Design,synthesis, and biological evaluation of novel substituted thiourea derivatives as potential anticancer agents for NSCLC by blocking K-Ras protein-effectors interactions

Abstract

Mutation of the proto-oncogene K-Ras is one of the most common molecular mechanisms in non-small cell lung cancer. Many drugs for treating lung cancer have been developed, however, due to clinical observed K-Ras mutations, corresponding chemotherapy and targeted therapy for such mutation are not efficient enough. In this study, on the basis of the crystal structure of K-Ras, 21 analogues (TKR01–TKR21) containing urea or thiourea were rationally designed, which can effectively inhibit the lung cancer cell A549 growth. The designing of these compounds was based on the structure of K-Ras protein, and the related groups were replaced by bioisosteres to improve the affinity and selectivity. Biological testing revealed that compound TKR15 could significantly inhibit the proliferation of A549 cell with IC₅₀ of 0.21?µM. Docking analysis showed that the TKR15 can effectively bind to the hydrophobic cavity and form a hydrogen bond with the Glu37. In addition, through flow apoptosis assay and immunofluorescence staining assay, it confirmed that this compound can inhibit A549 cell proliferation with the mechanism of blocking K-Ras^G12V protein and effector proteins interactions through the apoptotic pathway. In conclusion, our studies in finding novel potent compound (TKR15) with confirmed mechanism showed great potential for further optimisation and other medicinal chemistry relevant studies.     

CARAGANA FABR. PROMOTES REVEGETATION AND SOIL REHABILITATION IN SALINE-ALKALI WASTELAND

To determine how plantations of Caragana microphylla shrubs affect saline-alkali soil amelioration and revegetation, we investigated the vegetation and sampled soils from saline-alkali wasteland (SAW), perennial Caragana forestland (PCF), Caragana forest after fire disturbance (CFF). Results showed that with the development of Caragana Fabr., highly dominant species of Poaceae family, including Elymus dahuricus, Thermopsis lanceolata, Stipa tianschanica, died out in PCF. Moreover, Papilionaceae family, including Lespedeza indica, Oxytropis psammocharis, and Astragalus scaberrimus, was established both in PCF and CFF. Phytoremediation of saline-alkali wasteland (SAW) was achieved by plantation, resulting in the reduced soil pH, sodium adsorption ratio, exchangeable sodium percentage, salinity, and Na⁺ concentration around Caragana shrubs. Greater amounts of soil organic, total nitrogen, ammonium nitrogen, available phosphorus, and available potassium were observed in PCF topsoil than in SAW topsoil. The concentration of mineralized N in PCF soil was significantly lower than that in SAW soil at all sampled depths, indicating that Caragana shrubs were just using N and therefore less measured in soils. Fire disturbance resulted in decreased soil pH and salinity, but increased organic content, total nitrogen, and ammonium nitrogen. The improved soil parameters and self-recovery of shrubs indicated that Caragana shrubs were well established after burning event.     

MicroRNA-92a Upholds Bmp Signaling by Targeting noggin3 during Pharyngeal Cartilage Formation

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Highlights? mir92a inactivation leads to loss of pharyngeal cartilages in zebrafish embryos ? mir92a is required for chondrogenic progenitor proliferation and survival ? Bmp signaling is positively regulated by mir92a in the pharyngeal region ? nog3 mRNA is degraded by mir92a to maintain Bmp activity     

Identification and Validation of Urinary Metabolite Biomarkers for Major Depressive Disorder

Major depressive disorder (MDD) is a widespread and debilitating mental disorder. However, there are no biomarkers available to aid in the diagnosis of this disorder. In this study, a nuclear magnetic resonance spectroscopy–based metabonomic approach was employed to profile urine samples from 82 first-episode drug-naïve depressed subjects and 82 healthy controls (the training set) in order to identify urinary metabolite biomarkers for MDD. Then, 44 unselected depressed subjects and 52 healthy controls (the test set) were used to independently validate the diagnostic generalizability of these biomarkers. A panel of five urinary metabolite biomarkers—malonate, formate, N-methylnicotinamide, m-hydroxyphenylacetate, and alanine—was identified. This panel was capable of distinguishing depressed subjects from healthy controls with an area under the receiver operating characteristic curve (AUC) of 0.81 in the training set. Moreover, this panel could classify blinded samples from the test set with an AUC of 0.89. These findings demonstrate that this urinary metabolite biomarker panel can aid in the future development of a urine-based diagnostic test for MDD.Major depressive disorder (MDD)¹ is a debilitating mental disorder affecting up to 15% of the general population and accounting for 12.3% of the global burden of disease (1, 2). Currently, the diagnosis of MDD still relies on the subjective identification of symptom clusters rather than empirical laboratory tests. The current diagnostic modality results in a considerable error rate (3), as the clinical presentation of MDD is highly heterogeneous and the current symptom-based method is not capable of adequately characterizing this heterogeneity (4). An approach that can be used to circumvent these limitations is to identify disease biomarkers to support objective diagnostic laboratory tests for MDD.Metabonomics, which can measure the small molecules in given biosamples such as plasma and urine without bias (5), has been extensively used to characterize the metabolic changes of diseases and thus facilitate the identification of novel disease-specific signatures as putative biomarkers (6–10). Nuclear magnetic resonance (NMR) spectroscopy–based metabonomic approaches characterized by sensitive, high-throughput molecular screening have been employed previously in identifying novel biomarkers for a variety of neuropsychiatric disorders, including stroke, bipolar disorder, and schizophrenia (11–13).Specifically with regard to MDD, several animal studies have already characterized the metabolic changes in the blood and urine (14–19). These studies provide valuable clues as to the pathophysiological mechanism of MDD. However, no study has been designed with the aim of diagnosing this disease. Recently, using an NMR-based metabonomic approach, this research group identified a unique plasma metabolic signature that enables the discrimination of MDD from healthy controls with both high sensitivity and specificity (20). These findings motivated further study on urinary diagnostic metabolite biomarkers for MDD, which would be more valuable from a clinical applicability standpoint, as urine can be more non-invasively collected. Moreover, previous studies have also demonstrated the feasibility of identifying diagnostic metabolite biomarkers of psychiatric disorders in the urine. For example, using an NMR-based metabonomics approach, Yap et al. (21) identified a unique urinary metabolite signature that clearly discriminated autism patients from healthy controls. As systemic metabolic disturbances have been observed in the urine of a depressed animal model, it is likely that diagnostic metabolite markers for MDD can be detected in human urine.Therefore, in this study, NMR spectroscopy combined with multivariate pattern recognition techniques were used to profile 82 first-episode drug-naïve MDD subjects and 82 healthy controls (the training set) in order to identify potential metabolite biomarkers for MDD. Furthermore, 44 unselected MDD subjects and 52 healthy controls (the test set) were employed to independently validate the diagnostic performance of these urinary metabolite biomarkers.     

Molecular characterization of the sans fille gene in Antheraea pernyi: A highly conserved gene during the evolution of animals

Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B″, and plays an important role in sex determination in Drosophila melanogaster. In this study, the snf gene from Antheraea pernyi (Lepidoptera: Saturniidae), an economically important insect, was isolated and characterized. The obtained 925 bp cDNA sequence contains an open reading frame of 669 bp encoding a polypeptide of 222 amino acids, showing 78% sequence identity to that from D. melanogaster. A database search revealed that SNF protein homologs are present in many animals, including invertebrates and vertebrates, with more than 70% amino acid sequence identities, suggesting that they were highly conserved during the evolution of animals. Phylogenetic analysis revealed that A. pernyi SNF was closely related to Bombyx mori SNF. Quantitative real-time PCR (qRT-PCR) analysis showed that the A. pernyi snf gene was transcribed during five larval developmental stages, and in six tested tissues (ovaries, testes, silk glands, fat body, integument, and hemolymph), with the most abundance determined in the gonads (ovaries or testes). Investigation of expression changes throughout embryonic development indicated that A. pernyi snf mRNA was expressed at a low level from days 0 to 4, and reached a maximum level at day 10, but decreased to a low level before hatching. These results suggest that the product of the snf gene may play important roles in the development of A. pernyi.     

Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy

A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans was cloned and expressed in Escherichia coli BL21 (DE3). A downstream gene adjacent to the β-subunit was necessary for the functional expression of the recombinant NHase. The structural gene order of the Co-type NHase was α-subunit beyond β-subunit, different from the order typically reported for Co-type NHase genes. The NHase exhibited adequate thermal stability, with a half-life of 1.5 h at 50 °C. The NHase efficiently hydrated 3-cyanopyridine to produce nicotinamide. In a 1-L reaction mixture, 3.6 mol of 3-cyanopyridine was completely converted to nicotinamide in four feedings, exhibiting a productivity of 187 g nicotinamide/g dry cell weight/h. An industrial auto-induction medium was applied to produce the recombinant NHase in 10-L fermenter. A glycerol-limited feeding method was performed, and a final activity of 2170 U/mL culture was achieved. These results suggested that the recombinant NHase was efficiently cloned and produced in E. coli.     

Efficient secretion of lipase r27RCL in Pichia pastoris by enhancing the disulfide bond formation pathway in the endoplasmic reticulum

The lipase r27RCL from Rhizopus chinensis CCTCC M201021 was heterologously expressed in Pichia pastoris GS115 by simultaneous co-expression with two secretion factors ERO1p and PDI involved in the endoplasmic reticulum (ER). Compared to the expression of the lipase alone (12,500 U/ml), co-expression with these two proteins resulted in the production of larger total quantities of enzymes. The largest increase was seen when the combined ERO1p/PDI system was co-expressed, resulting in approximately 30 % higher enzyme yields (16,200 U/ml) than in the absence of co-expressed secretion factors. The extracellular protein concentration of the recombinant strain Co XY RCL-5 reached 9.39 g/l in the 7-l fermentor. Simultaneously, the fermentation time was also shortened by about 8 h compared to that of the control. The substrate-specific consumption rate (Qs) and the product-specific production rate (Qp) were both investigated in this research. In conclusion, the space–time yield was improved by co-expression with ERO1p and PDI. This is a potential strategy for high level expression of other heterologous proteins in P. pastoris.     

Caloric restriction augments radiation efficacy in breast cancer

Dietary modification such as caloric restriction (CR) has been shown to decrease tumor initiation and progression. We sought to determine if nutrient restriction could be used as a novel therapeutic intervention to enhance cytotoxic therapies such as radiation (IR) and alter the molecular profile of triple-negative breast cancer (TNBC), which displays a poor prognosis. In two murine models of TNBC, significant tumor regression is noted with IR or diet modification, and a greater regression is observed combining diet modification with IR. Two methods of diet modification were compared, and it was found that a daily 30% reduction in total calories provided more significant tumor regression than alternate day feeding. At the molecular level, tumors treated with CR and IR showed less proliferation and more apoptosis. cDNA array analysis demonstrated the IGF-1R pathway plays a key role in achieving this physiologic response, and multiple members of the IGF-1R pathway including IGF-1R, IRS, PIK3ca and mTOR were found to be downregulated. The innovative use of CR as a novel therapeutic option has the potential to change the biology of tumors and enhance the opportunity for clinical benefit in the treatment of patients with TNBC.     

Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

Background

Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism.

Methodology/Principal Findings

Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats.

Conclusions/Significance

In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future.     

Different Distribution of HIV-1 Subtype and Drug Resistance Were Found among Treatment Na?ve Individuals in Henan,Guangxi, and Yunnan Province of China

Yunnan, Guangxi and Henan are the provinces with the most severe HIV epidemic in China, which were also among the first group of areas providing free ART in 2004. However, little comprehensive data are available on prevalence of HIV subtype and baseline drug resistance in drug-naïve populations. In this study, 1746 treatment-naïve HIV-positive individuals were randomly selected from new-reported cases in Henan, Guangxi and Yunnan. Among of them, subtypes and drug resistance of 1159 strains were determined by amplifying and sequencing full-length pol genes. Significantly different distributions of HIV subtypes prevalent in three provinces were identified (P<0.01). CRF08_BC was found dominant in Yunnan (59.8%), while CRF01_AE was dominant in Guangxi (77.3%) and subtype B was dominant in Henan province (93.9%). The total prevalence of drug resistance was 7.1%. The highest prevalence of HIV drug resistance was found in Henan (12.2%), followed by Yunnan (5.6%) and Guangxi (3.3%). The results of this study suggest that genetic drug-resistance should be tested before initiation of ART in China, especially in Henan province. Furthermore, the prevalence of HIV drug resistant strains should be considered separately in different areas in China before the change of different free ART regimens.