Isolation of the inorganic phosphorus-solubilizing bacteria Lysinibacillus sphaericus and assessing its role in promoting rice growth.

Soluble phosphorus scarcity severely limits plant growth and crop yield. In this study, a strain of inorganic phosphorus-solubilizing bacteria, Lysinibacillus sphaericus, was isolated from rice rhizosphere soil. The available phosphorus content in liquid inorganic phosphorus identification medium and in L. sphaericus-inoculated soil increased from 204.28 mg/L to 1124.68 mg/L and from 4.75 mg/kg to 7.04 mg/kg, respectively. The pH decreased significantly from 6.87 to 6.14. Incubation with L. sphaericus significantly increased malic and succinic acid content in the liquid inorganic phosphorus identification medium and increased acid phosphatase and alkaline phosphatase activity in the soil. Inoculation with L. sphaericus significantly increased rice growth, chlorophyll a/b content, and photosynthesis by increasing the soluble phosphorus content in the rice rhizosphere soil under phosphorus-deficient conditions. Further analysis revealed that L. sphaericus improved soil phosphorus release by decreasing soil pH and promoting acid phosphatase and alkaline phosphatase activity. This study supports the production of microbial fertilizers to improve rice yield in phosphorus-deficient conditions.

Interplay Impact of Exogenous Application of Abscisic Acid (ABA) and Brassinosteroids (BRs) in Rice Growth, Physiology, and Resistance under Sodium Chloride Stress.

The hormonal imbalances, including abscisic acid (ABA) and brassinosteroid (BR) levels, caused by salinity constitute a key factor in hindering spikelet development in rice and in reducing rice yield. However, the effects of ABA and BRs on spikelet development in plants subjected to salinity stress have been explored to only a limited extent. In this research, the effect of ABA and BRs on rice growth characteristics and the development of spikelets under different salinity levels were investigated. The rice seedlings were subjected to three different salt stress levels: 0.0875 dS m-1 (Control, CK), low salt stress (1.878 dS m-1, LS), and heavy salt stress (4.09 dS m-1, HS). Additionally, independent (ABA or BR) and combined (ABA+BR) exogenous treatments of ABA (at 0 and 25 µM concentration) and BR (at 0 and 5 µM concentration) onto the rice seedlings were performed. The results showed that the exogenous application of ABA, BRs, and ABA+BRs triggered changes in physiological and agronomic characteristics, including photosynthesis rate (Pn), SPAD value, pollen viability, 1000-grain weight (g), and rice grain yield per plant. In addition, spikelet sterility under different salt stress levels (CK, LS, and HS) was decreased significantly through the use of both the single phytohormone and the cocktail, as compared to the controls. The outcome of this study reveals new insights about rice spikelet development in plants subjected to salt stress and the effects on this of ABA and BR. Additionally, it provides information on the use of plant hormones to improve rice yield under salt stress and on the enhancement of effective utilization of salt-affected soils.

Unearthing the Alleviatory Mechanisms of Brassinolide in Cold Stress in Rice.

Cold stress inhibits rice germination and seedling growth. Brassinolide (BR) plays key roles in plant growth, development, and stress responses. In this study, we explored the underlying mechanisms whereby BR helps alleviate cold stress in rice seedlings. BR application to the growth medium significantly increased seed germination and seedling growth of the early rice cultivar "Zhongzao 39" after three days of cold treatment. Specifically, BR significantly increased soluble protein and soluble sugar contents after three days of cold treatment. Moreover, BR stimulated the activity of superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase; thereby alleviating cold-induced damage and increasing glutathione content and the GSH/GSSG ratio while concomitantly reducing H2O2 content. BR upregulated the expression levels of cold-response-related genes, including OsICE1, OsFer1, OsCOLD1, OsLti6a, OsSODB, OsMyb, and OsTERF2, and downregulated that of OsWRKY45, overall alleviating cold stress symptoms. Thus, BR not only upregulated cellular osmotic content and the antioxidant enzyme system to maintain the physiological balance of reactive oxygen species under cold but, additionally, it regulated the expression of cold-response-related genes to alleviate cold stress symptoms. These results provide a theoretical basis for rice breeding for cold resistance using young seedlings.

The important role of P450 monooxygenase for the biosynthesis of new benzophenones from Cytospora rhizophorae.

Benzophenones are polyketides with diverse biological activities. Novel cytotoxic benzophenones cytosporaphenones A-C and cytorhizins A-D, which contain a new skeleton, were previously extracted from endophytic fungus Cytospora rhizophorae A761. However, the mechanism for the biosynthesis of these compounds remains unknown. Cytosporaphenone A was assumed to be the precursor for the biosynthesis of cytorhizins A-D. In this study, we sequenced the genome of C. rhizophorae A761 and characterized a benzoate 4-monooxygenase cytochrome P450(BAM). CRISPR/Cas9-mediated gene knockout and overexpression studies in C. rhizophorae confirmed the vital function of BAM in the biosynthesis of cytosporaphenones and cytorhizins. Overexpression of BAM also enhanced the yield of cytosporaphenone A by 1.868 folds. The in vitro function and enzymatic properties of BAM were also described. This study demonstrates the important role of BAM for the biosynthesis of cytosporaphenone A and cytorhizins and is also the first to provide approaches for the CRISPR-Cas9-mediated gene deletion and gene overexpression studies in C. rhizophoarae, thus laying a foundation for the elucidation of the biosynthetic mechanism of cytorhizins and the discovery of new benzophenones mediated by BAM.Key points• The novel bam gene encoding BAM protein in C. rhizophorae was firstly deleted using CRIPSR/Cas9 system.• The in vitro oxidation function of novel BAM protein and enzymatic properties was characterized.• The over expression of bam gene enhanced the yield of cytosporaphone A in C. rhizophorae significantly.

Characterization of novel gliotoxin biosynthesis-related genes from deep-sea-derived fungus Geosmithia pallida FS140.

Gliotoxins are epipolythiodioxopiperazine toxins produced by the filamentous fungi, which show great potential in the treatment of liver and lung cancer because of its cytotoxicity. In this study, three novel genes related to gliotoxin biosynthesis, gliT, gliM and gliK encoding thioredoxin reductase, O-methyltransferase and gamma-glutamyl cyclotransferase, respectively, from the deep-sea-derived fungus Geosmithia pallida were cloned from G. pallida and expressed in Escherichia coli. The recombinant GliT, GliM and GliK proteins were expressed and purified by Ni affinity column, which was demonstrated by SDS-PAGE and Western blot analysis. The inclusion bodies of GliT were renatured and the corresponding enzymatic properties of the two enzymes were further investigated. Using DTNB as a substrate, GliT showed the highest enzymatic activity of 11041 mU/L at pH 7.0, and the optimal reaction temperature was 40 °C. Using EGCG as a substrate, GliM showed the highest enzymatic activity of 239.19 mU/mg at pH 7.0, the optimum temperature was 35 °C. GliK from G. pallida was firstly reported to show bi-function of glutymal cyclotransferase and acetyltransfearse actvity with highest enzymatic activity of 615.5 U/mg in this study. The results suggested the important enzymatic function of GliT, GliM and GliK in the gliotoxin biosynthesis in G. pallida, which would lay a foundation for the mechanism elucidation of the gliotoxin biosynthesis in G. pallida and the exploitation of novel gliotoxin derivaties.

Effects of nitric oxide on nitrogen metabolism and the salt resistance of rice (Oryza sativa L.) seedlings with different salt tolerances.

Salt stress inhibits rice productivity seriously. Nitric oxide (NO) is an endogenous signaling molecule in plants that can improve the resistance of rice to abiotic stresses. Previous studies also showed that nitrogen metabolism is essential for rice stress-tolerance. However, the physiological and molecular mechanisms by how NO affects the nitrogen metabolisms of rice seedlings remain unclear. A hydroponic experiment with two rice varieties, Jinyuan85 (salt tolerant) and Liaojing763 (salt sensitive), was carried out to explore whether NO could alleviate the negative effects of salt stress on nitrogen metabolism and increase salt resistance of rice seedlings. The results showed that (1) the application of NO alleviated the inhibitory effects of salt stress on plant height and biomass accumulation, and increased the nitrogen content of rice leaf. (2) the accumulation of the sucrose and proline was markedly increased in salt stress after application of NO, and peroxidase activities was increased by 107% and 67.7% for Jinyuan85 and Liaojing763, respectively. (3) NO significantly increased the activities of glutamate dehydrogenase, sucrose synthase and sucrose phosphate synthase in both rice varieties under salt stress. (4) Additionally, NO regulated the expression levels of AMT, NIA and SUT genes, but these regulation effects are different with rice varieties and treatments. The results suggested that NO mainly increased the glutamate dehydrogenase and peroxidase activities and sucrose accumulation to enhance the nitrogen metabolism and antioxidative capacity, and alleviated the negative effects of salt stress on rice performance.

Pyridoxal 5'-phosphate enhances the growth and morpho-physiological characteristics of rice cultivars by mitigating the ethylene accumulation under salinity stress.

Salinity-induced ethylene accumulation caused by high production of 1-aminocyclopropane-1-carboxylic acid (ACC) hinders rice plant growth and development. Nevertheless, ACC deaminase may alleviate salt stress and high ethylene production in rice cultivars under salinity stress. Pyridoxal 5'-phosphate (PLP), an ACC deaminase co-factor, could be a useful ACC inhibitor in plants; however, it has not been studied before. In the present study, the effects of PLP on the growth and morphophysiological characteristics of rice cultivars (Jinyuan 85 (JY85) and Nipponbare (NPBA) were investigated under salinity stress (control (CK), low salinity (LS), and high salinity (HS) in hydroponic conditions. The experiment was laid out in a completely randomized design (CRD) under factorial arrangement of treatments. The results showed that, compared with no PLP, exogenous application of PLP significantly inhibited ACC and ethylene production in the roots, leaves and panicles of both cultivars under salinity, and PLP was more effective at improving the physiological characteristics of both cultivars under salinity stress. Further, root morphophysiological traits and pollen viability were triggered in the PLP treatment compared to the no-PLP treatment under various salinity levels. ACC production inhibited by PLP was useful for improving the 1000-grain weight, grain yield per plant, and total plant biomass under the CK, LS and HS treatments in both rice cultivars. These results revealed that PLP, as an ACC deaminase cofactor, is a key tool for mitigating ethylene-induced effects under salinity stress and for enhancing the agronomic and morphophysiological traits of rice under saline conditions.

DNA Stable-Isotope Probing Delineates Carbon Flows from Rice Residues into Soil Microbial Communities Depending on Fertilization.

Decomposition of crop residues in soil is mediated by microorganisms whose activities vary with fertilization. The complexity of active microorganisms and their interactions utilizing residues is impossible to disentangle without isotope applications. Thus, 13C-labeled rice residues were employed, and DNA stable-isotope probing (DNA-SIP) combined with high-throughput sequencing was applied to identify microbes active in assimilating residue carbon (C). Manure addition strongly modified microbial community compositions involved in the C flow from rice residues. Relative abundances of the bacterial genus Lysobacter and fungal genus Syncephalis were increased, but abundances of the bacterial genus Streptomyces and fungal genus Trichoderma were decreased in soils receiving mineral fertilizers plus manure (NPKM) compared to levels in soils receiving only mineral fertilizers (NPK). Microbes involved in the flow of residue C formed a more complex network in NPKM than in NPK soils because of the necessity to decompose more diverse organic compounds. The fungal species (Jugulospora rotula and Emericellopsis terricola in NPK and NPKM soils, respectively) were identified as keystone species in the network and may significantly contribute to residue C decomposition. Most of the fungal genera in NPKM soils, especially Chaetomium, Staphylotrichum, Penicillium, and Aspergillus, responded faster to residue addition than those in NPK soils. This is connected with the changes in the composition of the rice residue during degradation and with fungal adaptation (abundance and activity) to continuous manure input. Our findings provide fundamental information about the roles of key microbial groups in residue decomposition and offer important cues on manipulating the soil microbiome for residue utilization and C sequestration in soil.IMPORTANCE Identifying and understanding the active microbial communities and interactions involved in plant residue utilization are key questions to elucidate the transformation of soil organic matter (SOM) in agricultural ecosystems. Microbial community composition responds strongly to management, but little is known about specific microbial groups involved in plant residue utilization and, consequently, microbial functions under different methods of fertilization. We combined DNA stable-isotope (13C) probing and high-throughput sequencing to identify active fungal and bacterial groups degrading residues in soils after 3 years of mineral fertilization with and without manure. Manuring changed the active microbial composition and complexified microbial interactions involved in residue C flow. Most fungal genera, especially Chaetomium, Staphylotrichum, Penicillium, and Aspergillus, responded to residue addition faster in soils that historically had received manure. We generated a valuable library of microorganisms involved in plant residue utilization for future targeted research to exploit specific functions of microbial groups in organic matter utilization and C sequestration.

Disclosure of the Molecular Mechanism of Wheat Leaf Spot Disease Caused by Bipolaris sorokiniana through Comparative Transcriptome and Metabolomics Analysis.

Wheat yield is greatly reduced because of the occurrence of leaf spot diseases. Bipolaris sorokiniana is the main pathogenic fungus in leaf spot disease. In this study, B. sorokiniana from wheat leaf (W-B. sorokiniana) showed much stronger pathogenicity toward wheat than endophytic B. sorokiniana from Pogostemon cablin (P-B. sorokiniana). The transcriptomes and metabolomics of the two B. sorokiniana strains and transcriptomes of B. sorokiniana-infected wheat leaves were comparatively analyzed. In addition, the expression levels of unigenes related to pathogenicity, toxicity, and cell wall degradation were predicted and validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Results indicated that pathogenicity-related genes, especially the gene encoding loss-of-pathogenicity B (LopB) protein, cell wall-degrading enzymes (particularly glycosyl hydrolase-related genes), and killer and Ptr necrosis toxin-producing related unigenes in the W-B. sorokiniana played important roles in the pathogenicity of W-B. sorokiniana toward wheat. The down-regulation of cell wall protein, photosystem peptide, and rubisco protein suggested impairment of the phytosynthetic system and cell wall of B. sorokiniana-infected wheat. The up-regulation of hydrolase inhibitor, NAC (including NAM, ATAF1 and CUC2) transcriptional factor, and peroxidase in infected wheat tissues suggests their important roles in the defensive response of wheat to W-B. sorokiniana. This is the first report providing a comparison of the transcriptome and metabolome between the pathogenic and endophytic B. sorokiniana strains, thus providing a molecular clue for the pathogenic mechanism of W-B. sorokiniana toward wheat and wheat's defensive response mechanism to W-B. sorokiniana. Our study could offer molecular clues for controlling the hazard of leaf spot and root rot diseases in wheat, thus improving wheat yield in the future.

Marine Toxins Detection by Biosensors Based on Aptamers.

Marine toxins cause great harm to human health through seafood, therefore, it is urgent to exploit new marine toxins detection methods with the merits of high sensitivity and specificity, low detection limit, convenience, and high efficiency. Aptasensors have emerged to replace classical detection methods for marine toxins detection. The rapid development of molecular biological approaches, sequencing technology, material science, electronics and chemical science boost the preparation and application of aptasensors. Taken together, the aptamer-based biosensors would be the best candidate for detection of the marine toxins with the merits of high sensitivity and specificity, convenience, time-saving, relatively low cost, extremely low detection limit, and high throughput, which have reduced the detection limit of marine toxins from nM to fM. This article reviews the detection of marine toxins by aptamer-based biosensors, as well as the selection approach for the systematic evolution of ligands by exponential enrichment (SELEX), the aptamer sequences. Moreover, the newest aptasensors and the future prospective are also discussed, which would provide thereotical basis for the future development of marine toxins detection by aptasensors.

Long-term fertilization regimes change soil nitrification potential by impacting active autotrophic ammonia oxidizers and nitrite oxidizers as assessed by DNA stable isotope probing.

Chemoautotrophic ammonia-oxidizers and nitrite-oxidizers are responsible for a significant amount of soil nitrate production. The identity and composition of these active nitrifiers in soils under different long-term fertilization regimes remain largely under-investigated. Based on that soil nitrification potential significantly decreased in soils with chemical fertilization (CF) and increased in soils with organic fertilization (OF), a microcosm experiment with DNA stable isotope probing was further conducted to clarify the active nitrifiers. Both ammonia-oxidizing archaea (AOA) and bacteria (AOB) were found to actively respond to urea addition in soils with OF and no fertilizer (CK), whereas only AOB were detected in soils with CF. Around 98% of active AOB were Nitrosospira cluster 3a.1 in all tested soils, and more than 90% of active AOA were Nitrososphaera subcluster 1.1 in unfertilized and organically fertilized soils. Nitrite oxidation was performed only by Nitrospira-like bacteria in all soils. The relative abundances of Nitrospira lineage I and VI were 32% and 61%, respectively, in unfertilized soils, and that of Nitrospira lineage II was 97% in fertilized soils, indicating long-term fertilization shifted the composition of active Nitrospira-like bacteria in response to urea. This finding indicates that different fertilizer regimes impact the composition of active nitrifiers, thus, impacting soil nitrification potential.

An Easy and Efficient Strategy for the Enhancement of Epothilone Production Mediated by TALE-TF and CRISPR/dcas9 Systems in Sorangium cellulosum.

Epothilones are a kind of macrolides with strong cytotoxicity toward cancer cells and relatively lower side effects compared with taxol. Epothilone B derivate ixabepilone has been used for the clinical treatment of advanced breast cancer. However, the low yield of epothilones and the difficulty in the genetic manipulation of Sorangium cellulosum limited their wider application. Transcription activator-like effectors-Trancriptional factor (TALE-TF)-VP64 and clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-VP64 have been demonstrated as effective systems for the transcriptional improvement. In this study, a promoter for the epothilone biosynthesis cluster was obtained and the function has been verified. The TALE-TF-VP64 and CRISPR/dcas9-VP64 target P3 promoter were electroporated into S. cellulosum strain So ce M4, and the transcriptional levels of epothilone biosynthesis-related genes were significantly upregulated. The yield of epothilone B was improved by 2.89- and 1.53-fold by the introduction of recombinant TALE-TF-VP64-P3 and dCas9-VP64-P3 elements into So ce M4, respectively. The epothilone D yield was also improved by 1.12- and 2.18-fold in recombinant dCas9-So ce M4 and TALE-VP64 strains, respectively. The transcriptional regulation mechanism of TALE-TF-VP64 and the competition mechanism with endogenous transcriptional factor were investigated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP), demonstrating the combination of the P3 promoter and TALE-TF element and the competition between TALE-TF and endogenous transcriptional protein. This is the first report on the transcriptional regulation of the epothilone biosynthetic gene cluster in S. cellulosum using the TALE-TF and dCas9-VP64 systems, and the regulatory mechanism of the TALE-TF system for epothilone biosynthesis in S. cellulosum was also firstly revealed, thus shedding light on the metabolic engineering of S. cellulosum to improve epothilone yields substantially and promoting the application of epothilones in the biomedical industry.

Interaction of a Novel Zn2Cys6 Transcription Factor DcGliZ with Promoters in the Gliotoxin Biosynthetic Gene Cluster of the Deep-Sea-Derived Fungus Dichotomomyces cejpii.

Gliotoxin is an important epipolythiodioxopiperazine, which was biosynthesized by the gli gene cluster in Aspergillus genus. However, the regulatory mechanism of gliotoxin biosynthesis remains unclear. In this study, a novel Zn2Cys6 transcription factor DcGliZ that is responsible for the regulation of gliotoxin biosynthesis from the deep-sea-derived fungus Dichotomomyces cejpii was identified. DcGliZ was expressed in Escherichia coli and effectively purified from inclusion bodies by refolding. Using electrophoretic mobility shift assay, we demonstrated that purified DcGliZ can bind to gliG, gliM, and gliN promoter regions in the gli cluster. Furthermore, the binding kinetics and affinity of DcGliZ protein with different promoters were measured by surface plasmon resonance assays, and the results demonstrated the significant interaction of DcGliZ with the gliG, gliM, and gliN promoters. These new findings would lay the foundation for the elucidation of future gliotoxin biosynthetic regulation mechanisms in D. cejpii.

Disruption of STAT3-DNMT1 interaction by SH-I-14 induces re-expression of tumor suppressor genes and inhibits growth of triple-negative breast tumor.

Epigenetic regulation of gene expression is an emerging target to treat several human diseases including cancers. In cancers, expressions of many tumor suppressor genes are suppressed by hyper-methylation in their regulatory regions. Herein, we describe a novel carbazole SH-I-14 that decreased the level of the acetyl-STAT3 at the K685 residue. Mutation analysis revealed that SH-I-14 disrupted STAT3-DNMT1 interaction by removing acetyl group from K685 of STAT3. Finally, the inhibition of STAT3-DNMT1 interaction by SH-I-14 resulted in re-expression of tumor suppressor genes such as VHL and PDLIM4 through de-methylation of their promoter regions. In addition, SH-I-14 showed anti-proliferative effect in triple-negative breast cancer (TNBC) cell lines in vitro and anti-tumor effect in a mouse xenograft model of MDA-MB-231 tumor. Taken together, our results suggest that targeting acetyl-STAT3 (K685) provides potential therapeutic opportunity to treat a subset of human cancers.

Improving rice population productivity by reducing nitrogen rate and increasing plant density.

In terms of tillering potential, the aboveground portions of rice are significantly influenced by the nitrogen level (NL) and transplant density (TD). To obtain a suitable combination of NL and TD, five NLs (0, 90, 180, 270 and 360 kg ha-1) and two TDs [high density (HD), 32.5×104 hills ha-1; low density (LD), 25.5×104 hills ha-1] were used in the rice experiments during 2012 to 2014, in Jiangsu, China. The results showed the highest grain yield of rice obtained at HD and LD when N supply was 180 and 270 kg ha-1, respectively. That's because there are more tillers per unit area, a larger leaf biomass fraction of total aboveground biomass, a larger leaf area index (LAI) and a larger canopy photosynthesis potential (CPP) at HD. It can be concluded that, higher rice planting densities resulted in less N inputs, while more N is needed to improve single plant actual tiller ability under low density to offset the reduced planting density. When the NL was more than 180 kg ha-1, the actual tillering ability of a single plant at LD was 20% more than that at HD. Based on these results, the supply of 1 kg N can be replaced by adding approximately 1000 planting hills per hectare. Therefore, adjusting the transplant density could be an efficient method to reduce the amount of nitrogen fertilizer and increase the nitrogen fertilizer use efficiency, which is very conducive to the sustainable development of agriculture.

Development of a novel near-infrared fluorescent theranostic combretastain A-4 analogue, YK-5-252, to target triple negative breast cancer.

The treatment of triple negative breast cancer (TNBC) is a significant challenge to cancer research. The lack of hormone receptors limits the treatment options available to patients with this diagnosis, forcing them to endure prolonged radiation and chemotherapy. Anti-angiogenesis is a chemotherapeutic strategy that targets the vasculature of tumors. Combretastatin A-4 (CA-4) is a well-known vasculature-disrupting agent, which has been shown to effectively kill a variety of cancers through inhibition of tubulin polymerization. Due to its toxicity, small molecule analogues of CA-4 have been sought out. We have designed a novel dual action CA-4 prodrug, YK-5-252, which releases the drug through a disulfide bond cleavage mechanism and contains a near-infrared (NIR) fluorophore, which allows fluorescence monitoring of cleavage. This disulfide linkage causes CA-4 to become effective only when released by glutathione (GSH) reducing the toxicity of the drug while simultaneously releasing the NIR fluorophore. Therefore the prodrug, YK-5-252, represents a novel CA-4 analogue which has reduced toxicity and can be used for theranostics imaging.

Insulin deficiency induces rat renal mesangial cell dysfunction via activation of IGF-1/IGF-1R pathway.

AIM: Diabetic nephropathy is one of the major complications of diabetes and the major cause of end-stage renal disease. In this study we investigated the insulin deficiency (ID) induced changes in renal mesangial cells (MCs) and in the kidney of STZ-induced diabetic rats. METHODS: Cultured rat renal MCs were incubated in ID media. Cell proliferation was analyzed using BrdU incorporation assay. The expression of insulin receptor (IR), insulin-like growth factor-1 receptor (IGF-1R), phosphorylated IGF-1R, fibronectin, and collagen IV was determined with Western blot analysis. STZ-induced diabetic rats were treated with an IGF-1R antagonist picropodophyllin (PPP, 20 mg·kg(-1)·d(-1), po) for 8 weeks. After the rats were euthanized, plasma and kidneys were collected. IGF-1 levels in renal cortex were measured with RT-PCR or ELISA. The morphological changes in the kidneys were also examined. RESULTS: Incubation in ID media significantly increased cell proliferation, the synthesis of fibronectin and collagen IV, and the expression of IGF-1 and IGF-1R and phosphorylated IGF-1R in renal MCs. Pretreatment of the cells with PPP (50 nmol/L) blocked ID-induced increases in cell proliferation and the synthesis of fibronectin and collagen IV; knockdown of IGF-1R showed a similar effect as PPP did. In contrast, treatment of the cells with IGF-1 (50 ng/mL) exacerbated ID-induced increases in cell proliferation. In the kidneys of diabetic rats, the expression of IGF-1, IGF-1R and phosphorylated IGF-1R were significantly elevated. Treatment of diabetic rats with PPP did not lower the blood glucose levels, but significantly suppressed the expression of TGF-ß, fibronectin and collagen IV in the kidneys, the plasma levels of urinary nitrogen and creatinine, and the urinary protein excretion. CONCLUSION: Insulin deficiency increases the expression of IGF-1 and IGF-1R in renal MCs and the kidney of diabetic rats, which contributes to the development of diabetic nephropathy.

Total Synthesis of 7-Hydroxymurrayazolinine, Murrayamine D, and Mahanine via m-Nitro Group Activated Pyran Annulation.

The facile total synthesis of the natural product (±)-mahanine was obtained in eight steps with an overall 52% yield from readily accessible known nitrophenol derivative 6. After a one-step, acid-catalyzed annulation, two additional natural products were formed including 7-hydroxymurrayazolinine, representing its first reported total synthesis. In the whole process, the introduction of the m-nitro group significantly enhanced the key pyran annulation reaction through inductive effects.

Preclinical studies of the potent and selective nicotinic α4ß2 receptor ligand VMY-2-95.

The discovery and development of small molecules that antagonize neuronal nicotinic acetylcholine receptors may provide new ligands for evaluation in models of depression or addiction. We discovered a small molecule, VMY-2-95, a nAChR ligand with picomolar affinity and high selectivity for α4ß2 receptors. In this study, we investigated its preclinical profile in regards to solubility, lipophilicity, metabolic stability, intestinal permeability, bioavailability, and drug delivery to the rat brain. Metabolic stability of VMY-2-95·2HCl was monitored on human liver microsomes, and specific activity of VMY-2-95·2HCl on substrate metabolism by CYP1A2, 2C9, 2C19, 2D6, and 3A4 was tested in a high-throughput manner. The intestinal transport of VMY-2-95·2HCl was studied through Caco-2 cell monolayer permeability. VMY-2-95·2HCl was soluble in water and chemically stable, and the apparent partition coefficient was 0.682. VMY-2-95·2HCl showed significant inhibition of CYP2C9 and 2C19, but weak or no effect on 1A2, 2D6, and 3A4. The Caco-2 cell model studies revealed that VMY-2-95·2HCl was highly permeable with efflux ratio of 1.11. VMY-2-95·2HCl achieved a maximum serum concentration of 0.56 mg/mL at 0.9 h and was orally available with a half-life of ∼9 h. Furthermore, VMY-2-95·2HCl was detected in the rat brain after 3 mg/kg oral administration and achieved a maximal brain tissue concentration of 2.3 µg/g within 60 min. Overall, the results demonstrate that VMY-2-95·2HCl has good drug like properties and can penetrate the blood-brain barrier with oral administration.

A small molecule inhibitor of ETV1, YK-4-279, prevents prostate cancer growth and metastasis in a mouse xenograft model.

BACKGROUND: The erythroblastosis virus E26 transforming sequences (ETS) family of transcription factors consists of a highly conserved group of genes that play important roles in cellular proliferation, differentiation, migration and invasion. Chromosomal translocations fusing ETS factors to promoters of androgen responsive genes have been found in prostate cancers, including the most clinically aggressive forms. ERG and ETV1 are the most commonly translocated ETS proteins. Over-expression of these proteins in prostate cancer cells results in a more invasive phenotype. Inhibition of ETS activity by small molecule inhibitors may provide a novel method for the treatment of prostate cancer. METHODS AND FINDINGS: We recently demonstrated that the small molecule YK-4-279 inhibits biological activity of ETV1 in fusion-positive prostate cancer cells leading to decreased motility and invasion in-vitro. Here, we present data from an in-vivo mouse xenograft model. SCID-beige mice were subcutaneously implanted with fusion-positive LNCaP-luc-M6 and fusion-negative PC-3M-luc-C6 tumors. Animals were treated with YK-4-279, and its effects on primary tumor growth and lung metastasis were evaluated. YK-4-279 treatment resulted in decreased growth of the primary tumor only in LNCaP-luc-M6 cohort. When primary tumors were grown to comparable sizes, YK-4-279 inhibited tumor metastasis to the lungs. Expression of ETV1 target genes MMP7, FKBP10 and GLYATL2 were reduced in YK-4-279 treated animals. ETS fusion-negative PC-3M-luc-C6 xenografts were unresponsive to the compound. Furthermore, YK-4-279 is a chiral molecule that exists as a racemic mixture of R and S enantiomers. We established that (S)-YK-4-279 is the active enantiomer in prostate cancer cells. CONCLUSION: Our results demonstrate that YK-4-279 is a potent inhibitor of ETV1 and inhibits both the primary tumor growth and metastasis of fusion positive prostate cancer xenografts. Therefore, YK-4-279 or similar compounds may be evaluated as a potential therapeutic tool for treatment of human prostate cancer at different stages.