) in rat plasma with special consideration of ex vivo ZL-01, M1, and M7 stability. Several factors affecting the stability were investigated. EDTA and citric acid solution (1 M) were added to plasma to maintain the stability of analytes. The protein-precipitation method was selected with acetonitrile containing sofosbuvir as internal standard (IS). Adequate separation of ZL-01 and its metabolites was achieved on XSelect HSS T3 (3.5 µm, 4.6 × 150 mm) column by a gradient-elution with a mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.5 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 599.2→418.5 for ZL-01, m/z 529.7→398.2 for M1, m/z 330.5→182.0 for M7, m/z 260.3→112.1 for M8, m/z 261.3→113.2 for M9 and m/z 530.4→243.4 for IS. The calibration curves exhibited good linearity (r＞0.997) for all components. The lower limit of quantitation (LLOQ) was in the range of 1-2 ng/mL. The intra-day and inter-day precisions (RSD) at three different levels were both less than 10.2% and the accuracies (RE) ranged from -3.7-7.6%. The matrix effect and extraction recovery of them ranged from 84% to 110.3% and 88.3-106.3%. This LC-MS/MS method for the simultaneous quantitation of ZL-01 and its metabolites was developed successfully and applied in the pharmacokinetic studies of these in rats. Pharmacokinetic results indicated ZL-01 would be metabolized rapidly and M8 might be the main metabolites after oral absorption.

Metabolomics based comprehensive investigation of Gardeniae Fructus induced hepatotoxicity.

Gardeniae Fructus (Zhizi in Chinese, ZZ in brief), a commonly used herbal medicine, has aroused wide concern for hepatotoxicity, but the mechanism remains to be investigated. This study was aimed at investigating the mechanism of ZZ-induced liver injury in vivo and in vitro based on metabolomics and evaluating the hepatotoxicity prediction ability of the in vitro model. SD rats were administered with extracted ZZ and HepG2 cells were treated with genipin, the major hepatotoxic metabolite of ZZ. Liver, plasma, intracellular and extracellular samples were obtained for metabolomics analysis. As a result, ZZ caused plasma biochemical and liver histopathological alterations in rats, and induced purine and amino acid metabolism disorder in the liver and pyrimidine, primary bile acids, amino acid metabolism and pantothenate and CoA biosynthesis disorder in the plasma. Pyrimidine, purine, amino acid metabolism and pantothenate and CoA biosynthesis were also found to be disturbed in the genipin-treated HepG2 cells, which exhibited similarity with the result in vivo. This study comprehensively illustrates the underlying mechanism involved in ZZ-related hepatotoxicity from the aspect of metabolome, and provides evidence that identifying hepatotoxicity can be achieved in cells, representing a non-animal alternative for systemic toxicology.

Isoflavones' effects on pharmacokinetic profiles of main iridoids from Gardeniae Fructus in rats.

Gardeniae Fructus (GF) and Semen Sojae Praeparatum (SSP) are both medicine food homologies and widely used in Chinese clinical prescriptions together. The research investigated the pharmacokinetics of four iridoids in normal rats and isolfavones-fed rats, which were administered with isolfavones from SSP for 7, 14, 21 and 28 consecutive days. A validated LC-MS/MS method was developed for determining shanzhiside, genipin-1-gentiobioside, geniposide and their metabolite genipin in rat plasma. Plasma samples were pretreated by solid-phase extraction using paeoniflorin as the internal standard. The chromatographic separation was performed on a Waters Atlantis T3 (4.6 mm × 150 mm, 3 µm) column using a gradient mobile phase consisting of acetonitril and water (containing 0.06% acetic acid). The mass detection was under the multiple reaction monitoring (MRM) mode via polarity switching between negative and positive ionization modes. The calibration curves exhibited good linearity (r > 0.997) for all components. The lower limit of quantitation was in the range of 1-10 ng/mL. The intra-day and inter-day precisions (RSD) at three different levels were both less than 12.2% and the accuracies (RE) ranged from -10.1% to 16.4%. The extraction recovery of them ranged from 53.8% to 99.7%. Pharmacokinetic results indicated the bioavailability of three iridoid glycosides and the metabolite, genipin in normal rats was higher than that in rats exposed to isoflavones. With the longer time of administration of isoflavones, plasma concentrations of iridoids decreased, while genipin sulfate, the phase Ⅱ metabolite of genposide and genipin-1-gentiobioside, appeared the rising exposure. The pharmacokinetic profiles of main iridoids from GF were altered by isoflavones.

Non-isoflavones Diet Incurred Metabolic Modifications Induced by Constipation in Rats via Targeting Gut Microbiota.

Isoflavones, presenting in leguminous plants and the normal chow diet, are known to alter intestinal microbiota, yet their deficiency has not been widely studied for its effect on constipation in biochemical state of rats. Our previous study discovered the differences in pharmacokinetic traits of isoflavones from Semen sojae praeparatum fed with normal chow diet (ISO) and non-isoflavones diet (NISO). To gain insight into the key role of intestinal microbiota in constipation and metabolic differences caused by isoflavones deficiency, we observed a significant decrease in fecal pellet numbers, fecal water content, intestinal transit rate together with the serum concentrations of substance P (SP) and vasoactive intestinal peptide (VIP) in NISO group, compared with those in the ISO group. Following 16S rRNA compositional sequencing, results excluded the changes in intestinal microbiota over time and highlighted that a total of 5 phyla and 21 genera changed significantly, among which Firmicutes, Bacteroidetes, Blautia, Prevotella, Lactobacillus and Bifidobacterium were closely related to constipation. In addition, Lactobacillus, produceing ß-glucosidase which contribute to biotransform glycosides into aglycons and exert the bioactivities consequently, was decreased after non-isoflavones diet intake. Meanwhile, predicted metagenomics indicated that the pathway of glycan biosynthesis and metabolism was markedly down-regulated after non-isoflavones diet intake. Taken together, the findings suggested that the changes in the dietary components could alter the biochemical state of rats, which may be triggered by the abnormal modifications facilitated by ß-glucosidase-producing bacteria. Our study shed a new strategy to explore the relationship among disease phenotypes (D), intestinal microbiota (I), enzymes (E) and traits of metabolism (T) named as "DIET," which can provide a reference for further study of the mechanism in regulation of intestinal bacteria-mediated diet on diseases.

Quantification of isoflavone glycosides and aglycones in rat plasma by LC-MS/MS: Troubleshooting of interference from food and its application to pharmacokinetic study of Semen Sojae Praeparatum extract.

The isoflavones widely exist in the daily diets and interferences are usually inevitable in the determination of the in vivo level of the same analytes. A new strategy to eliminate the dietary interference was established to evaluate the exposure of isoflavones including daidzin, glycitin, genistin, daidzein, glycitein, and genistein in rats fed with Semen Sojae Praeparatum (SSP) extract. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate using quercetin as the internal standard (IS). The chromatographic separation was achieved on a Symmetry C18 column (100 mm × 3.0 mm) using a gradient mobile phase consisting of acetonitril and water (containing 0.1% formic acid) with a run time of 13.0 min at a flow rate of 0.4ml/min. The detection was carried out by a triple-quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via polarity switching between negative (for and positive (for daidzin glycitin) ionization mode. All calibration curves exhibited good linearity (r> 0.99) over a wide concentration range for all components. The lower limit of quantitation (LLOQ) was in the range of 0.1-0.4 ng/ml. The intra-day and inter-day precisions (RSD) at three different levels were both less than 14.9% and the accuracies (RE) ranged from -9.3% to 14.5%. The extraction recoveries of the analytes and the IS ranged from 85.7% to 100.2%. The validated method was first successfully applied to pharmacokinetic study of the six isoflavones in rat plasma after oral administration of SSP extract. The dynamic baseline levels of six isoflavones in blank plasma from rats consuming food containing dietary isoflavones were measured for the correction of the plasma concentrations. The principle pharmacokinetic parameters were calculated from rats with or without regular commercial food, and found to be altered by the dietary food containing some isoflavones.

A new strategy for statistical analysis-based fingerprint establishment: Application to quality assessment of Semen sojae praeparatum.

Semen sojae praeparatum with homology of medicine and food is a famous traditional Chinese medicine. A simple and effective quality fingerprint analysis, coupled with chemometrics methods, was developed for quality assessment of Semen sojae praeparatum. First, similarity analysis (SA) and hierarchical clusting analysis (HCA) were applied to select the qualitative markers, which obviously influence the quality of Semen sojae praeparatum. 21 chemicals were selected and characterized by high resolution ion trap/time-of-flight mass spectrometry (LC-IT-TOF-MS). Subsequently, principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were conducted to select the quantitative markers of Semen sojae praeparatum samples from different origins. Moreover, 11 compounds with statistical significance were determined quantitatively, which provided an accurate and informative data for quality evaluation. This study proposes a new strategy for "statistic analysis-based fingerprint establishment", which would be a valuable reference for further study.