PEG-functionalized aliphatic polycarbonate brushes with self-polishing dynamic antifouling properties.

Hydrophilic antifouling polymers provide excellent antifouling effects under usual short-term use conditions, but the long-term accumulation of contaminants causes them to lose their antifouling properties. To overcome this drawback, surface-initiated ring-opening graft polymerization (SI-ROP) was performed on the surface of the material by applying the cyclic carbide monomer 4'-(fluorosulfonyl)benzyl-5-methyl-2-oxo-1,3-dioxane-5-carboxylate (FMC), which contains a sulfonylfluoride group on the side chain, followed by a "sulfur(IV)-fluorine exchange" (SuFEx) post click modification reaction to link the hydrophilic polyethylene glycol (PEG) to the polyFMC (PFMC) brush, and a novel antifouling strategy for self-polishing dynamic antifouling surfaces was developed. The experimental results showed that the antifouling surface could effectively prevent the adsorption of proteins such as bovine serum albumin (BSA, ∼96.4%), fibrinogen (Fg, ∼87.8%) and lysozyme (Lyz ∼69.4%) as well as the adhesion of microorganisms such as the bacteria Staphylococcus aureus (S. aureus) (∼87.5%) and HeLa cells (∼67.2%). Moreover, the enzymatically self-polished surface still has excellent antifouling properties. Therefore, this modification method has potential applications in the field of biosensors and novel antifouling materials.

NSUN2-Mediated m5C Methylation Impairs Endometrial Receptivity.

Impaired endometrial decidualization is the primary cause of recurrent implantation failure (RIF). RNA methylation modification, especially NSUN family mediated m5C, is crucial for various physiological events, such as maternal-to-zygotic transition, gametogenesis, embryonic development, organismal lifespan, and cell cycle. However, the regulatory mechanisms between NSUN family mediated m5C modification and RIF remain unknown. We acquired NSUN2 expression data of 15 human endometrium samples at proliferative and secretory stages from reproductive cell atlas. The overall pattern of m5C sites and genes was elucidated through m5C-BS-seq, whereas the overall m5C levels in different groups were revealed by dot blot assay. BrdU and western blotting assays were carried out to evaluate the role of NSUN2 in proliferation and autophagy. The effects of NSUN2-mediated m5C modification on embryo attachment were evaluated by an in vitro model of a confluent monolayer of Ishikawa cells cocultured with BeWo spheroids, and its downstream targets were evaluated by real-time reverse-transcription PCR and western blotting in Ishikawa cells. The molecular mechanism for NSUN2 regulating its downstream targets' expression was determined by Cut&Tag and coimmunoprecipitation assays. NSUN2 was increased in SOX9+ cells and widespread in epithelial cell type at the proliferative stage by previous single-cell RNA sequencing data. NSUN2 overexpression (NSUN2OE) in the Ishikawa cell line elevated m5C levels and promoted cell proliferation and autophagy. NSUN2OE reduced attachment efficiency of BeWo cell spheres. Overexpressed NSUN2 was found to increase STAT1 and MMP14 mRNA expressions by inducing exon skipping. NSUN2 interacted with CLDN4 through m5C modification, and NSUN2OE or NSUN2 knockdown resulted in a similar variation tendency of CLDN4. Overexpression of NSUN2 increased CLDN4 H3K9ac modification by downregulating SIRT4 expression at the protein level, leading to the upregulation of CLDN4 mRNA expression. Our results uncovered a novel intricate regulatory mechanism between NSUN2-mediated m5C and RIF and suggested a potential new therapeutic strategy for RIF.

A low-cost, low-input method establishment for m6A MeRIP-seq.

N6-methyladenosine (m6A) is a highly prevalent modification found in mammal mRNA molecules that plays a crucial role in the regulation of cellular function. m6A RNA immunoprecipitation sequencing (MeRIP-seq) has been frequently used in transcriptomics research to identify the location of m6A. MABE572 (Millipore) is the most widely utilized and efficient anti-m6A antibody for MeRIP-seq. However, due to the high dose and price of this antibody, which has also been taken off the market, we discovered that CST's anti-m6A antibody can be used instead of MABE572 to map the m6A transcriptome. In the present study, we performed different concentrations of the CST anti-m6A antibodies with the corresponding initiation RNA of HEK293T cells, 2.5 µg antibody with 1 µg total RNA, 1.25 µg antibody with 0.5 µg total RNA, and 1.25 µg antibody with 0.1 µg total RNA. By comparing the m6A peak calling, enriched motifs, alternative splicing events, and nuclear transcripts modified by m6A between the CST and Millipore libraries, it was found that the CST library presented similar data to Millipore, even at incredibly low doses. The volume and cost of antibodies are significantly reduced by this refined MeRIP-seq using CST antibody, making it convenient to map future large-scale sample m6A methylation.

Janus-type ionic conductive gels based on poly(N,N-dimethyl)acrylamide for strain/pressure sensors.

Strain/pressure sensors with high sensitivity and a wide operation range have broad application prospects in wearable medical equipment, human-computer interactions, electronic skin, and so on. In this work, based on the different solubilities of Zr4+ in the aqueous phase and the hydrophobic ionic liquid [BMIM][Tf2N], we used N,N-dimethylacrylamide (DMA) as a vinyl monomer to prepare a Janus-type ionic conductive gel with one-sided adhesion through "one-step" UV irradiation polymerization. The Janus-type gel has satisfactory mechanical properties (tensile strength: 217.06 kPa, elongation at break: 1121.01%), electrical conductivity (conductivity: 0.10 S m-1), one-sided adhesion (adhesion strength to glass: 72.35 kPa) and antibacterial properties. The sensor based on the Janus gel can be used not only for real-time monitoring of strain changes caused by various movements of the human body (such as finger bending, muscle contraction, smiling, and swallowing) but also for real-time monitoring of pressure changes (such as pressing, water droplets, and writing movements). Therefore, based on the simplicity of this method for constructing Janus-type ionic conductive gels and the excellent electromechanical properties of the prepared gel, we believe that the method provided in this study has broad application prospects in the field of multifunctional wearable sensors.

Transparent ionic conductive elastomers with high mechanical strength and strong tensile properties for strain sensors.

Stretchable ionic conductive elastomers have been extensively studied due to their great application potential in the fields of sensors, batteries, capacitors and flexible robots. However, it is still challenging to prepare multifunctional ionic conductive elastomers with high mechanical strength and excellent tensile properties by a green and efficient method. In this study, we prepared PDES-DMA ionic conductive elastomers by a "one step" rapid in situ polymerization of AA/ChCl-type polymerizable deep eutectic solvents (PDES) and N,N-dimethylacrylamide (DMA) under ultraviolet (UV) irradiation. In addition to the characteristics of high mechanical strength (tensile strength of 9.27 MPa) and excellent tensile properties (elongation at break of 1071%), the PDES-DMA elastomer also has high transparency (>80%), strong self-adhesion (adhesion strength with glass surface 133.8 kPa) and self-healing properties. In addition, sensors based on the ionic conductive elastomer can be used to detect human movements such as finger, wrist, elbow, ankle and knee bending. Considering the convenience of the preparation method and the excellent versatility of the prepared PDES-DMA ionic conductive elastomer, we believe that the method proposed in this study has potential application prospects in the field of flexible electronics.

Fat mass and obesity-associated factor (FTO)-mediated N6-methyladenosine regulates spermatogenesis in an age-dependent manner.

N6-methyladenosine (m6A) is the most prevalent reversible RNA modification in the mammalian transcriptome. It has recently been demonstrated that m6A is crucial for male germline development. Fat mass and obesity-associated factor (FTO), a known m6A demethylase, is widely expressed in human and mouse tissues and is involved in manifold biological processes and human diseases. However, the function of FTO in spermatogenesis and male fertility remains poorly understood. Here, we generated an Fto knockout mouse model using CRISPR/Cas9-mediated genome editing techniques to address this knowledge gap. Remarkably, we found that loss of Fto in mice caused spermatogenesis defects in an age-dependent manner, resulting from the attenuated proliferation ability of undifferentiated spermatogonia and increased male germ cell apoptosis. Further research showed that FTO plays a vital role in the modulation of spermatogenesis and Leydig cell maturation by regulating the translation of the androgen receptor in an m6A-dependent manner. In addition, we identified two functional mutations of FTO in male infertility patients, resulting in truncated FTO protein and increased m6A modification in vitro. Our results highlight the crucial effects of FTO on spermatogonia and Leydig cells for the long-term maintenance of spermatogenesis and expand our understanding of the function of m6A in male fertility.

Sertoli cell PUMILIO proteins modulate mouse testis size through translational control of cell cycle regulators.

Testis size determination is an important question of reproductive biology. Sertoli cells are known to be a key determinant of mammalian testis size but the underlying molecular mechanisms remain incompletely understood. Previously we showed that highly conserved germ cell RNA-binding proteins, PUMILIO1(PUM1) and PUMILIO2 (PUM2), control mouse organ and body size through translational regulation, but how different cell types of the organs contribute to their organ size regulation has not been established. Here, we report a somatic role of PUM in gonad size determination. PUM1 is highly expressed in the Sertoli cells of the developing testis from embryonic and postnatal mice as well as in germ cells. Removal of Sertoli cell, but not germ cell, Pum1 gene, led to reduced testis size without significantly affecting sperm number or fertility. Knockout of PUM1 target, Cdkn1b, rescued the phenotype of reduced testis size, supporting a key role of Sertoli cell PUM1 mediated Cdkn1b repression in the testis size control. Furthermore, removal of Pum2 or both Pum1 and Pum2 in the Sertoli cells also only affected the testis size, not sperm development, with the biggest size reduction in Pum1/2 double knockout mice. We propose that PUM1 and PUM2 modulate the testis size through their synergistic translational regulation of cell cycle regulators in the Sertoli cell. Further investigation of the ovary or other organs could reveal if PUM-mediated translational control of cell proliferation of the supporting cell represents a general mechanism for organ size modulation.

LRG1 is an adipokine that mediates obesity-induced hepatosteatosis and insulin resistance.

Dysregulation in adipokine biosynthesis and function contributes to obesity-induced metabolic diseases. However, the identities and functions of many of the obesity-induced secretory molecules remain unknown. Here, we report the identification of leucine-rich alpha-2-glycoprotein 1 (LRG1) as an obesity-associated adipokine that exacerbates high fat diet-induced hepatosteatosis and insulin resistance. Serum levels of LRG1 were markedly elevated in obese humans and mice compared with their respective controls. LRG1 deficiency in mice greatly alleviated diet-induced hepatosteatosis, obesity, and insulin resistance. Mechanistically, LRG1 bound with high selectivity to the liver and promoted hepatosteatosis by increasing de novo lipogenesis and suppressing fatty acid ß-oxidation. LRG1 also inhibited hepatic insulin signaling by downregulating insulin receptor substrates 1 and 2. Our study identified LRG1 as a key molecule that mediates the crosstalk between adipocytes and hepatocytes in diet-induced hepatosteatosis and insulin resistance. Suppressing LRG1 expression and function may be a promising strategy for the treatment of obesity-related metabolic diseases.

JTE-013 Alleviates Inflammatory Injury and Endothelial Dysfunction Induced by Sepsis In Vivo and In Vitro.

BACKGROUND: Nowadays, there is no approved targeted agent for lung injury induced by sepsis. S1PR2 is confirmed to be a promising diagnosis and treatment target. JTE-013 as S1PR2 antagonists may be an agent of great potential. In this research, we sought to determine the functional role of JTE-013 in lung injury induced by sepsis. MATERIALS AND METHODS: Seventy-two rats were assigned into normal group, sepsis model group and JTE-013 group. The animal model of lung injury induced by sepsis was constructed by cecal ligation and puncture. The human pulmonary microvascular endothelial cells (HPMECs) were divided into control, LPS and LPS + JTE-013 group. HPMECs induced by LPS served as the cell model of lung injury induced by sepsis. HE staining assay was performed for assessment of the pathological condition and Evans blue was applied for assessment of pulmonary tissue permeability. Wet/dry ratio was measured as indicators of pulmonary edema degree and neutrophil count was measured as indicators of infection status. The levels of inflammatory factors were detected by corresponding kits, cell survival by CCK-8 assay and protein expression level by western blot. RESULTS: S1PR2 was highly expressed in vivo model of lung injury induced by sepsis. It was observed that JTE-013 as antagonist of S1PR2 alleviated the lung tissue injury, endothelial dysfunction and pulmonary edema induced by sepsis. In addition, JTE-013 reduced neutrophil count and levels of inflammatory factors. Moreover, results confirmed that JTE-013 enhanced cell viability and mitigated inflammatory response in cell model of sepsis. CONCLUSIONS: Overall, JTE-013 as an antagonist of S1PR2 could relieve inflammatory injury and endothelial dysfunction induced by sepsis in vivo and vitro, resulting in attenuation of lung injury. These findings elucidated that JTE-013 may be a promising targeted agent for lung injury induced by sepsis.

Human placental mesenchymal stem cells ameliorate chemotherapy-induced damage in the testis by reducing apoptosis/oxidative stress and promoting autophagy.

BACKGROUND: The side effects of busulfan on male reproduction are serious, so fertility preservation in children undergoing busulfan treatment is a major worldwide concern. Human placental mesenchymal stem cells (hPMSCs) have advantages such as stable proliferation and lower immunogenicity that make them an ideal material for stimulating tissue repair, especially restoring spermatogenesis. The protective effects of hPMSCs in busulfan-induced Sertoli cells and in busulfan-treated mouse testes have not been determined. Our study aimed to elaborate the protective effect and potential mechanisms of hPMSCs in busulfan-treated testes and Sertoli cells. METHODS: First, we developed a mouse model of busulfan-induced testicular toxicity in vivo and a mouse Sertoli cell line treated with busulfan in vitro to assess the protective effect and mechanisms of hPMSC treatment on spermatogenesis. Then, the length, width, and weight of the testes were monitored using Vernier calipers. Furthermore, at 1 week and 4 weeks after the transplantation of hPMSCs, histological sections of testes were stained with hematoxylin-eosin, and the seminiferous tubules with fluid-filled cavities were counted. Through ELISA analysis, testosterone levels and MDA, SOD, LDH, and CAT activities, which are associated with ROS, were detected. Markers of ROS, proliferation (Ki67), and apoptosis (Annexin V) were evaluated by FACS. Next, the fluorescence intensity of proliferation markers (BrdU and SCP3), an antioxidant marker (SIRT1), a spermatogenesis marker (PLZF), and autophagy-related genes (P62 and LC3AB) were detected by fluorescence microscopy. The mRNA expression of γ-H2AX, BRCA1, PARP1, PCNA, Ki67, P62, and LC3 was determined by qRT-PCR. RESULTS: hPMSCs restored disrupted spermatogenesis, promoted improved semen parameters, and increased testosterone levels, testis size, and autophagy in the testis toxicity mouse model induced by busulfan. hPMSCs suppressed the apoptosis of Sertoli cells and enhanced their rate of proliferation in vitro. Additionally, hPMSCs protected against oxidative stress and decreased oxidative damage in the testis toxicity mouse model induced by busulfan. Furthermore, hPMSCs increased the expression of proliferation genes (PCNA and KI67) and decreased the mRNA levels of apoptotic genes such as γ-H2AX, BRCA1, and PARP1. CONCLUSIONS: This research showed that hPMSC injection ameliorated busulfan-induced damage in the testis by reducing apoptosis/oxidative stress and promoting autophagy. The present study offers an idea for a new method for clinical treatment of chemotherapy-induced spermatogenesis.

Circular RNAs from BOULE play conserved roles in protection against stress-induced fertility decline.

Circular RNAs (circRNAs) are a large family of newly identified transcripts, and their physiological roles and evolutionary significance require further characterization. Here, we identify circRNAs generated from a conserved reproductive gene, Boule, in species from Drosophila to humans. Flies missing circular Boule (circBoule) RNAs display decreased male fertility, and sperm of circBoule knockout mice exhibit decreased fertilization capacity, when under heat stress conditions. During spermatogenesis, fly circBoule RNAs interact with heat shock proteins (HSPs) Hsc4 and Hsp60C, and mouse circBoule RNAs in sperm interact with HSPA2. circBoule RNAs regulate levels of HSPs by promoting their ubiquitination. The interaction between HSPA2 and circBoule RNAs is conserved in human sperm, and lower levels of the human circBoule RNAs circEx3-6 and circEx2-7 are found in asthenozoospermic sperm. Our findings reveal conserved physiological functions of circBoule RNAs in metazoans and suggest that specific circRNAs may be critical modulators of male reproductive function against stresses in animals.

Identification and preclinical development of an anti-proteolytic uPA antibody for rheumatoid arthritis.

Blocking the proteolytic capacity of urokinase-type plasminogen activator (uPA) with a monoclonal antibody (mAb) reduces arthritis progression in the collagen-induced mouse arthritis model to an extent that is on par with the effect of blocking tumor necrosis factor-alpha by etanercept. Seeking to develop a novel therapy for rheumatoid arthritis, a humanized mAb, NNC0266-0043, was selected for its dual inhibition of both the zymogen activation and the proteolytic capacity of human uPA. The antibody revealed nonlinear elimination kinetics in cynomolgus monkeys consistent with binding to and turnover of endogenous uPA. At a dose level of 20.6 mg kg-1, the antibody had a plasma half-life of 210 h. Plasma uPA activity, a pharmacodynamic marker of anti-uPA therapy, was reduced to below the detection limit during treatment, indicating that an efficacious plasma concentration was reached. Pharmacokinetic modeling predicted that sufficient antibody levels can be sustained in arthritis patients dosed subcutaneously once weekly. The anti-uPA mAb was also well tolerated in cynomolgus monkeys at weekly doses up to 200 mg kg-1 over 4 weeks. The data from cynomolgus monkeys and from human material presented here indicates that anti-uPA mAb NNC0266-0043 is suitable for clinical testing as a novel therapeutic for rheumatic diseases. KEY MESSAGES: Background: Anti-uPA therapy is on par with etanercept in a mouse arthritis model. A new humanized antibody blocks activation and proteolytic activity of human uPA. The antibody represents a radically novel mode-of-action in anti-rheumatic therapy. The antibody has PK/PD properties in primates consistent with QW clinical dosing.

LYPD4, mouse homolog of a human acrosome protein, is essential for sperm fertilizing ability and male fertility†.

Fertilization is one of the fundamental biological processes, but so far, we still do not have a full understanding of the underlying molecular mechanism. We have identified a human acrosome protein, LY6/PLAUR domain containing 4 (LYPD4), expressed specifically in human testes and sperm, and conserved within mammals. Mouse Lypd4, also specific to the testis and sperm, is essential for male fertility. LYPD4 protein first appeared in round spermatids during acrosome biogenesis and became part of acrosomes during spermatogenesis and in mature sperm. Lypd4 knockout mice are infertile with normal sperm number and motility. Mutant sperm, however, failed to reach oviduct during sperm migration inside the female reproductive tract, leading to fertilization failure and infertility. In addition, Lypd4 mutant sperms were unable to fertilize denuded egg via IVF (in vitro fertilization) but could fertilize eggs within intact Cumulus-Oocyte Complex, supporting an additional role in sperm-zona interaction. Out of more than five thousand spermatozoa proteins identified by mass spectrometry analysis, only a small subset of proteins (26 proteins) was changed in the absence of LYPD4, revealing a whole proteome picture of mutant sperm defective in sperm migration and sperm-zona binding. ADAM3, a key component of fertilization complex, as well as other sperm ADAM proteins are significantly reduced. We hence propose that LYPD4 plays an essential role in mammalian fertilization, and further investigation of its function and its interaction with other sperm membrane complexes may yield insights into human fertilization and novel strategy to improve IVF success.

Solvent and Surface/Interface Effect on the Hierarchical Assemblies of Chiral Aggregation-Induced Emitting Molecules.

The core of aggregation-induced emitting (AIE) molecules was their aggregation behavior. It was, in essence, a self-assembly process driven by noncovalent interactions, which were governed not only by the chemical structures of the molecules but also by the conditions where the self-assemblies were formed. The self-assemblies of two AIE molecules, tetraphenylethene (TPE) derivatives carrying one valine attachment (TPE-Val) and two valine attachments (TPE-2Val), were studied. Both kinds of molecules self-assembled into supramolecular helical fibers with different handedness upon the addition of poor solvent to their solution. However, when deposited on air/water interface, both kinds of molecules formed aligned elementary helical fibers instead of supramolecular fibers. The lateral solvophobic effect exerted by water molecules caused a shift of the original noncovalent balance between molecules and solvent; thus, the supramolecular helical assemblies were unraveled into aligned helical elementary fibers. Similar elementary assemblies were formed on the surface of 3-aminopropyl triethoxysilane-modified mica, confirming the lateral solvophobic effect on the self-assemblies of the molecules.

Discovery of a New Pterocarpan-Type Antineuroinflammatory Compound from Sophora tonkinensis through Suppression of the TLR4/NFκB/MAPK Signaling Pathway with PU.1 as a Potential Target.

Neuroinflammation underlies many neuro-degenerative diseases. In this paper, we report the identification of a new pterocarpan-type anti-inflammatory compound named sophotokin isolated from Sophora tonkinensis. S. tonkinensis has been used traditionally for treatment of conditions related to inflammation. Our initial screening showed that sophotokin dose-dependently inhibits lipopolysaccharide (LPS)-stimulated production of NO, TNF-α, PGE2, and IL-1ß in microglial cells. This antineuroinflammatory effect was associated with sophotokin's blockade of LPS-induced production of the inflammatory mediators iNOS and COX-2. Western blot and qPCR analysis demonstrated that sophotokin inhibits both the p38-MAPK and NF-κB signal pathways. Further studies revealed that sophotokin also suppresses the expression of cluster differentiation 14 (CD14) in the toll-like receptor 4 (TLR4) signaling pathway. Following down-regulation of MyD88 and TRAF6, sophotokin inhibits the activation of the NF-κB and MAPK signal pathways in LPS-induced BV-2 cells. In silico studies suggested that sophotokin could interact with PU.1-DNA complex through hydrogen binding at sites 1 and 2 of the complex, blocking the DNA binding. This suggests that PU.1 may be a potential target of sophotokin. Taken together, these results suggest that sophotokin may have therapeutic potential for diseases related to neuroinflammation. The mechanism of antineuroinflammatory effects involves inhibition of the TLR4 signal pathway at the sites of NF-κB and MAPK with PU.1 as a likely upstream target.

Essential requirement of mammalian Pumilio family in embryonic development.

Mouse PUMILIO1 (PUM1) and PUMILIO2 (PUM2) belong to the PUF (Pumilio/FBF) family, a highly conserved RNA binding protein family whose homologues play critical roles in embryonic development and germ line stem cell maintenance in invertebrates. However, their roles in mammalian embryonic development and stem cell maintenance remained largely uncharacterized. Here we report an essential requirement of the Pum gene family in early embryonic development. A loss of both Pum1 and Pum2 genes led to gastrulation failure, resulting in embryo lethality at E8.5. Pum-deficient blastocysts, however, appeared morphologically normal, from which embryonic stem cells (ESCs) could be established. Both mutant ESCs and embryos exhibited reduced growth and increased expression of endoderm markers Gata6 and Lama1, making defects in growth and differentiation the likely causes of gastrulation failure. Furthermore, ESC Gata6 transcripts could be pulled down via PUM1 immunoprecipitation and mutation of conserved PUM-binding element on 3'UTR (untranslated region) of Gata6 enhanced the expression of luciferase reporter, implicating PUM-mediated posttranscriptional regulation of Gata6 expression in stem cell development and cell lineage determination. Hence, like its invertebrate homologues, mouse PUM proteins are conserved posttranscriptional regulators essential for embryonic and stem cell development.

Design, synthesis, and biological evaluation of compounds with a new scaffold as anti-neuroinflammatory agents for the treatment of Alzheimer's disease.

Twenty-eight compounds with a new scaffold were designed and synthesized by assembling fragments derived from known agents such as stilbenes and piperazinyl pyrimidines. Many strategies have been explored to improve the druggability of these series of compounds, such as increasing the distance between two benzene rings in the scaffold and introducing functional groups at designated positions. These compounds were validated for their anti-neuroinflammatory activity in BV2 cells. Experimental results reveal that the most active compound 8b can inhibit nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) production with IC50 values of 1.0, 2.6, and 0.5 µM, respectively. The compound can also significantly modulate the MAPK pathways through inhibiting the phosphorylation of JNK, ERK1/2, and p38 MAPK without disturbing NF-κB pathway. Parallel artificial membrane permeation assay demonstrated that the most active compound can overcome the blood-brain barrier (BBB). Therefore, this compound can be a promising lead for the treatment of Alzheimer's disease.

Diterpenoids with diverse scaffolds from Vitex trifolia as potential topoisomerase I inhibitor.

Eleven new compounds, including six labdane (1-6), three halimane (7-9), and two clerodane (10-11) diterpenoids and 16 known analogues (12-27), were isolated from the leaves of Vitex trifolia. The structures of 1-11 were established by extensive 1D- and 2D-NMR and HRMS spectroscopic data. The absolute configurations of compounds 3, 7, and 10 were assigned using X-ray diffraction. Compounds 1-27 were evaluated for DNA topoisomerases I (Top1) inhibitory activity and cytotoxicity against HCT 116 cells. Compounds 8 and 11 exhibited equipotent Top1 inhibitory activity to the positive control, camptothecin (CPT), at 100µM. Compounds 8, 9, 16, and 27 showed moderate cytotoxicity at low micromolar concentrations.

Vitepyrroloids A-D, 2-Cyanopyrrole-Containing Labdane Diterpenoid Alkaloids from the Leaves of Vitex trifolia.

Vitepyrroloids A-D (1-4), four new 2-cyano-substituted pyrrole-ring-containing labdane diterpenoids, were isolated from the leaves of Vitex trifolia. Their structures were elucidated based on spectroscopic data analysis. The absolute configuration of compound 1 was determined by X-ray diffraction. Compounds 1-4 are unprecedented labdane diterpenoids featuring a 2-cyano-substituted pyrrole ring. Compound 1 showed cytotoxic activity against a human nasopharyngeal carcinoma cell line (CNE1) with an IC50 value of 8.7 µM.

Modulation of the pentose phosphate pathway alters phase I metabolism of testosterone and dextromethorphan in HepG2 cells.

AIM: The pentose phosphate pathway (PPP) is involved in the activity of glucose-6-phosphate dehydrogenase (G6PD) and generation of NADPH, which plays a key role in drug metabolism. The aim of this study was to investigate the effects of modulation of the PPP on drug metabolism capacity in vitro. METHODS: A pair of hepatic cell lines, ie, the cancerous HepG2 cells and normal L02 cells, was used. The expression of CYP450 enzymes, p53 and G6PD in the cells were analyzed. The metabolism of testosterone (TEST, 10 µmol/L) and dextromethorphan (DEM, 1 µmol/L), the two typical substrates for CYP3A4 and CYP2D6, in the cells was examined in the presence of different agents. RESULTS: Both the expression and metabolic activities of CYP3A4 and CYP2D6 were considerably higher in HepG2 cells than in L02 cells. The metabolism of TEST and DEM in HepG2 cells was dose-dependently inhibited by the specific CYP3A4 inhibitor ketoconazole and CYP2D6 inhibitor quinidine. Addition of the p53 inhibitor cyclic PFT-α (5, 25 µmol/L) in HepG2 cells dose-dependently enhanced the metabolism of DEM and TEST, whereas addition of the p53 activator NSC 66811 (3, 10, 25 µmol/L) dose-dependently inhibited the metabolism. Furthermore, addition of the G6PD inhibitor 6-aminonicotinamide (5, 15 µmol/L) in HepG2 cells dose-dependently inhibited the metabolism of DEM and TEST, whereas addition of the PPP activity stimulator menadione (1, 5, 15 µmol/L) dose-dependently enhanced the metabolism. CONCLUSION: Modulation of p53 and the PPP alters the metabolism of DEM and TEST, suggesting that the metabolic flux pattern of PPP may be closely involved in drug metabolism and the individual variance.