Design,synthesis, cytotoxicity,HuTopoIIα inhibitory activity and molecular docking studies of pyrazole derivatives as potential anticancer agents

In an attempt to find potential anticancer agents, a series of novel ethyl 4-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylates 5a-i and 5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamides 6a-i were designed, synthesized and evaluated for their topoisomerase IIα inhibitory activity and in vitro cytotoxicity against a panel of cancerous cell lines (MCF-7, NCI-H460, HeLa) and a normal cell line (HEK-293T). Molecular docking studies of all the synthesized compounds into the binding site of topoisomerase IIα protein (PDB ID: 1ZXM) were performed to gain a comprehensive understanding into plausible binding modes. These compounds were also screened for in silico drug-likeliness properties on the basis of the absorption, distribution, metabolism and excretion (ADME) prediction. Among all the synthesized compounds, analogue 5d showed superior cytotoxicity with an IC₅₀ value of 7.01 ± 0.60 μM for HeLa, 8.55 ± 0.35 μM for NCI-H460 and 14.31 ± 0.90 for MCF-7 cancer cell lines. Further, compound 5d showed 70.82% inhibition of topoisomerase IIα at a concentration of 100 μM with maximum docking score of −8.24. Results of ADME prediction revealed that most of these compounds showed in silico drug-likeliness properties within the ideal range.     

Tet-on inducible system combined with in ovo electroporation dissects multiple roles of genes in somitogenesis of chicken embryos

The in ovo electroporation technique in chicken embryos has enabled investigators to uncover the functions of numerous developmental genes. In this technique, the ubiquitous promoter, CAGGS (CMV base), has often been used for overexpression experiments. However, if a given gene plays a role in multiple steps of development and if overexpression of this gene causes fatal consequences at the time of electroporation, its roles in later steps of development would be overlooked. Thus, a technique with which expression of an electroporated DNA can be controlled in a stage-specific manner needs to be formulated. Here we show for the first time that the tetracycline-controlled expression method, "tet-on" and "tet-off", works efficiently to regulate gene expression in electroporated chicken embryos. We demonstrate that the onset or termination of expression of an electroporated DNA can be precisely controlled by timing the administration of tetracycline into an egg. Furthermore, with this technique we have revealed previously unknown roles of RhoA, cMeso-1 and Pax2 in early somitogenesis. In particular, cMeso-1 appears to be involved in cell condensation of a newly forming somite by regulating Pax2 and NCAM expression. Thus, the novel molecular technique in chickens proposed in this study provides a useful tool to investigate stage-specific roles of developmental genes.     

The appendage role of insect disco genes and possible implications on the evolution of the maggot larval form

Though initially identified as necessary for neural migration, Disconnected and its partially redundant paralog, Disco-related, are required for proper head segment identity during Drosophila embryogenesis. Here, we present evidence that these genes are also required for proper ventral appendage development during development of the adult fly, where they specify medial to distal appendage development. Cells lacking the disco genes cannot contribute to the medial and distal portions of ventral appendages. Further, ectopic disco transforms dorsal appendages toward ventral fates; in wing discs, the medial and distal leg development pathways are activated. Interestingly, this appendage role is conserved in the red flour beetle, Tribolium (where legs develop during embryogenesis), yet in the beetle we found no evidence for a head segmentation role. The lack of an embryonic head specification role in Tribolium could be interpreted as a loss of the head segmentation function in Tribolium or gain of this function during evolution of flies. However, we suggest an alternative explanation. We propose that the disco genes always function as appendage factors, but their appendage nature is masked during Drosophila embryogenesis due to the reduction of limb fields in the maggot style Drosophila larva.     

Overexpression of SR proteins and splice variants modulates chondrogenesis

Fibronectin alternative exon EIIIA is largely included in undifferentiated mesenchymal cells of the developing limb bud, whereas the exon is excluded in differentiated chondrocytes. Inclusion of exon EIIIA in chondrocytic cells is increased by overexpression of SRp40, and, to a lesser extent, SRp75, but not SRp55. RT-PCR analysis using real-time PCR revealed that the levels of the mRNAs for these three proteins did not vary significantly in chick chondrocytes versus mesenchymal cells of the developing limb bud. However, a variant spliced form of SRp40, termed, SRp40LF, is detected preferentially in chondrocytes and in chondrifying mesenchymal cells. Forced overexpression of SRp40 or SRp75, but not SRp55, enhanced chondrogenic differentiation of chick limb mesenchymal cells in a high-density micromass assay. Overexpression of SRp40LF, which produces a truncated form of SRp40, also was strongly pro-chondrogenic. In a HeLa cell-based assay, SRp40LF fails to substitute for SRp40 in mediating an increase in exon EIIIA inclusion, suggesting that the latter event is not essential for the pro-chondrogenic effect. These results demonstrate the ability of these highly conserved splicing factors to modulate chondrogenesis and are consistent with earlier results that implicated exon EIIIA-containing isoforms of fibronectin in formation of chondrogenic condensations.     

Synthetic biology and metabolic engineering of actinomycetes for natural product discovery

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications.     

传染性法氏囊病病毒中国强毒株A节段cDNA基因的克隆和序列分析

以来自哈尔滨传染性法氏囊病病毒（ＩＢＤＶ） 强毒株（Ｈａｒｂｉｎ 毒株,Ｈ） 的基因组ＲＮＡ为模板,用反转录聚合酶链反应（ＲＴ－ ＰＣＲ） 的方法得到了其Ａ 节段的全长ｃＤＮＡ 片段,分５＇端（１ ６５９ｂｐ） 和３＇端（１ ４４４ｂｐ） 上下两段分别克隆到ｐＧＥＭＢ○Ｒ － Ｔ 载体上,测定了其核苷酸顺序,在长为３ １０１ ｂｐ 中含有两个阅读框ＯＲＦＡ１ 和ＯＲＦＡ２ ,分别编码１ ０１２ 个氨基酸的前体蛋白（ＶＰ２ － ４ －３） 和１４５ 个氨基酸的ＶＰ５,ＯＲＦＡ１ 和ＯＲＦＡ２ 有部分的重叠。将核苷酸序列及推测出的氨基酸序列与已报道的ＩＢＤＶ 血清Ⅰ型和Ⅱ型毒株的相应序列进行了比较,结果表明：Ｈ 毒株与其它血清Ⅰ型毒株之间,在核苷酸水平上存在２５ｂｐ － ２６７ｂｐ 的差异;在氨基酸水平上存在１７ ～４０ 个氨基酸的差异。在ＶＰ２ － ４ － ３ 内比较显示,Ｈ 毒株与Ｐ２ 、Ｃｕ－ １ 之间氨基酸的差异最小为１ ．７％ ,Ｈ 毒株与ＵＫ６６１ 之间氨基酸的差异最大为３ ．９ ％ 。变异主要发生在ＶＰ２ 的可变区（２０６ － ３５０ 位氨基酸） ,在Ｈ 毒株所特有的１２ 个氨基酸当中,该区就占５ 个,代表１ ．７６ ％ 的变异。ＶＰ４、ＶＰ３ 和ＶＰ５区各有     

Progression into and out of mitosis

Progression through mitosis is controlled by cyclin-dependent kinases, which drive cells into metaphase, and by the anaphase-promoting complex/cyclosome, a ubiquitin ligase that triggers sister chromatid separation and exit from mitosis. Recent work has shown how the mutual regulation between cyclin-dependent kinases and the anaphase-promoting complex/cyclosome ensures that cell-cycle events occur in the right order. The analysis of complexes required for sister chromatid cohesion and chromosome condensation has revealed how cyclin-dependent kinases and the anaphase-promoting complex/cyclosome control the behaviour of chromosomes.     

Synthesis of amyloid β-peptides in solution employing chloroform-phenol mixed solvent for facile segment condensation of sparingly soluble protected peptides

Segment condensation reaction of sparingly soluble protected peptides proceeded smoothly in CHCl₃-phenol mixed solvent without danger of epimerization or of significant ester formationwith the carboxyl component when 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was employedin the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine(HOOBt). The optimal conditions for enhancement of peptide coupling mediated by EDC/HOOBt in CHCl₃-phenol were determined and successfully applied to the synthesis of amyloid -peptide (1-42), (1-43) and [Pyr³]-(3-42). These peptides of high homogeneity were used to examine the relation between structure and amyloidogenesis by means of CD spectra andfluorimetric assay.     

Dynamics of chromosome compaction during mitosis

We have quantitatively studied the space-time dynamics of mitotic chromosome compaction in cultured amphibian cells. After collecting digital phase-contrast images we have done digital image analysis to study spatial correlations in density. We find a characteristic distance at which the strongest correlations occur, which provides a quantitative measure of the size of patches of dense chromatin during interphase and early prophase. Later in mitosis, this length corresponds to the thickness of prophase and metaphase chromosomes. We find that during interphase strong correlations exist at a few-micrometer length; during prophase this correlation length progressively drops as the chromosomes are compacted. Our data are explained by a model based on assembly of chromatin loops onto already fiberlike interphase chromosomes. To test this model we have microinjected cobalt hexamine trichloride into interphase nuclei and have observed the rapid condensation of the interphase chromatin into thick fibers with a spacing similar to the native-state interphase correlation length determined from our image analysis.