A fluorene derivative inhibits human hepatocellular carcinoma cells by ROS-mediated apoptosis, anoikis and autophagy.

Fluorene was previously reported to have anticancer activity against human cancer cells. In this study, we examined the in vitro function of 9-methanesulfonylmethylene-2, 3-dimethoxy-9 H -fluorene (MSDF), a novel fluorene derivative, its anticancer potential in human hepatocellular carcinoma (HCC) cells and its underlying molecular mechanism. The disruption of cellular homeostasis caused by MSDF was found to promote reactive oxygen species (ROS) generation, leading to the activation of cellular apoptosis. As a survival strategy, cells undergo autophagy during oxidative stress. MSDF-induced apoptosis occurred through both receptor-mediated extrinsic and mitochondrial-mediated intrinsic routes. The development of acidic vesicular organelles and the accumulation of LC3-II protein suggest an increase in the autophagic process. Apoptosis was detected by double staining. The MAPK/ERK and PI3K/Akt signaling pathways were indeed suppressed during treatment. Along with elevated ROS generation and apoptosis, MSDF also caused anoikis and cell death by causing cells to lose contact with their extracellular matrix. ROS production was induced by MSDF and sustained by an NAC scavenger. MSDF-induced apoptosis led to increased autophagy, as shown by the suppression of apoptosis by Z-VAD-FMK. However, inhibition of autophagy by inhibitor 3-MA increased MSDF-induced apoptosis. More evidence shows that MSDF downregulated the expression of immune checkpoint proteins, suggesting that MSDF could be used in the future as an adjuvant to improve the effectiveness of HCC immunotherapy. Altogether, our results highlight the potential of MSDF as a multitarget drug for the treatment of HCC.

Pre-arrayed Pan-AAV Peptide Display Libraries for Rapid Single-Round Screening.

Display of short peptides on the surface of adeno-associated viruses (AAVs) is a powerful technology for the generation of gene therapy vectors with altered cell specificities and/or transduction efficiencies. Following its extensive prior use in the best characterized AAV serotype 2 (AAV2), recent reports also indicate the potential of other AAV isolates as scaffolds for peptide display. In this study, we systematically explored the respective capacities of 13 different AAV capsid variants to tolerate 27 peptides inserted on the surface followed by production of reporter-encoding vectors. Single-round screening in pre-arrayed 96-well plates permitted rapid and simple identification of superior vectors in >90 cell types, including T cells and primary cells. Notably, vector performance depended not only on the combination of capsid, peptide, and cell type, but also on the position of the inserted peptide and the nature of flanking residues. For optimal data availability and accessibility, all results were assembled in a searchable online database offering multiple output styles. Finally, we established a reverse-transduction pipeline based on vector pre-spotting in 96- or 384-well plates that facilitates high-throughput library panning. Our comprehensive illustration of the vast potential of alternative AAV capsids for peptide display should accelerate their in vivo screening and application as unique gene therapy vectors.

Photosynthetic index and nitrogen assimilation in rapeseed seedlings transplanted in soil with ammonium glufosinate / Índice fotossintético e assimilação de nitrogênio em plântulas de colza transplantadas em resposta a resíduos de glufosinato presentes no solo

ABSTRACT: Herbicide application is an effective weed control method for mitigating crop yield loss; however, herbicide overuse can cause toxicity in non-target plants. The present study evaluated the effects of glufosinate at recommended dose for agricultural application (0.45 kg ha-1) and at overuse dose (0.90 kg ha-1) glufosinate application on photosynthetic performance and nitrogen assimilation of the rapeseed varieties D148 and Zhongshuang 11 (ZS11). Both glufosinate concentrations significantly decreased the content of chlorophyll and nitrogenous compounds, except free proline, and the activity of glutamine synthetase and glutamate synthase, and increased the activity of glutamic acid dehydrogenase in both varieties. When the concentration of glyphosate was 0.45kg ha-1, the nitrogen assimilation of the two varieties decreased, which indicated that the recommended dosage inhibited the nitrogen assimilation of the two varieties; however, the increase of net photosynthetic rate of D148 and the decrease of that of ZS11 mean that D148 is more tolerant to the recommended dose of glyphosate than ZS11. The 0.90 kg ha-1 dosage was toxic to both rapeseed varieties. Overall, our results indicated that herbicide overuse inhibited the photosynthetic rate and nitrogen assimilation in rapeseed seedlings, and it is essential to apply a suitable glufosinate dose based on the variety grown to minimize adverse effects on crops and environment.

RESUMO: A aplicação de herbicidas é um método eficaz de controle de ervas daninhas para mitigar a perda de produtividade das culturas. No entanto, o uso excessivo de herbicidas pode causar toxicidade em plantas não alvo. O presente estudo avaliou os efeitos da dose recomendada para aplicação agrícola (0.45 kg ha-1) e dose excessiva (0.90 kg ha-1) de glufosinato no desempenho fotossintético e assimilação de nitrogênio das variedades de colza D148 e Zhongshuang 11 (ZS11). Ambas as concentrações de glutamato diminuíram significativamente o teor de clorofila e compostos azotados, exceto a prolina livre, e a atividade de síntese da glutamina e de síntese de glutamato, e aumentaram a atividade de desidrogenase do ácido glutâmico em ambas as variedades. Quando a concentração de glifosato foi 0.45 kg ha-1, a assimilação de azoto das duas variedades diminuiu, o que indicou que a dosagem recomendada de glifosato inibiu a assimilação de azoto das duas variedades de colza. Entretanto, a taxa fotosintética líquida do D148 aumentou enquanto o do ZS11 diminuiu, o que significa que o D148 é mais tolerante a dose recomendada de glifosato do que o ZS11. A dose de 0.90 kg ha-1 de glifosato foi prejudicial para as mudas de duas variedades de colza. Em geral, os nossos resultados indicam que o uso excessivo de glufosinato inibe a taxa fotossintética e a assimilação de nitrogênio em mudas de colza, sendo essencial aplicar uma dose adequada deste herbicida com base na variedade cultivada para minimizar os efeitos adversos nas culturas e no meio ambiente.

Ionic Liquids Enhanced Alkynyl Schiff Bases Derivatives of Fipronil Synthesis and Their Cytotoxicity Studies.

To obtain highly selective toxic derivatives of fipronil, a series of Schiff bases with an alkynyl group (3a-3k) were designed and synthesized from 4-ethynylbenzaldehyde (2) and 4-substituted 5-amino-N-arylpyrazole (1a-1k) via a nucleophilic addition elimination reaction in ionic liquids. Utilization of ionic liquids was demonstrated to endow the yield of each compound beyond 50%, which was enhanced over 1.5 times of the synthetic productive rates comparing the conventional method by which longer reactive time was consumed. The derivatives were characterized via nuclear magnetic resonance hydrogen spectroscopy (1H-NMR), carbon-13 nuclear magnetic resonance spectroscopy (13C-NMR), and electrospray ionization high resolution mass spectrometry (ESI-HRMS). The cytotoxicity of these derivatives on Trichoplusia ni (Hi-5) cell and Spodoptera litura cell (SL cell) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) bioassays. The results indicated that several compounds had potential cytotoxicity on Hi-5 cell, especially a 4-ethyl substituted alkynyl Schiff base derivative (3f) that was demonstrated to possess high selective toxicity to the Hi-5 cell than the SL cell. In addition, 3f exhibited comparable toxic activity to commercial fipronil on a Hi-5 cell while a little toxic effect on the SL cell, which satisfied the expectation for selective toxicity screening.

Undecaprenyl Phosphate Phosphatase Activity of Undecaprenol Kinase Regulates the Lipid Pool in Gram-Positive Bacteria.

Bacteria cell walls contain many repeating glycan structures, such as peptidoglycans, lipopolysaccharides, teichoic acids, and capsular polysaccharides. Their synthesis starts in the cytosol, and they are constructed from a glycan lipid carrier, undecaprenyl phosphate (C55P), which is essential for cell growth and survival. The lipid derivative undecaprenol (C55OH) is predominant in many Gram-positive bacteria but has not been detected in Gram-negative bacteria; its origin and role have thus remained unknown. Recently, a homologue of diacylglycerol kinase (DgkA) in Escherichia coli (E. coli) was demonstrated to be an undecaprenol kinase (UK) in the Gram-positive bacterium Streptococcus mutans (S. mutans). In this study, we found that S. mutans UK was not only an undecaprenol kinase but also a Mg-ADP-dependent undecaprenyl phosphate phosphatase (UpP), catalyzing the hydrolysis of C55P to C55OH and a free inorganic phosphate. Furthermore, the naturally undetectable C55OH was observed in E. coli cells expressing S. mutans dgkA, supporting the phosphatase activity of UK/UpP in vivo. These two activities were indispensable to each other and utilized identical essential residues binding to their substrates, suggesting that both activities share the same active site and might involve a direct phosphoryl transfer mechanism. This study revealed a unique membrane enzyme displaying bifunctional activities determined by substrate binding and C55OH production. The reciprocal conversion of C55P and the undecaprenol pool efficiently regulate cell wall synthesis, especially in Gram-positive bacteria.

Characterization of the Adeno-Associated Virus 1 and 6 Sialic Acid Binding Site.

UNLABELLED: The adeno-associated viruses (AAVs), which are being developed as gene delivery vectors, display differential cell surface glycan binding and subsequent tissue tropisms. For AAV serotype 1 (AAV1), the first viral vector approved as a gene therapy treatment, and its closely related AAV6, sialic acid (SIA) serves as their primary cellular surface receptor. Toward characterizing the SIA binding site(s), the structure of the AAV1-SIA complex was determined by X-ray crystallography to 3.0 Å. Density consistent with SIA was observed in a pocket located at the base of capsid protrusions surrounding icosahedral 3-fold axes. Site-directed mutagenesis substitution of the amino acids forming this pocket with structurally equivalent residues from AAV2, a heparan sulfate binding serotype, followed by cell binding and transduction assays, further mapped the critical residues conferring SIA binding to AAV1 and AAV6. For both viruses five of the six binding pocket residues mutated (N447S, V473D, N500E, T502S, and W503A) abolished SIA binding, whereas S472R increased binding. All six mutations abolished or decreased transduction by at least 50% in AAV1. Surprisingly, the T502S substitution did not affect transduction efficiency of wild-type AAV6. Furthermore, three of the AAV1 SIA binding site mutants-S472R, V473D, and N500E-escaped recognition by the anti-AAV1 capsid antibody ADK1a. These observations demonstrate that common key capsid surface residues dictate both virus binding and entry processes, as well as antigenic reactivity. This study identifies an important functional capsid surface "hot spot" dictating receptor attachment, transduction efficiency, and antigenicity which could prove useful for vector engineering. IMPORTANCE: The adeno-associated virus (AAV) vector gene delivery system has shown promise in several clinical trials and an AAV1-based vector has been approved as the first gene therapy treatment. However, limitations still exist with respect to transduction efficiency and the detrimental effects of preexisting host antibodies. This study aimed to identify key capsid regions which can be engineered to overcome these limitations. A sialic glycan receptor recognition pocket was identified in AAV1 and its closely related AAV6, using X-ray crystallography. The site was confirmed by mutagenesis followed by cell binding and transduction assays. Significantly, residues controlling gene expression efficiency, as well as antibody escape variants, were also identified. This study thus provides, at the amino acid level, information for rational structural engineering of AAV vectors with improved therapeutic efficacy.

Site-Directed Mutagenesis of Surface-Exposed Lysine Residues Leads to Improved Transduction by AAV2, But Not AAV8, Vectors in Murine Hepatocytes In Vivo.

The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of recombinant adeno-associated virus 2 (AAV2) vectors, which negatively impacts the transduction efficiency of these vectors. Because ubiquitination occurs on lysine (K) residues, we performed site-directed mutagenesis where we replaced each of 10 surface-exposed K residues (K258, K490, K507, K527, K532, K544, K549, K556, K665, and K706) with glutamic acid (E) because of similarity of size and lack of recognition by modifying enzymes. The transduction efficiency of K490E, K544E, K549E, and K556E scAAV2 vectors increased in HeLa cells in vitro up to 5-fold compared with wild-type (WT) AAV2 vectors, with the K556E mutant being the most efficient. Intravenous delivery of WT and K-mutant ssAAV2 vectors further corroborated these results in murine hepatocytes in vivo. Because AAV8 vectors transduce murine hepatocytes exceedingly well, and because some of the surface-exposed K residues are conserved between these serotypes, we generated and tested two single mutants (K547E and K569E), and one double-mutant (K547 + 569E) AAV8 vector. However, no significant increase in the transduction efficiency of any of these mutant AAV8 vectors was observed in murine hepatocytes in vivo. These studies suggest that although targeting the surface-exposed K residues is yet another strategy to improve the transduction efficiency of AAV vectors, phenotypic outcome is serotype specific.

Enzymatic synthesis of lipid II and analogues.

The emergence of antibiotic resistance has prompted active research in the development of antibiotics with new modes of action. Among all essential bacterial proteins, transglycosylase polymerizes lipid II into peptidoglycan and is one of the most favorable targets because of its vital role in peptidoglycan synthesis. Described in this study is a practical enzymatic method for the synthesis of lipid II, coupled with cofactor regeneration, to give the product in a 50-70% yield. This development depends on two key steps: the overexpression of MraY for the synthesis of lipid I and the use of undecaprenol kinase for the preparation of polyprenol phosphates. This method was further applied to the synthesis of lipid II analogues. It was found that MraY and undecaprenol kinase can accept a wide range of lipids containing various lengths and configurations. The activity of lipid II analogues for bacterial transglycolase was also evaluated.

New continuous fluorometric assay for bacterial transglycosylase using Förster resonance energy transfer.

The emergence of antibiotic resistance has prompted scientists to search for new antibiotics. Transglycosylase (TGase) is an attractive target for new antibiotic discovery due to its location on the outer membrane of bacteria and its essential role in peptidoglycan synthesis. Though there have been a few molecules identified as TGase inhibitors in the past thirty years, none of them have been developed into antibiotics for humans. The slow pace of development is perhaps due to the lack of continuous, quantitative, and high-throughput assay available for the enzyme. Herein, we report a new continuous fluorescent assay based on Förster resonance energy transfer, using lipid II analogues with a dimethylamino-azobenzenesulfonyl quencher in the lipid chain and a coumarin fluorophore in the peptide chain. During the process of transglycosylation, the quencher-appended polyprenol is released and the fluorescence of coumarin can be detected. Using this system, the substrate specificity and affinity of lipid II analogues bearing various numbers and configurations of isoprene units were investigated. Moreover, the inhibition constants of moenomycin and two previously identified small molecules were also determined. In addition, a high-throughput screening using the new assay was conducted to identify potent TGase inhibitors from a 120,000 compound library. This new continuous fluorescent assay not only provides an efficient and convenient way to study TGase activities, but also enables the high-throughput screening of potential TGase inhibitors for antibiotic discovery.

Solution structure of the oncogenic MIEN1 protein reveals a thioredoxin-like fold with a redox-active motif.

The novel tumor biomarker MIEN1, identified by representational difference analysis, is overexpressed in breast cancer and prostate cancer. MIEN1 is considered an oncogenic protein, because MIEN1 overexpression functionally enhances migration and invasion of tumor cells via modulating the activity of AKT. However, the structure and molecular function of MIEN1 is little understood. Here, we report the solution structure of MIEN1, which adopts a thioredoxin-like fold with a redox-active motif. Comparison of backbone chemical shifts showed that most of the residues for both oxidized and reduced MIEN1 possessed the same backbone conformation, with differences limited to the active motif and regions in proximity. The redox potential of this disulfide bond was measured as -225 mV, which compares well with that of disulfides for other thioredoxin-like proteins. Overall, our results suggest that MIEN1 may have an important regulatory role in phosphorylation of AKT with its redox potential.

Total synthesis of polyprenyl N-glycolyl lipid II as a mycobacterial transglycosylase substrate.

A feasible synthetic approach toward the Mycobacterium tuberculosis (Mtb) N-glycolyl lipid II-like molecule 1 is described. Compound 1 bears pendant undecaprenol and l-lysin moieties instead of the naturally occurring decaprenol and meso-diaminopimelic acid, which are not readily available. Functionalization of 1 with a fluorophore on the peptide side chain gave 14, which was found to be recognized as an Mtb TGase substrate. This result suggests it has tremendous utility for mechanistic studies, the characterization of mycobacterial enzymes, and mycobacterial TGase inhibitor evaluation.

Resonance assignments of human C35 (C17orf37) protein, a novel tumor biomarker.

A new tumor-specific target, termed C35 (C17orf37), has been identified by representational difference analysis of tumor and normal human mammary cell lines. C35 protein is considered to be an important target for cancer therapy, since the over expression of C35 functionally enhances migration and invasion of tumor cells. Here we report the NMR resonance assignments of C35 protein for further structural determination and functional studies.

A novel siRNA validation system for functional screening and identification of effective RNAi probes in mammalian cells.

Small interfering RNAs (siRNAs) have become the most powerful and widely used gene silencing reagents for reverse functional genomics and molecular therapeutics. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily dependent on the effectiveness and specificity of the RNAi targeting sequence. However, only a limited number of siRNAs is capable of inducing highly effective and sequence-specific gene silencing by RNA interference (RNAi) mechanism. In addition, the efficacy of siRNA-induced gene silencing can only be experimentally measured based on inhibition of the target gene expression. Therefore, it is important to establish a fully robust and comparative validating system for determining the efficacy of designed siRNAs. In this study, we have developed a reliable and quantitative reporter-based siRNA validation system that consists of a short synthetic DNA fragment containing an RNAi targeting sequence of interest and two expression vectors for targeting reporter and triggering siRNA expression. The efficacy of the siRNAs is measured by their abilities to inhibit expression of the targeting reporter gene with easily quantified readouts including enhanced green fluorescence protein (EGFP) and firefly luciferase. Using fully analyzed siRNAs against human hepatitis B virus (HBV) surface antigen (HBsAg) and tumor suppressor protein p53, we have demonstrated that this system could effectively and faithfully report the efficacy of the corresponding siRNAs. In addition, we have further applied this system for screening and identification of the highly effective siRNAs that could specifically inhibit expression of mouse matrix metalloproteinase-7 (MMP-7), Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and human serine/threonine kinase AKT1. Since only a readily available short synthetic DNA fragment is needed for constructing this novel reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective siRNAs but also implicates in the use of RNAi for studying novel gene function in mammals.