Blockade of spinal dopamine D1/D2 receptor heteromers by levo-Corydalmine suppressed calcium signaling cascade in spinal neurons to alleviate bone cancer pain in rats.

Background: Dopamine receptors have been reported to be involved in pain, while the exact effects and mechanism in bone cancer pain have not been fully explored. Methods: Bone cancer pain model was created by implanting walker 256 mammary gland carcinoma into right tibia bone cavity. Primary cultured spinal neurons were used for in vitro evaluation. FLIPR, western-blot, immunofluorescence, and Co-IP were used to detect cell signaling pathway. Results: Our results indicated that spinal dopamine D1 receptor (D1DR) and spinal dopamine D2 receptor (D2DR) could form heteromers in TCI rats, and antagonizing spinal D1DR and D2DR reduced heteromers formation and alleviated TCI-induced bone cancer pain. Further results indicated that D1DR or D2DR antagonist induced antinociception in TCI rats could be reversed by D1DR, D2DR, and D1/D2DR heteromer agonists. And Gq, IP3, and PLC inhibitors also attenuated TCI-induced bone cancer pain. In vitro results indicated that D1DR or D2DR antagonist decreased the Ca2+ oscillations upregulated by D1DR, D2DR, and D1/D2DR heteromer agonists in activated primary cultured spinal neurons. Moreover, inhibition of D1/D2DR heteromers induced antinociception in TCI rats was partially mediated by the CaMKII and MAPKs pathway. In addition, a natural compound levo-Corydalmine (l-CDL), could inhibit D1/D2DR heteromers and attenuate bone cancer pain. Results: Inhibition of spinal D1/D2DR heteromers via l-CDL decreases excitability in spinal neurons, which might present new therapeutic strategy for bone cancer pain.

Illumination of Hydroxyl Radical in Kidney Injury and High-Throughput Screening of Natural Protectants Using a Fluorescent/Photoacoustic Probe.

The hydroxyl radical (•OH) is shown to play a crucial role in the occurrence and progression of acute kidney injury (AKI). Therefore, the development of a robust •OH probe holds great promise for the early diagnosis of AKI, high-throughput screening (HTS) of natural protectants, and elucidating the molecular mechanism of intervention in AKI. Herein, the design and synthesis of an activatable fluorescent/photoacoustic (PA) probe (CDIA) for sensitive and selective imaging of •OH in AKI is reported. CDIA has near-infrared fluorescence/PA channels and fast activation kinetics, enabling the detection of the onset of •OH in an AKI model. The positive detection time of 12 h using this probe is superior to the 48-hour detection time for typical clinical assays, such as blood urea nitrogen and serum creatinine detection. Furthermore, a method is established using CDIA for HTS of natural •OH inhibitors from herbal medicines. Puerarin is screened out by activating the Sirt1/Nrf2/Keap1 signaling pathway to protect renal cells in AKI. Overall, this work provides a versatile and dual-mode tool for illuminating the •OH-related pathological process in AKI and screening additional compounds to prevent and treat AKI.

Two New Amides from Physochlainae Radix.

In this article, two undescribed amides (1-2) with an unusual (2-formyl-5-hydroxymethyl)pyrroyl-butylamine moiety were obtained from the Physochlainae Radix. Comprehensive spectroscopic studies, including NMR and HR-ESI-MS, coupling with spectroscopic data comparisons were used to determine structures. Anti-inflammatory assay results showed that new amides possessed significant inhibitory activities of the NO production of LPS-induced RAW 264.7 cells, with IC50 values of 17.52±1.68 µM and 20.37±2.42 µM, respectively.

Research Progress of Exosomal microRNA in Cardiovascular Disease and Its Forensic Application Prospects.

Exosomal microRNAs (miRNAs) are miRNAs that are mediated by exosomes to achieve cell-to-cell communication, and they are widespread in organisms. In recent years, the key role of the multiple biological functions of exosomal miRNAs in the occurrence and development of cardiovascular diseases has been confirmed by a large number of studies, which has become a hot spot in clinical and basic research. Sudden cardiac death caused by cardiovascular disease is one of the important contents in forensic medical identification. This article introduces the research progress of cardiovascular disease prediction, treatment and prognosis on exosomal miRNA. The prospects of the application in forensic medical identification are discussed.

Role of 5-HT degradation in acute liver injury induced by carbon tetrachloride.

5-hydroxytryptamine (5-HT) is involved in the pathological processes of several liver diseases. Acute liver injury underlies the development of many liver diseases, but the mechanism remains unclear. We aimed to investigate the role of 5-HT in carbon tetrachloride (CCl4)-induced acute liver injury. Acute liver injury was induced with CCl4 (10 mg/kg) in mice pretreated with the 5-HT2A receptor antagonist sarpogrelate hydrochloride (SH) and the 5-HT synthesis inhibitor carbidopa (CDP). LO2 cells were treated with CCl4, 5-HT or 2,5-dimethoxy-4-idopametamine and pretreated with SH, CDP or the monoamine oxidase A (MAO-A) inhibitor clorgyline. Hematoxylin-eosin staining, immunohistochemistry, Real-time quantitative PCR, western blotting, fluorescent probe and biochemical markers were used to evaluate liver compromise. 5-HT2A receptor, 5-HT synthetase and MAO-A were expressed in hepatocytes; their gene and protein expression were upregulated by CCl4, which led to the degradation of mitochondrial 5-HT and overproduction of reactive oxygen species (ROS). Hepatic injury may be aggravated by ROS, which induce oxidative stress and the phosphorylation of p38 mitogen-activated protein kinase, Jun N-terminal kinase, extracellular regulated protein kinase, signal transducer and activator of transcription 3 and nuclear factor kappa-B. 5-HT2A receptor may contribute to acute liver injury by modulating 5-HT synthase and MAO-A expression. The synergistic action of SH and CDP treatment may inhibit CCl4-induced acute liver injury in a dose-dependent manner. Hence, CCl4-induced acute liver injury is due to an increase in mitochondrial ROS production caused by increased 5-HT degradation and probably involves increases in 5-HT2A receptor expression and 5-HT synthesis.

Identification of NHXs in Gossypium species and the positive role of GhNHX1 in salt tolerance.

BACKGROUND: Plant Na+/H+ antiporters (NHXs) are membrane-localized proteins that maintain cellular Na+/K+ and pH homeostasis. Considerable evidence highlighted the critical roles of NHX family in plant development and salt response; however, NHXs in cotton are rarely studied. RESULTS: The comprehensive and systematic comparative study of NHXs in three Gossypium species was performed. We identified 12, 12, and 23 putative NHX proteins from G. arboreum, G. raimondii, and G. hirsutum, respectively. Phylogenetic study revealed that repeated polyploidization of Gossypium spp. contributed to the expansion of NHX family. Gene structure analysis showed that cotton NHXs contain many introns, which will lead to alternative splicing and help plants to adapt to high salt concentrations in soil. The expression changes of NHXs indicate the possible differences in the roles of distinct NHXs in salt response. GhNHX1 was proved to be located in the vacuolar system and intensively induced by salt stress in cotton. Silencing of GhNHX1 resulted in enhanced sensitivity of cotton seedlings to high salt concentrations, which suggests that GhNHX1 positively regulates cotton tolerance to salt stress. CONCLUSION: We characterized the gene structure, phylogenetic relationship, chromosomal location, and expression pattern of NHX genes from G. arboreum, G. raimondii, and G. hirsutum. Our findings indicated that the cotton NHX genes are regulated meticulously and differently at the transcription level with possible alternative splicing. The tolerance of plants to salt stress may rely on the expression level of a particular NHX, rather than the number of NHXs in the genome. This study could provide significant insights into the function of plant NHXs, as well as propose promising candidate genes for breeding salt-resistant cotton cultivars.

Comparative acetylome analysis of wild-type and fuzzless-lintless mutant ovules of upland cotton (Gossypium hirsutum Cv. Xu142) unveils differential protein acetylation may regulate fiber development.

Protein acetylation (KAC) is a significant post-translational modification, which plays an essential role in the regulation of growth and development. Unfortunately, related studies are inadequately available in angiosperms, and to date, there is no report providing insight on the role of protein acetylation in cotton fiber development. Therefore, we first compared the lysine-acetylation proteome (acetylome) of upland cotton ovules in the early fiber development stages by using wild-type as well as its fuzzless-lintless mutant to identify the role of KAC in the fiber development. A total of 1696 proteins with 2754 acetylation sites identified with the different levels of acetylation belonging to separate subcellular compartments suggesting a large number of proteins differentially acetylated in two cotton cultivars. About 80% of the sites were predicted to localize in the cytoplasm, chloroplast, and mitochondria. Seventeen significantly enriched acetylation motifs were identified. Serine and threonine and cysteine located downstream and upstream to KAC sites. KEGG pathway enrichment analysis indicated oxidative phosphorylation, fatty acid, ribosome and protein, and folate biosynthesis pathways enriched significantly. To our knowledge, this is the first report of comparative acetylome analysis to compare the wild-type as well as its fuzzless-lintless mutant acetylome data to identify the differentially acetylated proteins, which may play a significant role in cotton fiber development.

WITHDRAWN: Identification of abnormally expressed genes and their roles in invasion and migration of hepatocellular carcinoma.

This paper was originally published in Aging Advance Online Publications on February 2, 2020. In compliance with Aging's withdrawal policy, the paper was withdrawn in its entirety. It will not appear in Aging internal or any external indexes or archives.

A cancer-specific activatable theranostic nanodrug for enhanced therapeutic efficacy via amplification of oxidative stress.

Rationale: Despite considerable advances, the reactive oxygen species (ROS)-mediated cancer treatment suffers from the problems of up-regulation of adaptive antioxidants in cancer cells as well as side effects to normal cells. Therefore, development of a new generation of cancer-specific nanomedicine capable of amplifying oxidative stress would be of great interest for accurate and effective cancer treatment. Methods: Herein, transferrin (Tf)-decorated, dihydroartemisinin (DHA), L-buthionine-sulfoximine (BSO), and CellROX-loaded liposomal nanoparticles (Tf-DBC NPs) were developed for precise cancer theranositcs. Tf-DBC NPs could specifically recognize cancer cells via Tf-Tf receptor binding and be uptaken into the lysosomes of cancer cells, where Tf-DBC NPs were activated to release Fe(II), DHA, and BSO. ROS was generated by DHA in the presence of Fe(II), and GSH was depleted by BSO to disrupt the redox balance in cancer cells. Furthermore, CellROX, as a fluorescent probe for imaging of intracellular oxidative stress, was used to monitor the therapeutic efficacy. Results: The integration of Tf, DHA, and BSO into the acidic pH-responsive liposomes selectively and effectively killed cancer cells and prevented the oxidative injury to normal cells. The high oxidative state was visualized at the tumor site and the amplification of oxidative stress enabled tumor eradication by Tf-DBC NPs, demonstrating the successful implementation of this novel strategy in vivo. Conclusion: Our study provides a new paradigm for the design of ROS-mediated therapeutics and offers a promising perspective for precise cancer treatment.

Construction of Cu2O/In2O3 Hybrids with p-n Heterojunctions for Enhanced Photocatalytic Performance.

Fabrication of p-n heterojunction materials is an efficient strategy for improving the photocatalytic activity by suppressing the recombination of photogenerated electrons and holes. In this study, a p-n heterojunction consisting of Cu2O and In2O3 was synthesized using the hydrothermal in situ deposition method. The Cu2O/In2O3 hybrid exhibited enhanced performance in the photocatalytic hydrogen evolution reaction and degradation of organic pollutants compared with pure Cu2O and In2O3. The sensitization of In2O3 by Cu2O considerably enhanced the light response of the Cu2O/In2O3 hybrid, and the close contact between In2O3 and Cu2O led to the generation of the p-n heterojunction. The photoelectrochemical properties test proved that the successful fabrication of the p-n heterojunction in the Cu2O/In2O3 hybrid promoted the transfer of photoinduced electrons and holes and inhibited the recombination of photogenerated carriers efficiently. The present strategy provides an efficient approach to improve the performance of photocatalysts through the fabrication of p-n heterojunctions.

Targeted Myocardial Hypoxia Imaging Using a Nitroreductase-Activatable Near-Infrared Fluorescent Nanoprobe.

Development of a highly selective and sensitive imaging probe for accurate detection of myocardial hypoxia will be helpful to estimate the degree of ischemia and subsequently guide personalized treatment. However, an efficient optical approach for hypoxia monitoring in myocardial ischemia is still lacking. In this work, a cardiomyocyte-specific and nitroreductase-activatable near-infrared nanoprobe has been developed for selective and sensitive imaging of myocardial hypoxia. The nanoprobe is a liposome-based nanoarchitecture which is functionalized with a peptide (GGGGDRVYIHPF) for targeting heart cells and encapsulating a nitrobenzene-substituted BODIPY for nitroreductase imaging. The nanoprobe can specifically recognize and bind to angiotensin II type 1 receptor that is overexpressed on the ischemic heart cells by the peptide and is subsequently uptaken into heart cells, in which the probe is released and activated by hypoxia-related nitroreductase to produce fluorescence emission at 713 nm. The in vitro response of the nanoprobe toward nitroreductase resulted in 55-fold fluorescence enhancement with the limit of detection as low as 7.08 ng/mL. Confocal fluorescence imaging confirmed the successful uptake of nanoprobe by hypoxic heart cells and intracellular detection of nitroreductase. More significantly, in vivo imaging of hypoxia in a murine model of myocardial ischemia was achieved by the nanoprobe with high sensitivity and good biocompatibility. Therefore, this work presents a new tool for targeted detection of myocardial hypoxia and will promote the investigation of the hypoxia-related physiological and pathological process of ischemic heart disease.

Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression.

BACKGROUND: When developing CRISPR/Cas9 systems for crops, it is crucial to invest time characterizing the genome editing efficiency of the CRISPR/Cas9 cassettes, especially if the transformation system is difficult or time-consuming. Cotton is an important crop for the production of fiber, oil, and biofuel. However, the cotton stable transformation is usually performed using Agrobacterium tumefaciens taking between 8 and 12 months to generate T0 plants. Furthermore, cotton is a heterotetraploid and targeted mutagenesis is considered to be difficult as many genes are duplicated in this complex genome. The application of CRISPR/Cas9 in cotton is severely hampered by the long and technically challenging genetic transformation process, making it imperative to maximize its efficiency. RESULTS: In this study, we provide a new system to evaluate and validate the efficiency of CRISPR/Cas9 cassettes in cotton using a transient expression system. By using this system, we could select the most effective CRISPR/Cas9 cassettes before the stable transformation. We have also optimized the existing cotton CRISPR/Cas9 system to achieve vastly improved mutagenesis efficiency by incorporating an endogenous GhU6 promoter that increases sgRNA expression levels over the Arabidopsis AtU6-29 promoter. The 300 bp GhU6.3 promoter was cloned and validated using the transient expression system. When sgRNAs were expressed under the control of the GhU6.3 promoter in CRISPR/Cas9 cassettes, expression levels were 6-7 times higher than those provided by the AtU6-29 promoter and CRISPR/Cas9-mediated mutation efficiency was improved 4-6 times. CONCLUSIONS: This study provides essential improvements to maximize CRISPR/Cas9-mediated mutation efficiency by reducing risk and workload for the application of CRISPR/Cas9 approaches in the targeted mutagenesis of cotton.

Calcium-dependent protein kinases in cotton: insights into early plant responses to salt stress.

BACKGROUND: Soil salinization is one of the major environmental constraints to plant growth and agricultural production worldwide. Signaling components involving calcium (Ca2+) and the downstream calcium-dependent protein kinases (CPKs) play key roles in the perception and transduction of stress signals. However, the study of CPKs in cotton and their functions in response to salt stress remain unexplored. RESULTS: A total of 98 predicted CPKs were identified from upland cotton (Gossypium hirsutum L. 'TM-1'), and phylogenetic analyses classified them into four groups. Gene family distribution studies have revealed the substantial impacts of the genome duplication events to the total number of GhCPKs. Transcriptome analyses showed a wide distribution of CPKs' expression among different organs. A total of 19 CPKs were selected for their rapid responses to salt stress at the transcriptional level, most of which were also incduced by the thylene-releasing chemical ethephon, suggesting a partal overlap of the salinity and ethylene responses. Silencing of 4 of the 19 CPKs (GhCPK8, GhCPK38, GhCPK54, and GhCPK55) severely compromised the basal cotton resistance to salt stress. CONCLUSIONS: Our genome-wide expression analysis of CPK genes from up-land cotton suggests that CPKs are involved in multiple developmental responses as well as the response to different abiotic stresses. A cluster of the cotton CPKs was shown to participate in the early signaling events in cotton responses to salt stress. Our results provide significant insights on functional analysis of CPKs in cotton, especially in the context of cotton adaptions to salt stress.

Succinate/NLRP3 Inflammasome Induces Synovial Fibroblast Activation: Therapeutical Effects of Clematichinenoside AR on Arthritis.

Clematichinenoside AR (C-AR) is a triterpene saponin isolated from the root of Clematis manshurica Rupr., which is a herbal medicine used in traditional Chinese medicine for the treatment of arthritis. C-AR exerts anti-inflammatory and immunosuppressive properties, but little is known about its action in the suppression of fibroblast activation. Low oxygen tension and transforming growth factor-ß (TGF-ß1) induction in the synovium contribute to fibrosis in arthritis. This study was designed to investigate the effect of C-AR on synovial fibrosis from the aspects of hypoxic TGF-ß1 and hypoxia-inducible transcription factor-1α (HIF-1α) induction. In the synovium of rheumatoid arthritis (RA) rats, hypoxic TGF-ß1 induction increased succinate accumulation due to the reversal of succinate dehydrogenase (SDH) activation and induced NLRP3 inflammasome activation in a manner dependent on HIF-1α induction. In response to NLRP3 inflammasome activation, the released IL-1ß further increased TGF-ß1 induction, suggesting the forward cycle between inflammation and fibrosis in myofibroblast activation. In the synovium of RA rats, C-AR inhibited hypoxic TGF-ß1 induction and suppressed succinate-associated NLRP3 inflammasome activation by inhibiting SDH activity, and thereby prevented myofibroblast activation by blocking the cross-talk between inflammation and fibrosis. Taken together, these results showed that succinate worked as a metabolic signaling, linking inflammation with fibrosis through NLRP3 inflammasome activation. These findings suggested that synovial succinate accumulation and HIF-1α induction might be therapeutical targets for the prevention of fibrosis in arthritis.

Pharmacological activation of AMPK prevents Drp1-mediated mitochondrial fission and alleviates endoplasmic reticulum stress-associated endothelial dysfunction.

BACKGROUND AND PURPOSE: This study aims to investigate whether and how pharmacological activation of AMP-activated protein kinase (AMPK) improves endothelial function by suppressing mitochondrial ROS-associated endoplasmic reticulum stress (ER stress) in the endothelium. Experimental approach Palmitate stimulation induced mitochondrial fission and ER stress-associated endothelial dysfunction. The effects of AMPK activators salicylate and AICA riboside (AICAR) on mitochondrial ROS production, Drp1 phosphorylation, mitochondrial fission, ER stress, thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation, inflammation, cell apoptosis and endothelium-dependent vasodilation were observed. Key results "Silencing" of TXNIP by RNA interference inhibited NLRP3 inflammasome activation in response to ER stress, indicating that TXNIP was a key link between ER stress and NLRP3 inflammasome activation. AMPK activators salicylate and AICAR prevented ROS-induced mitochondrial fission by enhancing dynamin-related protein 1 (Drp1) phosphorylation (Ser 637) and thereby attenuated IRE-1α and PERK phosphorylation, but their actions were blocked by knockdown of AMPK. Salicylate and AICAR reduced TXNIP induction and inhibited NLRP3 inflammasome activation by reducing NLRP3 and caspase-1 expression, leading to a reduction in IL-1ß secretion. As a result, salicylate and AICAR inhibited inflammation and reduced cell apoptosis. Meanwhile, salicylate and AICAR enhanced eNOS phosphorylation and restored the loss of endothelium-dependent vasodilation in the rat aorta. Immunohistochemistry staining showed that AMPK activation inhibited ER stress and NLRP3 inflammasome activation in the vascular endothelium. CONCLUSION AND IMPLICATIONS: Pharmacological activation of AMPK regulated mitochondrial morphology and ameliorated endothelial dysfunction by suppression of mitochondrial ROS-associated ER stress and subsequent TXNIP/NLRP3 inflammasome activation. These findings suggested that regulation of Drp1 phosphorylation by AMPK activation contributed to suppression of ER stress and thus presented a potential therapeutic strategy for AMPK activation in the regulation of endothelium homeostasis.

Mouse myosin-19 is a plus-end-directed, high-duty ratio molecular motor.

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament.

Studies on the stability of salvianolic acid B as potential drug material.

INTRODUCTION: Salvianolic acid B (Sal B) is one of the major water-soluble compounds isolated from the roots of Salvia miltiorrhiza, which is widely used as a traditional Chinese medicine. Although much research on the general stability of Sal B has been undertaken and reported, there is still a need for further study of the stability required as a potential drug material. OBJECTIVE: To study the stability of Sal B in the solid state and in normal saline (NS) solution during storage, as required in the ICH guidelines (2003) and Chinese Pharmacopoeia (2005). METHODOLOGY: Sal B stability was analysed using the high-performance liquid chromatography (HPLC) method described in the Chinese Pharmacopoeia. HPLC coupled with time-of-flight mass spectrometry (HPLC-TOFMS) was applied for the separation and identification of the degradation products of Sal B. RESULTS: In the solid state, Sal B packaged in aluminium foil bags was stable for 6 months under 'accelerated conditions' (40°C, 75% relative humidity, RH). However, solid Sal B degradation was observed under open exposure to stress conditions of high temperature (60°C) or high humidity (92.5 or 75% RH). In NS solution, Sal B underwent severe degradation under accelerated conditions. Through HPLC-TOFMS, nine degradation products were identified and the possible degradation pathway was deduced. CONCLUSION: The results demonstrate that the potential drug material Sal B could be used in a solid formulation, but is not suitable for use as a liquid formulation.