Genome-wide association study reveals the genetic basis for petal-size formation in rapeseed (Brassica napus) and CRISPR-Cas9-mediated mutagenesis of BnFHY3 for petal-size reduction.

Petals in rapeseed (Brassica napus) serve multiple functions, including protection of reproductive organs, nutrient acquisition, and attraction of pollinators. However, they also cluster densely at the top, forming a thick layer that absorbs and reflects a considerable amount of photosynthetically active radiation. Breeding genotypes with large, small, or even petal-less varieties, requires knowledge of primary genes for allelic selection and manipulation. However, our current understanding of petal-size regulation is limited, and the lack of markers and pre-breeding materials hinders targeted petal-size breeding. Here, we conducted a genome-wide association study on petal size using 295 diverse accessions. We identified 20 significant single nucleotide polymorphisms and 236 genes associated with petal-size variation. Through a cross-analysis of genomic and transcriptomic data, we focused on 14 specific genes, from which molecular markers for diverging petal-size features can be developed. Leveraging CRISPR-Cas9 technology, we successfully generated a quadruple mutant of Far-Red Elongated Hypocotyl 3 (q-bnfhy3), which exhibited smaller petals compared to the wild type. Our study provides insights into the genetic basis of petal-size regulation in rapeseed and offers abundant potential molecular markers for breeding. The q-bnfhy3 mutant unveiled a novel role of FHY3 orthologues in regulating petal size in addition to previously reported functions.

Immunological nanomaterials to combat cancer metastasis.

Metastasis causes greater than 90% of cancer-associated deaths, presenting huge challenges for detection and efficient treatment of cancer due to its high heterogeneity and widespread dissemination to various organs. Therefore, it is imperative to combat cancer metastasis, which is the key to achieving complete cancer eradication. Immunotherapy as a systemic approach has shown promising potential to combat metastasis. However, current clinical immunotherapies are not effective for all patients or all types of cancer metastases owing to insufficient immune responses. In recent years, immunological nanomaterials with intrinsic immunogenicity or immunomodulatory agents with efficient loading have been shown to enhance immune responses to eliminate metastasis. In this review, we would like to summarize various types of immunological nanomaterials against metastasis. Moreover, this review will summarize a series of immunological nanomaterial-mediated immunotherapy strategies to combat metastasis, including immunogenic cell death, regulation of chemokines and cytokines, improving the immunosuppressive tumour microenvironment, activation of the STING pathway, enhancing cytotoxic natural killer cell activity, enhancing antigen presentation of dendritic cells, and enhancing chimeric antigen receptor T cell therapy. Furthermore, the synergistic anti-metastasis strategies based on the combinational use of immunotherapy and other therapeutic modalities will also be introduced. In addition, the nanomaterial-mediated imaging techniques (e.g., optical imaging, magnetic resonance imaging, computed tomography, photoacoustic imaging, surface-enhanced Raman scattering, radionuclide imaging, etc.) for detecting metastasis and monitoring anti-metastasis efficacy are also summarized. Finally, the current challenges and future prospects of immunological nanomaterial-based anti-metastasis are also elucidated with the intention to accelerate its clinical translation.

Intermittent Theta-Burst Stimulation for Stroke: Primary Motor Cortex Versus Cerebellar Stimulation: A Randomized Sham-Controlled Trial.

BACKGROUND: Stroke survivors with impaired balance and motor function tend to have relatively poor functional outcomes. The cerebellum and primary motor cortex (M1) have been suggested as targets for neuromodulation of balance and motor recovery after stroke. This study aimed to compare the efficacy and safety of intermittent theta-burst stimulation (iTBS) to the cerebellum or M1 on balance and motor recovery in patients with stroke. METHODS: In this randomized, double-blind, sham-controlled clinical trial, patients with subacute stroke were randomly divided into 3 groups: M1-, cerebellar-, and sham-iTBS (n=12 per group; 15 sessions, 3 weeks). All outcomes were evaluated before intervention (T0), after 1 week of intervention (T1), after 3 weeks of intervention (T2), and at follow-up (T3). The primary outcome was the Berg balance scale score at T2. Secondary outcomes include the Fugl-Meyer assessment scale for lower extremities, the trunk impairment scale, the Barthel index, the modified Rankin Scale, the functional ambulation categories, and cortical excitability. RESULTS: A total of 167 inpatients were screened, 36 patients (age, 57.50±2.41 years; 10 women, 12 ischemic) were enrolled between December 2020 and January 2023. At T2, M1- or cerebellar-iTBS significantly improved Berg balance scale scores by 10.7 points ([95% CI, 2.7-18.6], P=0.009) and 14.2 points ([95% CI, 1.2-27.2], P=0.032) compared with the sham-iTBS group. Moreover, the cerebellar-iTBS group showed a significantly greater improvement in Fugl-Meyer assessment scale for lower extremities scores by 5.6 points than the M1-iTBS ([95% CI, 0.3-10.9], P=0.037) and by 7.8 points than the sham-iTBS ([95% CI, 1.1-14.5], P=0.021) groups at T2. The motor-evoked potential amplitudes of the M1- and cerebellar-iTBS groups were higher than those of the sham-iTBS group (P<0.001). CONCLUSIONS: Both M1- and cerebellar-iTBS could improve balance function. Moreover, cerebellar-iTBS, but not M1-iTBS, induced significant effects on motor recovery. Thus, cerebellar-iTBS may be a valuable new therapeutic option in stroke rehabilitation programs. REGISTRATION: URL: https://www.chictr.org.cn/; Unique identifier: ChiCTR2100047002.

Selinexor With Anti-PD-1 Antibody as a Potentially Effective Regimen for Patients With Natural Killer/T-Cell Lymphoma Failing Prior L-Asparaginase and PD-1 Blockade.

BACKGROUND: Natural killer/T-cell lymphoma (NKTCL) is a rare and heterogeneous tumor type of non-Hodgkin's lymphoma (NHL) with a poor clinical outcome. There is no standardized salvage treatment failing l-asparaginase-based regimens. Here we report our retrospective results of the combined use of selinexor and PD-1 blockade (tislelizumab) in 5 patients with NKTCL who had exhausted almost all available treatments. PATIENTS AND METHODS: A total of 5 patients with relapsed/refractory(R/R) NK/T-cell lymphomas failing prior l-asparaginase and anti-PD-1 antibody were retrospectively collected. They were treated with at least one cycle of XPO1 inhibitor plus the same anti-PD-1 antibody. Anti-PD-1 antibody (Tislelizumab) was administrated at 200 mg on day 1 every 3 weeks and selinexor doses and schedules ranged from 40 mg weekly for 2 weeks per 21-day cycle to 60 mg weekly per cycle. RESULTS: Five patients with relapsed NKTCL with extensive organ involvement including 4 central nervous system (CNS) infiltration patients were included. Four patients achieved objective responses including 3 complete responses (CR) and 1 partial response (PR). After a median follow-up time of 14.5 (range, 5-22) months, 1 patient was still in remission with CR, and the other 4 patients discontinued due to disease progression with a median progression-free survival (PFS) of 6 months and median overall survival (OS) of 12 months. Four patients with CNS involvement achieved a median OS of 8 months. Our data suggest that selinexor in combination with an anti-PD-1 antibody is a promising small molecule and immunotherapy combination regimen for patients with relapsed or refractory NKTCL.

The effect of aromatic side chains on the dilational rheological properties of N-acyltaurate amphiphiles at water-decane interfaces.

To elucidate the effect of aromatic side chains on dilational rheological properties of N-acyltaurate amphiphiles at the decane-water interface, the interfacial rheological properties of sodium N-2-(2-naphthoxy)-tetradecanoyltaurinate (12+N-T) and sodium N-2-(p-butylphenoxy)-tetradecanoyltaurinate (12+4B-T) were investigated utilizing the drop shape analysis method. The effects of adsorption time, interfacial pressure, oscillating frequency, and bulk concentration on the interfacial dilational modulus and phase angle were explored. The results show that the 12+4B-T molecule with a longer hydrophobic chain shows a higher ability for reducing the interfacial tension (IFT). In addition, the interfacial films of both 12+N-T and 12+4B-T are dominated by diffusion exchange at high concentrations. The rigidity of molecules controls the diffusion exchange at low concentrations, while the molecular hydrodynamic radius determines the diffusion exchange at high concentrations. Thus, at low concentrations, the stronger intermolecular interaction between 12+4B-T molecules results in higher dilational modulus values than 12+N-T. When approaching the CMC (critical micelle concentration) value, the rapid diffusion exchange of 12+4B-T between the sublayer micelles and the interface causes a significant decrease in the dilational modulus, while the relatively rigid structure of 12+N-T enables a higher dilational modulus than 12+4B-T. What's more, the longer hydrophobic chain allows 12+4B-T molecules to escape from the interface more easily, resulting in a higher phase angle at low concentrations. However, the diffusion exchange of 12+4B-T is slower than that of 12+N-T, which results in lower phase angles for 12+4B-T than 12+N-T at high concentrations. In general, the introduction of a rigid naphthalene ring in the molecular structure gives the interfacial film greater strength at high concentration. The research results in this paper provide a new technique for the strength regulation of interfacial surfactant adsorption films.

Prediction of heterosis in the recent rapeseed (Brassica napus) polyploid by pairing parental nucleotide sequences.

The utilization of heterosis is a successful strategy in increasing yield for many crops. However, it consumes tremendous manpower to test the combining ability of the parents in fields. Here, we applied the genomic-selection (GS) strategy and developed models that significantly increase the predictability of heterosis by introducing the concept of a regional parental genetic-similarity index (PGSI) and reducing dimension in the calculation matrix in a machine-learning approach. Overall, PGSI negatively affected grain yield and several other traits but positively influenced the thousand-seed weight of the hybrids. It was found that the C subgenome of rapeseed had a greater impact on heterosis than the A subgenome. We drew maps with overviews of quantitative-trait loci that were responsible for the heterosis (h-QTLs) of various agronomic traits. Identifications and annotations of genes underlying high impacting h-QTLs were provided. Using models that we elaborated, combining abilities between an Ogu-CMS-pool member and a potential restorer can be simulated in silico, sidestepping laborious work, such as testing crosses in fields. The achievements here provide a case of heterosis prediction in polyploid genomes with relatively large genome sizes.

Per- and polyfluoroalkyl substances inhibit human and rat 17ß-hydroxysteroid dehydrogenase 1: Quantitative structure-activity relationship and molecular docking analysis.

Per- and polyfluoroalkyl (PFAS) substances are enduring industrial materials. 17ß-Hydroxysteroid dehydrogenase isoform 1 (17ß-HSD1) is an estrogen metabolizing enzyme, which transforms estrone into estradiol in human placenta and rat ovary. Whether PFAS inhibit 17ß-HSD1 and what the structure-activity relationship (SAR) remains unexplored. We screened 18 PFAS for inhibiting human and rat 17ß-HSD1 in microsomes and studied their SAR and mode of action(MOA). Of the 11 perfluorocarboxylic acids (PFCAs), C8-C14 PFCAs at a concentration of 100 µM substantially inhibited human 17ß-HSD1, with order of C11 (half-maximal inhibition concentration, IC50, 8.94 µM) > C10 (10.52 µM) > C12 (14.90 µM) > C13 (30.97 µM) > C9 (43.20 µM) > C14 (44.83 µM) > C8 (73.38 µM) > others. Of the 7 per- and poly-fluorosulfonic acids (PFSAs), the potency was C8S (IC50, 14.93 µM) > C7S (80.70 µM) > C6S (177.80 µM) > others. Of the PFCAs, C8-C14 PFCAs at 100 µM markedly reduced rat 17ß-HSD1 activity, with order of C11 (IC50, 9.11 µM) > C12 (14.30 µM) > C10 (18.24 µM) > C13 (25.61 µM) > C9 (67.96 µM) > C8 (204.39 µM) > others. Of the PFSAs, the potency was C8S (IC50, 37.19 µM) > C7S (49.38 µM) > others. In contrast to PFOS (C6S), the partially fluorinated compound 6:2 FTS with an equivalent number of carbon atoms demonstrated no inhibition of human and rat 17ß-HSD1 activity at a concentration of 100 µM. The inhibition of human and rat enzymes by PFAS followed a V-shaped trend from C4 to C14, with a nadir at C11. Moreover, human 17ß-HSD1 was more sensitive than rat enzyme. PFAS inhibited human and rat 17ß-HSD1 in a mixed mode. Docking analysis revealed that they bind to the NADPH and steroid binding site of both 17ß-HSD1 enzymes. The 3D quantitative SAR (3D-QSAR) showed that hydrophobic region, hydrogen bond acceptor and donor are key factors in binding to 17ß-HSD1 active sites. In conclusion, PFAS exhibit inhibitory effects on human and rat 17ß-HSD1 depending on factors such as carbon chain length, degree of fluorination, and the presence of carboxylic acid or sulfonic acid groups, with a notable V-shaped shift observed at C11.

Prenatal diethylhexylphthalate exposure disturbs adult Leydig cell function via epigenetic downregulation of METTL4 expression in male rats.

Prenatal exposure to diethylhexyl phthalate (DEHP) has been linked with a decline in testosterone levels in adult male rats, but the underlying mechanism remains unclear. We investigated the potential epigenetic regulation, particularly focusing on N6-methyladenosine (m6A) modification, as a possible mechanism. Dams were gavaged with DEHP (0, 10, 100, and 750 mg/kg/day) from gestational day 14 to day 21. The male offspring were examined at the age of 56 days. Prenatal DEHP administration at 750 mg/kg/day caused a decline in testosterone concentrations, an elevation in follicle-stimulating hormone, a downregulated expression of CYP11A1 HSD3B2, without affecting Leydig cell numbers. Interestingly, Methyltransferase Like 4 (METTL4), an m6A methyltransferase, was downregulated, while there were no changes in METTL3 and METTL14. Moreover, CYP11A1 showed m6A reduction in response to prenatal DEHP exposure. Additionally, METTL4 expression increased postnatally, peaking in adulthood. Knockdown of METTL4 resulted in the downregulation of CYP11A1 and HSD3B2 and an increase in SCARB1 expression. Furthermore, the increase in autophagy protection in adult Leydig cells induced by prenatal DEHP exposure was not affected by 3-methyladenosine (3MA) treatment, indicating a potential protective role of autophagy in response to DEHP exposure. In conclusion, prenatal DEHP exposure reduces testosterone by downregulating CYP11A1 and HSD3B2 via m6A epigenetic regulation and induction of autophagy protection in adult Leydig cells as a response to DEHP exposure.

Halogenated bisphenol A derivatives potently inhibit human and rat 11ß-hydroxysteroid dehydrogenase 1: Structure-activity relationship and molecular docking.

Chlorinated bisphenol A (BPA) derivatives are formed during chlorination process of drinking water, whereas bisphenol S (BPS) and brominated BPA and BPS (TBBPA and TBBPS) were synthesized for many industrial uses such as fire retardants. However, the effect of halogenated BPA and BPS derivatives on glucocorticoid metabolizing enzyme 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) remains unclear. The inhibitory effects of 6 BPA derivatives in the inhibition of human and rat 11ß-HSD1 were investigated. The potencies for inhibition on human 11ß-HSD1 were TBBPA (IC50, 3.87 µM) = monochloro BPA (MCBPA, 4.08 µM) = trichloro BPA (TrCBPA, 4.41 µM) > tetrachloro BPA (TCBPA, 9.75 µM) > TBBPS (>100 µM) = BPS (>100 µM), and those for rat 11ß-HSD1 were TrCBPA (IC50, 2.76 µM) = MCBPA (3.75 µM) > TBBPA (39.58 µM) > TCBPA = TBBPS = BPS. All these BPA derivatives are mixed/competitive inhibitors of both human and rat enzymes. Molecular docking studies predict that MCBPA, TrCBPA, TCBPA, and TBBPA all bind to the active site of human 11ß-HSD1, forming hydrogen bonds with catalytic residue Ser170 except TCBPA. Regression of the lowest binding energy with IC50 values revealed a significant inverse linear regression. In conclusion, halogenated BPA derivatives are mostly potent inhibitors of human and rat 11ß-HSD1, and there is structure-dependent inhibition.

Redox-Active Ferrocene Quencher-Based Supramolecular Nanomedicine for NIR-II Fluorescence-Monitored Chemodynamic Therapy.

Real-time monitoring of hydroxyl radical (⋅OH) generation is crucial for both the efficacy and safety of chemodynamic therapy (CDT). Although ⋅OH probe-integrated CDT agents can track ⋅OH production by themselves, they often require complicated synthetic procedures and suffer from self-consumption of ⋅OH. Here, we report the facile fabrication of a self-monitored chemodynamic agent (denoted as Fc-CD-AuNCs) by incorporating ferrocene (Fc) into ß-cyclodextrin (CD)-functionalized gold nanoclusters (AuNCs) via host-guest molecular recognition. The water-soluble CD served not only as a capping agent to protect AuNCs but also as a macrocyclic host to encapsulate and solubilize hydrophobic Fc guest with high Fenton reactivity for in vivo CDT applications. Importantly, the encapsulated Fc inside CD possessed strong electron-donating ability to effectively quench the second near-infrared (NIR-II) fluorescence of AuNCs through photoinduced electron transfer. After internalization of Fc-CD-AuNCs by cancer cells, Fenton reaction between redox-active Fc quencher and endogenous hydrogen peroxide (H2 O2 ) caused Fc oxidation and subsequent NIR-II fluorescence recovery, which was accompanied by the formation of cytotoxic ⋅OH and therefore allowed Fc-CD-AuNCs to in situ self-report ⋅OH generation without undesired ⋅OH consumption. Such a NIR-II fluorescence-monitored CDT enabled the use of renal-clearable Fc-CD-AuNCs for efficient tumor growth inhibition with minimal side effects in vivo.

A non-metal single atom nanozyme for cutting off the energy and reducing power of tumors.

Enzymes are considered safe and effective therapeutic tools for various diseases. With the increasing integration of biomedicine and nanotechnology, artificial nanozymes offer advanced controllability and functionality in medical design. However, several notable gaps, such as catalytic diversity, specificity and biosafety, still exist between nanozymes and their native counterparts. Here we report a non-metal single-selenium (Se)-atom nanozyme (SeSAE), which exhibits potent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mimetic activity. This novel single atom nanozyme provides a safe alternative to conventional metal-based catalysts and effectively cuts off the cellular energy and reduction equivalents through its distinctive catalytic function in tumors. In this study, we have demonstrated the substantial efficacy of SeSAE as an antitumor nanomedicine across diverse mouse models without discernible systemic adverse effects. The mechanism of the NADPH oxidase-like activity of the non-metal SeSAE was rationalized by density functional theory calculations. Furthermore, comprehensive elucidation of the biological functions, cell death pathways, and metabolic remodeling effects of the nanozyme was conducted, aiming to provide valuable insights into the development of single atom nanozymes with clinical translation potential.

Engineering Single-Atom Nanozymes for Catalytic Biomedical Applications.

Nanomaterials with enzyme-mimicking properties, coined as nanozymes, are a promising alternative to natural enzymes owing to their remarkable advantages, such as high stability, easy preparation, and favorable catalytic performance. Recently, with the rapid development of nanotechnology and characterization techniques, single atom nanozymes (SAzymes) with atomically dispersed active sites, well-defined electronic and geometric structures, tunable coordination environment, and maximum metal atom utilization are developed and exploited. With superior catalytic performance and selectivity, SAzymes have made impressive progress in biomedical applications and are expected to bridge the gap between artificial nanozymes and natural enzymes. Herein, the recent advances in SAzyme preparation methods, catalytic mechanisms, and biomedical applications are systematically summarized. Their biomedical applications in cancer therapy, oxidative stress cytoprotection, antibacterial therapy, and biosensing are discussed in depth. Furthermore, to appreciate these advances, the main challenges, and prospects for the future development of SAzymes are also outlined and highlighted in this review.

Genome-wide-association study and transcriptome analysis reveal the genetic basis controlling the formation of leaf wax in Brassica napus.

Cuticular wax protects plants from various biotic and abiotic stresses. However, the genetic network of wax biosynthesis and the environmental factors influencing leaf wax production in rapeseed (Brassica napus) remains unclear. Here, we demonstrated the role of leaf wax in the resistance to Sclerotinia infection in rapeseed. We found that leaves grown under high light intensity had higher expression of genes involved in wax biosynthesis, and produced more wax on the leaf surface, compared with those grown under low light conditions. Genome-wide association study (GWAS) identified 89 single nucleotide polymorphisms significantly associated with leaf wax coverage. A cross-analysis between GWAS and differentially expressed genes (DEGs) in the leaf epidermis of the accessions with contrasting differences in wax content revealed 17 candidate genes that control this variation in rapeseed. Selective sweep analysis combined with DEG analysis unveiled 510 candidate genes with significant selective signatures. From the candidate genes, we selected BnaA02.LOX4, a putative lipoxygenase, and BnaCnn.CER1, BnaA02.CER3, BnaC02.CER3, and BnaA01.CER4 (ECERIFERUM1-4) that were putatively responsible for wax biosynthesis, to analyse the allelic forms and haplotypes corresponding to high or low leaf wax coverage. These data enrich our knowledge about wax formation, and provide a gene pool for breeding an ideal leaf wax content in rapeseed.

Structural variations and environmental specificities of flowering time-related genes in Brassica napus.

KEY MESSAGE: We found that the flowering time order of accessions in a genetic population considerably varied across environments, and homolog copies of essential flowering time genes played different roles in different locations. Flowering time plays a critical role in determining the life cycle length, yield, and quality of a crop. However, the allelic polymorphism of flowering time-related genes (FTRGs) in Brassica napus, an important oil crop, remains unclear. Here, we provide high-resolution graphics of FTRGs in B. napus on a pangenome-wide scale based on single nucleotide polymorphism (SNP) and structural variation (SV) analyses. A total of 1337 FTRGs in B. napus were identified by aligning their coding sequences with Arabidopsis orthologs. Overall, 46.07% of FTRGs were core genes and 53.93% were variable genes. Moreover, 1.94%, 0.74%, and 4.49% FTRGs had significant presence-frequency differences (PFDs) between the spring and semi-winter, spring and winter, and winter and semi-winter ecotypes, respectively. SNPs and SVs across 1626 accessions of 39 FTRGs underlying numerous published qualitative trait loci were analyzed. Additionally, to identify FTRGs specific to an eco-condition, genome-wide association studies (GWASs) based on SNP, presence/absence variation (PAV), and SV were performed after growing and observing the flowering time order (FTO) of plants in a collection of 292 accessions at three locations in two successive years. It was discovered that the FTO of plants in a genetic population changed a lot across various environments, and homolog copies of some key FTRGs played different roles in different locations. This study revealed the molecular basis of the genotype-by-environment (G × E) effect on flowering and recommended a pool of candidate genes specific to locations for breeding selection.

Homochiral Chemistry Strategy to Trigger Second-Harmonic Generation and Dual Dielectric Switches.

Switchable materials have attracted enormous interest due to their promising applications in important fields such as sensing, electronic components, and information storage. Nevertheless, obtaining multifunctional switching materials is still a problem worth investigating. Herein, by incorporating (Rac-, L-, D-2-amino-1-propanol) as the templating cation, we have obtained (Rac-, L-, D-HTMPA)CdCl3 (HTMPA = 1-hydroxy-N, N, N-trimethyl-2-propanaminium). We have adopted a chiral chemistry strategy that causes (Rac-HTMPA)CdCl3 in the central symmetric space to crystallize in the chiral space group. Based on the modulation of the homochiral strategy, (L-, D-HTMPA)CdCl3 shows a dual phasic transition at 269 and 326 K and a switchable second-harmonic generation response. In addition, (L-, D-HTMPA)CdCl3 is chiral switchable material to exhibit stable dual dielectric and second-harmonic generation (SHG) switches. This work provides an approach to exploring multifunctional chiral switchable materials.

Multiblock Poly-ε-Caprolactones: One-Step Synthesis toward Programmable Properties.

Control of monomer sequence enables predictable structure-property relationships in versatile polymeric materials. The facile synthesis of multiblock copolymers (MBCPs) with controlled chain structure is highly challenging, particularly for those prepared via one-pot copolymerization of mixed monomers. Herein, poly-ε-caprolactone MBCPs, a series of thermoplastic elastomers with tailored thermal, mechanical, rheological, and degradable properties, are synthesized by Janus polymerization. Melting temperature, tensile strength, ductility, viscosity, and enzymatic degradability are governed by block length which is in turn dictated by the monomer-to-catalyst feed ratio. The relationships between the physicochemical properties and the architectures are investigated in detail.

Tinopanoids K-T, clerodane diterpenoids with anti-inflammatory activity from Tinospora crispa.

A total of 17 structurally diverse clerodane diterpenoids, including ten undescribed clerodane diterpenoids (tinopanoids K-T, 1-10) and seven known compounds (11-17), were isolated from the vines and leaves of Tinospora crispa. Compound 3 has not only bear the dominant substituents of γ-hydroxy-α, ß-unsaturated-γ-lactone with anti-inflammatory activity, but also a ternary epoxy structure at C-3/C-4. The planar structures and relative configurations of the clerodane diterpenoids were elucidated by spectroscopic data interpretation. The absolute configurations of compounds 1, 4, 8 and 13 were determined by single-crystal X-ray crystallographic, while that of compound 3 was determined using computed ECD data and single crystal X-ray diffraction of related p-bromobenzoate ester (3a). Subsequently, all compounds were evaluated for their inhibitory effect on nitric oxide (NO) production of LPS-activated BV-2 cells, and compounds 3 and 8 exhibited better NO inhibitory potency, with IC50 values of 5.6 and 13.8 µM than the positive control minocycline (Mino, IC50 = 22.9 µM). The corresponding results of western blot analysis and qRT-PCR revealed that compound 3 can significantly inhibit the inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions, mRNA levels of pro-inflammatory cytokins of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and interleukin 1ß (IL-1ß). The underlying mechanism by which compound 3 exerted anti-neuroinflammatory effects was investigated by western blot and immunofluorescence assay, which suggested compound 3 inhibited LPS induced neuroinflammation via the suppression of toll-like receptor 4 (TLR4) dependent Signal Transducer and Activator of Transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) signaling pathways, and the activation of Heme Oxygenase-1 (HO-1) mediated signals.

Curcumin analogues exert potent inhibition on human and rat gonadal 3ß-hydroxysteroid dehydrogenases as potential therapeutic agents: structure-activity relationship and in silico docking.

Curcuminoids are functional food additives, and the effect on gonadal hormone biosynthesis remains unclear. Gonads contain 3ß-hydroxysteroid dehydrogenase isoforms, h3ß-HSD2 (humans) and r3ß-HSD1 (rats), which catalyse pregnenolone into progesterone. The potency and mechanisms of curcuminoids to inhibit 3ß-HSD activity were explored. The inhibitory potency was bisdemethoxycurcumin (IC50, 1.68 µM) >demethoxycurcumin (3.27 µM) > curcumin (13.87 µM) > tetrahydrocurcumin (109.0 µM) > dihydrocurcumin and octahydrocurcumin on KGN cell h3ß-HSD2, while that was bisdemethoxycurcumin (1.22 µM) >demethoxycurcumin (2.18 µM) > curcumin (4.12 µM) > tetrahydrocurcumin (102.61 µM) > dihydrocurcumin and octahydrocurcumin on testicular r3ß-HSD1. All curcuminoids inhibited progesterone secretion by KGN cells under basal and forskolin-stimulated conditions at >10 µM. Docking analysis showed that curcuminoids bind steroid-active site with mixed or competitive mode. In conclusion, curcuminoids inhibit gonadal 3ß-HSD activity and de-methoxylation of curcumin increases inhibitory potency and metabolism of curcumin by saturation of carbon chain losses inhibitory potency.

Distinct inhibitory strength of bisphenol A analogues on human and rat 11ß-hydroxysteroid dehydrogenase 1: 3D quantitative structure-activity relationship and in silico molecular docking analysis.

Bisphenol A (BPA) analogues are developed to replace BPA usage. However, their effects on 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) are largely unknown. The inhibitory effects of BPA and 10 BPA analogues with the substituents on the bridge moiety on human and rat 11ß-HSD1 were explored in human and rat liver microsomes. The strength of inhibiting human 11ß-HSD1 was bisphenol FL (IC50, 3.87 µM) > bisphenol Z (6.86 µM) > bisphenol AF (9.42 µM) > bisphenol C (16.14 µM) > bisphenol AP (32.14 µM) = bisphenol B (32.34 µM) > 4,4'-thiodiphenol (67.35 µM) > BPA (297.35 µM) > other BPA analogues (ineffective at 100 µM). The strength of inhibiting rat 11ß-HSD1 was bisphenol Z (IC50, 14.44 µM) > 4,4'-thiodiphenol (19.01 µM) > bisphenol B (20.13 µM) > bisphenol F (22.10 µM) > bisphenol E (33.04 µM) > bisphenol AF (49.67 µM) > bisphenol C > (56.97 µM) > bisphenol AP (62.71 µM) >bisphenol FL (96.31 µM) > other BPA analogues (ineffective at 100 µM). Bisphenol A, AF, AP, B, C, F, FL, Z, and 4,4'-thiodiphenol bind to the active sites of human and rat 11ß-HSD1. Regression of LogP and molecular weight with IC50 values revealed distinct inhibitory pattern (negative correlation for human 11ß-HSD1 vs. positive correlation for rat enzyme). Regression of the lowest binding energy with IC50 values revealed a significant positive regression. 3D QSAR pharmacophore analysis showed one hydrogen bond acceptor and two hydrogen bond donors for human 11ß-HSD1. In conclusion, most BPA analogues are more potent inhibitors of human and rat 11ß-HSD1 enzymes and there is structure-dependent and species-dependent inhibition.

Benzene ring bisphenol A substitutes potently inhibit human, rat, and mouse gonadal 3ß-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis.

Bisphenol A (BPA) is a chemical used in the production of certain plastics and resins. Recent research has found that BPA can inhibit the activity of 3ß-hydroxysteroid dehydrogenase/Δ5,4-isomerases (3ß-HSDs). Whether benzene ring BPA substitutes can inhibit human, rat, and mouse gonadal 3ß-HSDs, the structure-activity relationship and the underlying mechanism remain unclear. In this study, we compared 6 benzene ring BPA substitutes to BPA in the inhibition of human, rat, and mouse gonadal 3ß-HSDs and conducted structure-activity relationship and in silico docking analysis. The inhibitory activity (IC50) of human 3ß-HSD2 in KGN cells ranged from about 0.02 µM for bisphenol H to 8.75 µM for BPA, that of rat 3ß-HSD1 in testicular microsomes ranged from 0.099 µM for bisphenol H to 31.32 µM for BPA, and that of mouse 3ß-HSD6 ranged from 0.021 µM for BPH to ineffectiveness for 100 µM BPA. These compounds acted as mixed inhibitors with LogP inversely correlated with IC50 and ΔG positively correlated with IC50 value. Docking analysis showed that these compounds bind to the steroid active site of the 3ß-HSD enzymes. In conclusion, some benzene ring BPA substitutes potently inhibit gonadal 3ß-HSD in various species, and lipophilicity and binding affinity determine their inhibitory strength.