The complete chloroplast genome sequence and phylogenetic analysis of Strobilanthes dalzielii (W.W.Sm.) Benoist 1935 (Acanthaceae).

Strobilanthes dalzielii of Acanthaceae is an herb species with potentially extensive applications for its pharmaceutical and ornamental values. Due to taxonomic complications and limited genetic information, the structural characteristics, and phylogenetic relationships of the S. dalzielii chloroplast genome were assembled and characterized here for the first time. The complete chloroplast genome of S. dalzielii was 144,580 bp in length. The genome is quadripartite in structure and consists of a large single-copy region (92,137 bp) and a small single-copy region (17,669 bp), which are separated by a pair of inverted repeats (each 17,387 bp). A total of 125 genes were annotated, including 80 protein-coding, 37 transfer RNA, and eight ribosomal RNA genes. The overall GC content was 36.4%. Phylogenetic analysis based on the complete chloroplast genome sequence of 21 taxa within the tribe Ruellieae of Acanthaceae using the maximum likelihood and Bayesian inference methods revealed that Strobilanthes diverged after Ruellia; S. dalzielii is closely related to S. tonkinensis. The genomic data obtained from this study will serve as valuable information to the species delimitation and genetic classification of Strobilanthes.

A Radical-Generating Probe to Release Free Fluorophores and Identify Artemisinin-Sensitive Cancer Cells.

The smart light-up probes have been extensively developed to image various enzymes and other bioactive molecules. Upon activation, these probes result in light-up fluorophores that exist in a protein-bound or a free form. The difference between these two forms has not yet been reported. Here, we present a pair of smart light-up probes that generate a protein-bound fluorophore and a free fluorophore upon activation by heme. Probe 8 generated a radical-attached fluorophore that predominantly existed in the free form, while probe 10 generated an α,ß-unsaturated ketone-attached fluorophore that showed extensive labeling of proteins. In live-cell imaging, probe 8 showed greater fluorescence intensity than probe 10 when low concentrations (0.1-5 µM) of the probes were used, but probe 8 was less fluorescent than probe 10 when the concentrations of the probes were high (10 µM). Finally, probe 8 was used to reflect the activation level of the endoperoxide bond in cancer cells and to effectively distinguish ART-sensitive cancer cells from ART-insensitive ones.

Global Reactivity Profiling of the Catalytic Lysine in Human Kinome for Covalent Inhibitor Development.

Advances in targeted covalent inhibitors (TCIs) have been made by using lysine-reactive chemistries. Few aminophiles possessing balanced reactivity/stability for the development of cell-active TCIs are however available. We report herein lysine-reactive activity-based probes (ABPs; 2-14) based on the chemistry of aryl fluorosulfates (ArOSO2 F) capable of global reactivity profiling of the catalytic lysine in human kinome from mammalian cells. We concurrently developed reversible covalent ABPs (15/16) by installing salicylaldehydes (SA) onto a promiscuous kinase-binding scaffold. The stability and amine reactivity of these probes exhibited a broad range of tunability. X-ray crystallography and mass spectrometry (MS) confirmed the successful covalent engagement between ArOSO2 F on 9 and the catalytic lysine of SRC kinase. Chemoproteomic studies enabled the profiling of >300 endogenous kinases, thus providing a global landscape of ligandable catalytic lysines of the kinome. By further introducing these aminophiles into VX-680 (a noncovalent inhibitor of AURKA kinase), we generated novel lysine-reactive TCIs that exhibited excellent in vitro potency and reasonable cellular activities with prolonged residence time. Our work serves as a general guide for the development of lysine-reactive ArOSO2 F-based TCIs.

A pilot study of several environmental endocrine disrupting chemicals in children with autism spectrum disorder in south China.

Autism spectrum disorders (ASD) is a group of heterogeneous neurodevelopmental disorders. Evidence has implied that environmental pollutants are important factors related to ASD. In this study, several environmental endocrine-disrupting chemicals, including parabens, benzophenone-type ultraviolet filters, hydroxyl polycyclic aromatic hydrocarbons, triclosan and tetrabromobisphenol A were analyzed in blood plasma in ASD children (n = 34) and the control children (n = 28). The results showed that parabens were the most concentrated chemicals (2.18 ng/mL, median value), followed by hydroxyl polycyclic aromatic hydrocarbons (0.73 ng/mL), benzophenone-type ultraviolet filters (0.14 ng/mL), triclosan (0.13 ng/mL) and tetrabromobisphenol A (0.03 ng/mL). ASD children accumulated significantly lower 2-hydroxy-4-methoxybenzophenone, 2,4-dihydroxybenzophenone, 4-hydroxybenzophenone and triclosan but higher 2-hydroxyphenanthrene and tetrabromobisphenol A than the control children (0.02/0.09 ng/mL of 2-hydroxy-4-methoxybenzophenone, p < 0.05; 0.04/0.07 ng/mL of 2,4-dihydroxybenzophenone, p < 0.05; 0.03/0.04 ng/mL of 4-hydroxybenzophenone, p < 0.05; 0.13/1.22 ng/mL of triclosan, p < 0.01; 0.03 ng/mL/not detected of 2-hydroxyphenanthrene, p < 0.05; 0.03/0.004 ng/mL of tetrabromobisphenol A, p < 0.05). Gender differences in certain environmental endocrine-disrupting chemicals were evident, and the differences were more inclined toward boys. Positive associations between 2-hydroxy-4-methoxybenzophenone and triclosan, and tetrabromobisphenol A and 2-hydroxyphenanthrene were found in ASD boys. Binary logistic regression analysis showed that the adjusted odds ratio value of 2-hydroxyphenanthrene in ASD boys was 11.0 (1.45-84.0, p < 0.05). This is the first pilot study on multiple environmental endocrine-disrupting chemicals in children with ASD in China.

Stereochemistry and antimalarial activity of C-10 carba analogues of artemisinin.

Artemisinin is an endoperoxide bond-containing sesquiterpene lactone showing potent antimalarial effect as well as antitumor and antivirus activities. Inspired by this unique pharmacorphore, researchers around the world developed numerous Artemisinin derivatives. Among these derivatives, the C-10 carba analogues of artemisinin are frequently reported. However, the stereochemistry of C-10 carba analogues of artemisinin is overlooked and the corresponding mixture of stereoisomers are used. Herein, we reported for the first time stereochemistry and antimalarial activity of C-10 carba analogues of artemisinin. We employed two approaches to obtain the pure isomer of C-10 carba analogues and presented an interesting observation about their antimalarial activities. The minor isomer with large-sized substitute and S configuration at C-10 position had much lower antimalarial effect than the major isomer with R configuration. The study will shed light on the development of effective antimalarial drugs based on ART.

Bioequivalence Study of Miglitol Orally Disintegrating Tablets in Healthy Chinese Volunteers Under Fasting Condition Based on Pharmacodynamic and Pharmacokinetic Parameters.

To investigate the bioequivalence of miglitol orally disintegrating tablets in healthy Chinese volunteers based on pharmacodynamic (PD) and pharmacokinetic (PK) parameters. Additionally, the safety profile was estimated. Two randomized, open-label, single-dose, crossover trials were conducted under fasting conditions. In the PD trial (CTR20191811), 45 healthy volunteers were randomly divided into 3 groups in a 1:1:1 ratio and administered sucrose alone or coadministered with 50 mg of miglitol orally disintegrating tablet test or reference formulation/sucrose. In the PK trial (CTR20191696), 24 healthy volunteers were randomized (1:1) to receive the test or reference formulation (50 mg). Blood samples were collected at 15 and 17 sampling points per cycle in the PD and PK trials, respectively. Plasma miglitol and serum glucose concentrations were analyzed using a validated liquid chromatography-tandem mass spectrometry method. Serum insulin concentrations were measured using electrochemiluminescent immunoassay. Statistical analyses for the PD and PK parameters were subsequently performed. The volunteers' physical indicators were monitored and documented during the entire study to estimate drug safety. The PD and PK parameters of the two formulations were similar. The main PD and PK end points were both within the prespecified range of 80%-125%. The incidences of treatment-emergent adverse events (TEAEs) and drug-related TEAEs were similar between the test and reference formulation groups, and no serious TEAEs or deaths occurred during the 2 trials. These 2 formulations were demonstrated to be bioequivalent and well tolerated in healthy Chinese volunteers under fasting condition.

Orthogonal Strategies for Profiling Potential Cellular Targets of Anandamide and Cannabidiol.

The human endocannabinoid system regulates a myriad of physiological processes through a complex lipid signaling network involving cannabinoids and their respective receptors, cannabinoid receptor 1 (hCB1 R) and cannabinoid receptor 2 (hCB2 R). Anandamide (AEA) and cannabidiol (CBD) are classical examples of cannabinoids that elicit a variety of effects, both beneficial and detrimental, through these receptors. Mounting evidence suggested the presence of other potential cannabinoid targets that may be responsible for other observable effects. However, prior pharmacological studies on these cannabinoid compounds provided scant evidence of direct engagement to these proposed targets. Moreover, to the best of our knowledge, no chemoproteomic studies have been demonstrated on CBD. Here we showed that, by taking advantage of a recently developed 'label-free' 2D-TPP (2 Dimensional-Thermal Protein Profiling) approach, we have identified several new putative targets of both AEA and CBD. Comparison of these interaction landscapes with those obtained from well-established affinity-based protein profiling (AfBPP) platforms has led to the discovery of both shared and unique protein targets. Subsequent target validation of selected proteins led us to conclude that this 2D-TPP strategy complements well with AfBPP.

Occurrence and ecological risk assessment of 16 phthalates in surface water of the mainstream of the Yangtze River, China.

Phthalic acid esters (PAEs), a class of typical endocrine disruptors, have received considerable attention due to their widespread applications and adverse effects on biological health. In this study, 30 water samples, along the mainstream of the Yangtze River (YR), were collected from Chongqing (upper stream) to Shanghai (estuary) from May to June in 2019. The total concentrations of 16 targeted PAEs ranged from 0.437 to 20.5 µg/L, with an average of 1.93 µg/L, where dibutyl phthalate (DBP, 0.222-20.2 µg/L), bis (2-ethylhexyl) phthalate (DEHP, 0.254-7.03 µg/L), and diisobutyl phthalate (DIBP, 0.0645-0.621 µg/L) were the most abundant PAEs. According to the pollution level in the YR to assess the ecological risk posed by PAEs, the results showed medium risk level of PAEs in the YR, among which DBP and DEHP posed a high ecological risk to aquatic organisms. The optimal solution for DBP and DEHP is found in ten fitting curves. The PNECSSD of them is 2.50 µg/L and 0.34 µg/L, respectively.

Transformation from biofiltration unit to hybrid constructed wetland-microbial fuel cell: Improvement of wastewater treatment performance and energy recovery.

This study aimed to compare the performance of biofiltration, constructed wetland, and constructed wetland microbial fuel cell (CW-MFC). The transformation from a biofiltration unit to a hybrid CW-MFC was demonstrated with the advantages of improvement of wastewater treatment while generating electricity simultaneously. The introduction of plants to the upper region of the bioreactor enhanced the DO level by 0.8 mg/L, ammonium removal by 5 %, and COD removal by 1 %. The integration of electrodes and external circuits stimulated the degradation rate of organic matter in the anodic region (1 % without aeration and 3 % with aeration) and produced 5.13 mW/m3 of maximum power density. Artificial aeration improved the nitrification efficiency by 38 % and further removed the residual COD to an efficiency of 99 %. The maximum power density was also increased by 3.2 times (16.71 mW/m3) with the aid of aeration. In treating higher organic loading wastewater (3M), the maximum power density showed a significant increment to 78.01 mW/m3 (4.6-fold) and the COD removal efficiency was 98 %. The ohmic overpotential dominated the proportion of total loss (67-91 %), which could be ascribed to the low ionic conductivity. The reduction in activation and concentration loss contributed to the lower internal resistance with the additional aeration and higher organic loading. Overall, the transformation from biofiltration to a hybrid CW-MFC system is worthwhile since the systems quite resemble while CW-MFC could improve the wastewater treatment as well as recover energy from the treated wastewater.

The toxic natural product tutin causes epileptic seizures in mice by activating calcineurin.

Tutin, an established toxic natural product that causes epilepsy in rodents, is often used as a tool to develop animal model of acute epileptic seizures. However, the molecular target and toxic mechanism of tutin were unclear. In this study, for the first time, we conducted experiments to clarify the targets in tutin-induced epilepsy using thermal proteome profiling. Our studies showed that calcineurin (CN) was a target of tutin, and that tutin activated CN, leading to seizures. Binding site studies further established that tutin bound within the active site of CN catalytic subunit. CN inhibitor and calcineurin A (CNA) knockdown experiments in vivo proved that tutin induced epilepsy by activating CN, and produced obvious nerve damage. Together, these findings revealed that tutin caused epileptic seizures by activating CN. Moreover, further mechanism studies found that N-methyl-D-aspartate (NMDA) receptors, gamma-aminobutyric acid (GABA) receptors and voltage- and Ca2+- activated K+ (BK) channels might be involved in related signaling pathways. Our study fully explains the convulsive mechanism of tutin, which provides new ideas for epilepsy treatment and drug development.

A Late-Stage Aryl C-H Olefination Strategy and Its Application Towards Global Proteome Profiling of Δ8 -Tetrahydrocannabinol.

Drugs and bioactive natural products exert their pharmacological effects by engaging numerous cellular targets in our body. Identification of these protein targets is essential for understanding the mechanism-of-action of these compounds, thus contributing to improved drug design in drug discovery programs. Termed "in situ drug profiling", a common strategy for studying these bioactive compounds centralized on the covalent capture of protein targets along with a reporter tag to facilitate downstream proteomic analyses. Though highly successful, such reliance on innate electrophilic traps to facilitate covalent capture restricted its applications to covalent acting compounds. Late-stage C-H functionalization (LSF) may resolve this by substituting biologically inert C-H bonds with desired electrophilic groups. Herein, we demonstrated this concept by arming a diverse range of electron-rich aromatic drugs and natural products with α,ß-unsaturated esters, via late-stage C-H olefination with an arylthio-based carboxylic acid ligand developed by Ibanez and co-workers. We also showed that covalent probes generated from this LSF approach could be applied for "in situ drug profiling" of Δ8 -THC, as exemplified by the successful target engagement of α-4 db, a Δ8 -THC-based probe, to its native target hCB2 R. In combination with AfBP 7, a photoaffinity-based derivative of Δ8 -THC, we identified several novel putative targets that could account for some of the effects in THC consumption. We anticipate our C-H LSF strategy to be widely adopted for future studies of non-covalent drugs.

Rhodojaponin VI indirectly targets Cav2.2 channels via N-ethylmaleimide-sensitive fusion protein to alleviate neuropathic pain.

Neuropathic pain is a chronic disease that severely afflicts the life and emotional status of patients, but currently available treatments are often ineffective. Novel therapeutic targets for the alleviation of neuropathic pain are urgently needed. Rhodojaponin VI, a grayanotoxin from Rhododendron molle, showed remarkable antinociceptive efficacy in models of neuropathic pain, but its biotargets and mechanisms are unknown. Given the reversible action of rhodojaponin VI and the narrow range over which its structure can be modified, we perforwmed thermal proteome profiling of the rat dorsal root ganglion to determine the protein target of rhodojaponin VI. N-Ethylmaleimide-sensitive fusion (NSF) was confirmed as the key target of rhodojaponin VI through biological and biophysical experiments. Functional validation showed for the first time that NSF facilitated trafficking of the Cav2.2 channel to induce an increase in Ca2+ current intensity, whereas rhodojaponin VI reversed the effects of NSF. In conclusion, rhodojaponin VI represents a unique class of analgesic natural products targeting Cav2.2 channels via NSF.

Synthesis and structure-activity relationships of N - (3 - (1H-imidazol-2-yl) phenyl) - 3-phenylpropionamide derivatives as a novel class of covalent inhibitors of p97/VCP ATPase.

Noncovalent inhibitors of p97 have entered clinical studies. Compared with noncovalent inhibitors, covalent inhibitors have unique advantages in maintaining inhibitory effect and improving the resistance of the target. We previously employed the activity-based protein profiling to definitely identify p97 as the protein target of FL-18 that has a unique scaffold of benpropargylamide coupled with an imidazole. In this study, we report a thorough structure-activity-relationship study involving the new scaffold. A total of three rounds of optimization led to the discovery of the most potent covalent inhibitor of p97 to date. A chemical proteomics study indicated that the newly-synthesized compounds still targeted the C522 residue of p97 and retained selectivity among the complicated whole proteome. This study provides a suite of new covalent inhibitors of p97 to assist in its biological study and drug discovery.

Concentration-Dependent Enrichment Identifies Primary Protein Targets of Multitarget Bioactive Molecules.

Multitarget bioactive molecules (MBMs) are of increasing importance in drug discovery as they could produce high efficacy and a low chance of resistance. Several advanced approaches of quantitative proteomics were developed to accurately identify the protein targets of MBMs, but little study has been carried out in a sequential manner to identify primary protein targets (PPTs) of MBMs. This set of proteins will first interact with MBMs in the temporal order and play an important role in the mode of action of MBMs, especially when MBMs are at low concentrations. Herein, we describe a valuable observation that the result of the enrichment process is highly dependent on concentrations of the probe and the proteome. Interestingly, high concentrations of probe and low concentrations of incubated proteome will readily miss the hyper-reactive protein targets and thereby increase the probability of rendering PPTs with false-negative results, while low concentrations of probe and high concentrations of incubated proteome more than likely will capture the PPTs. Based on this enlightening observation, we developed a proof-of-concept approach to identify the PPTs of iodoacetamide, a thiol-reactive MBM. This study will deepen our understanding of the enrichment process and improve the accuracy of pull-down-guided target identification.

A meta-analysis and trial sequential analysis of high intensity focused ultrasound ablation combined with transhepatic arterial chemotherapy and embolization for hepatoma.

Objective: The efficacy of High Intensity Focused Ultrasound Ablation(HIFU) combined with Transhepatic Arterial Chemotherapy And Embolization(TACE) versus TACE alone in the treatment of hepatoma was evaluated by meta-analysis and trial sequential analyses(TSA). Methods: Pubmed, Cochrane, Embase, Web of Science, Scoups and CNKI, CQVIP, Wanfang Data(China National Knowledge Infrastructure) databases were searched from database construction to April 2022, and randomized controlled trials were included. Revman and Stata software were used for meta-analysis of tumor changes, survival rate, laboratory indicators and adverse reactions in the included studies, and TSA0.9 was used for sequential analysis. Grade Pro was also used to evaluate the included indicators. Results: Twelve studies were included with a sample size of 1025 cases. Meta-analysis showed that the tumor response rate in the combined treatment group was 1.54 times higher than that in TACE alone (OR: 2.54; 95%CI:1.81-3.57) and the 6-month to 5-year survival rate was 1-4 times higher, with statistically significant differences (P<0.05). Subgroup analysis showed that country, pathological type and study type were the sources of heterogeneity. Egger results showed that there was no publication bias (95%CI: -1.333, 3.552; Ppublication=0.276), and the sensitivity analysis results were reliable. TSA results suggest that there may be false positive results, which need to be further confirmed by more studies. Grade evaluation results indicated that the quality of evidence for response rate and one-year survival was low. Conclusion: HIFU combined with TACE has better efficacy in the treatment of hepatoma, which is worthy of promotion. However, there may be false positive results in this study, which needs to be further verified by more extensive and more tests.

Associations Between microRNA-related Genetic Polymorphisms and Clinical Response to Methotrexate in Chinese Rheumatoid Arthritis Patients.

BACKGROUND: Emerging evidence indicates that microRNA (miRNA)-related genetic polymorphisms are strongly involved in the post-transcriptional regulation of the expression of pharmacokinetics and pharmacodynamics- related genes, therefore contributing to the genetic variability of drug response. OBJECTIVE: To investigate the associations of miRNA-related genetic polymorphisms, including miRNA-5189 rs562929801, miRNA-595 rs4909237, SLCO1A2 rs4149009 and MTHFR rs3737966, and clinical response to methotrexate in Chinese rheumatoid arthritis patients. METHODS: One hundred patients treated with MTX for approximately 3 months were prospectively followed up to evaluate the clinical response according to European League Against Rheumatism (EULAR) good and moderate response, disease activity score in 28 joint counts - erythrocyte sedimentation rate (DAS28-ESR) low disease activity (LDA) and remission (REM), change in DAS28-ESR (ΔDAS28-ESR) and ΔDAS28-ESR > 0.6. Genetic polymorphisms were genotyped utilizing the HI-SNP technology. RESULTS: Of the 100 patients with a mean age of 52.23 ± 12.71 years, 81 patients were female (81.00%). After adjusting potential confounders, the major allele of miRNA-5189 rs562929801 was found to be significantly associated with EULAR response (A/A + A/G versus G/G, RR = 0.81, 95% CI = 0.67-0.99, P = 0.04) and ΔDAS28-ESR > 0.6 under dominant model (A/A + A/G versus G/G, RR = 0.83, 95% CI = 0.71-0.98, P = 0.03). However, nonsignificant evidence was detected for the remaining three miRNA-related genetic polymorphisms in neither univariable analysis nor multivariable analysis. CONCLUSION: Our results indicated that miRNA-5189 rs562929801 was significantly associated with clinical response to MTX, and this association warrants further replication studies with larger sample sizes.

Cell-Active, Reversible, and Irreversible Covalent Inhibitors That Selectively Target the Catalytic Lysine of BCR-ABL Kinase.

Despite recent interests in developing lysine-targeting covalent inhibitors, no general approach is available to create such compounds. We report herein a general approach to develop cell-active covalent inhibitors of protein kinases by targeting the conserved catalytic lysine residue using key SuFEx and salicylaldehyde-based imine chemistries. We validated the strategy by successfully developing (irreversible and reversible) covalent inhibitors against BCR-ABL kinase. Our lead compounds showed high levels of selectivity in biochemical assays, exhibited nanomolar potency against endogenous ABL kinase in cellular assays, and were active against most drug-resistant ABL mutations. Among them, the salicylaldehyde-containing A5 is the first-ever reversible covalent ABL inhibitor that possessed time-dependent ABL inhibition with prolonged residence time and few cellular off-targets in K562 cells. Bioinformatics further suggested the generality of our strategy against the human kinome.

A targeted covalent inhibitor of p97 with proteome-wide selectivity.

A resurging interest in targeted covalent inhibitors (TCIs) focus on compounds capable of irreversibly reacting with nucleophilic amino acids in a druggable target. p97 is an emerging protein target for cancer therapy, viral infections and neurodegenerative diseases. Extensive efforts were devoted to the development of p97 inhibitors. The most promising inhibitor of p97 was in phase 1 clinical trials, but failed due to the off-target-induced toxicity, suggesting the selective inhibitors of p97 are highly needed. We report herein a new type of TCIs (i.e., FL-18) that showed proteome-wide selectivity towards p97. Equipped with a Michael acceptor and a basic imidazole, FL-18 showed potent inhibition towards U87MG tumor cells, and in proteome-wide profiling, selectively modified endogenous p97 as confirmed by in situ fluorescence scanning, label-free quantitative proteomics and functional validations. FL-18 selectively modified cysteine residues located within the D2 ATP site of p97. This covalent labeling of cysteine residue in p97 was verified by LC‒MS/MS-based site-mapping and site-directed mutagenesis. Further structure-activity relationship (SAR) studies with FL-18 analogs were established. Collectively, FL-18 is the first known small-molecule TCI capable of covalent engagement of p97 with proteome-wide selectivity, thus providing a promising scaffold for cancer therapy.

Chemoproteomics-based target profiling of sinomenine reveals multiple protein regulators of inflammation.

Although sinomenine (SIN) has been used to treat several inflammation-related diseases in the clinic for decades, the detailed anti-inflammatory mechanism remains elusive. Here, we present a chemoproteomic study that supports a polypharmacological mode of action for SIN to inhibit inflammation. Notably, functional validation revealed multiple new protein regulators whose knockdown could significantly affect inflammation.