An improved Trizol method for extracting total RNA from Eleutherococcus senticosus (Rupr. & Maxim.) Maxim leaves.

INTRODUCTION: High-quality nucleic acids are the basis for molecular biology experiments. Traditional RNA extraction methods are not suitable for Eleutherococcus senticosus Maxim. OBJECTIVE: To find a suitable method to improve the quality of RNA extracted, we modified the RNA extraction methods of Trizol. METHODOLOGY: Based on the conventional Trizol method, the modified Trizol method 1 and modified Trizol method 2 were used as the control for extraction of RNA from E. senticosus Maxim leaves. The modified Trizol method 1 added ß-mercaptoethanol on the conventional Trizol method. After RNA was dissolved, a mixed solution of phenol, chloroform, and isoamyl alcohol was added to denature protein and inhibit the degradation of RNA. The modified Trizol method 2 adds PVPP to grind on the basis of modified Trizol method 1, so as to better remove phenols from leaves, and eliminates the step of incubation at -20°C to reduce extraction time and RNA degradation. Chloroform, CTAB, and CH3COONa were used instead of a phenol, chloroform, and isoamyl alcohol mixed solution to ensure complete separation of nucleic acid from plant tissues and to obtain high-purity RNA. RESULTS: The research results showed that the quality of RNA extracted by conventional Trizol method, modified Trizol method 1, was incomplete, accompanied with different degrees of contamination of polysaccharides, polyphenols, and DNA. The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. The ratio of A260/A280 was in the range of 1.8-2.0, and the yield of RNA was the highest, which was 1.68 and 1.15 times compared with that by conventional Trizol method and modified Trizol method 1 extraction, respectively. The reverse transcription cDNA was further tested through PCR with the specific primers. The amplified fragments are displayed in clear and bright bands in accordance with the expected size. CONCLUSION: The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. High-quality RNA has more advantages in molecular biology study of E. senticosus Maxim.

Differential Transcriptomic Signatures of Small Airway Cell Cultures Derived from IPF and COVID-19-Induced Exacerbation of Interstitial Lung Disease.

Idiopathic pulmonary fibrosis (IPF) is a pathological condition wherein lung injury precipitates the deposition of scar tissue, ultimately leading to a decline in pulmonary function. Existing research indicates a notable exacerbation in the clinical prognosis of IPF patients following infection with COVID-19. This investigation employed bulk RNA-sequencing methodologies to describe the transcriptomic profiles of small airway cell cultures derived from IPF and post-COVID fibrosis patients. Differential gene expression analysis unveiled heightened activation of pathways associated with microtubule assembly and interferon signaling in IPF cell cultures. Conversely, post-COVID fibrosis cell cultures exhibited distinctive characteristics, including the upregulation of pathways linked to extracellular matrix remodeling, immune system response, and TGF-ß1 signaling. Notably, BMP signaling levels were elevated in cell cultures derived from IPF patients compared to non-IPF control and post-COVID fibrosis samples. These findings underscore the molecular distinctions between IPF and post-COVID fibrosis, particularly in the context of signaling pathways associated with each condition. A better understanding of the underlying molecular mechanisms holds the promise of identifying potential therapeutic targets for future interventions in these diseases.

Supplementary UV-B Radiation Effects on Photosynthetic Characteristics and Important Secondary Metabolites in Eucommia ulmoides Leaves.

To explore the effects of ultraviolet light supplementation on the photosynthetic characteristics and content of secondary metabolites in the leaves of Eucommia ulmoides Oliver (E. ulmoides), the effects of supplementary UV-B (sUV-B) radiation on the medicinally active components of E. ulmoides were comprehensively evaluated. In our study, we selected leaves of five-year-old E. ulmoides seedlings as experimental materials and studied the effect of supplemental ultraviolet-B (sUV-B) radiation on growth, photosynthetic parameters, photosynthetic pigments, fluorescence parameters, and secondary metabolites of E. ulmoides using multivariate analysis. The results showed that the leaf area and the number of branches increased after sUV-B radiation, which indicated that sUV-B radiation was beneficial to the growth of E. ulmoides. The contents of chlorophyll a and chlorophyll b increased by 2.25% and 4.25%, respectively; the net photosynthetic rate increased by 5.17%; the transpiration rate decreased by 35.32%; the actual photosynthetic efficiency increased by 10.64%; the content of the secondary metabolite genipin increased by 12.9%; and the content of chlorogenic acid increased by 75.03%. To identify the genes that may be related to the effects of sUV-B radiation on the growth and development of E. ulmoides leaves and important secondary metabolites, six cDNA libraries were prepared from natural sunlight radiation and sUV-B radiation in E. ulmoides leaves. Comparative analysis of both transcriptome databases revealed a total of 3698 differential expression genes (DEGs), including 1826 up-regulated and 1872 down-regulated genes. According to the KOG database, the up-regulated unigenes were mainly involved in signal transduction mechanisms [T] and cell wall/membrane biogenesis [M]. It is also involved in plant hormone signal transduction and phenylpropanoid biosynthesis metabolic pathways by the KEGG pathway, which might further affect the physiological indices and the content of chlorogenic acid, a secondary metabolite of E. ulmoides. Furthermore, 10 candidate unigenes were randomly selected to examine gene expression using qRT-PCR, and the six libraries exhibited differential expression and were identical to those obtained by sequencing. Thus, the data in this study were helpful in clarifying the reasons for leaf growth after sUV-B radiation. And it was beneficial to improve the active components and utilization rate of E. ulmoides after sUV-B radiation.

Discovery and optimization of 2,4-diaminoquinazoline derivatives as a new class of potent dengue virus inhibitors.

The results of a high-throughput screening assay using the DENV-2 replicon showed that the 2,4-diaminoquinazoline derivative 4a has a high dengue virus inhibitory activity (EC(50) = 0.15 µM). A series of 2,4-diaminoquinazoline derivatives based on 4a as a lead compound were synthesized and subjected to structure-antidengue activity relationship studies. Among the series of 2,4-diaminoquinazoline derivative probed, 4o was observed to display both the highest antiviral potency (EC(50) = 2.8 nM, SI > 1000) and an excellent pharmacokinetic profile.

Synthesis and biological evaluation of 2,4,5-substituted pyrimidines as a new class of tubulin polymerization inhibitors.

Members of a series of 2,4,5-substituted pyrimidine derivatives were synthesized, and their interactions with tubulin and their antiproliferative activities against the human hepatocellular carcinoma cells of liver (BEL-7402) were evaluated. One member of this family, the indole-pyrimidine 4k, having an indole-aryl-substituted aminopyrimidine structure, was observed to be an excellent inhibitor of tubulin polymerization (IC(50) = 0.79 µM) and to display significantly high antiproliferative activities against several cancer cell lines with IC(50) values ranging from 16 to 62 nM. This substance displayed a high propensity to arrests cells at the G(2)/M phase of the cell cycle (EC(50) = 20 nM). In addition, 4k was found to competitively inhibit colchicine binding to tubulin, indicating that it binds to the colchicine-binding site of tubulin. The observations made in this investigation demonstrate that 2,4,5-substituted pyrimidines represent a new class of tubulin polymerization inhibitors with significant antiproliferative activity.

Three-component one-pot approach to synthesize benzopyrano[4,3-d]pyrimidines.

A novel benzopyrano[4,3-d]pyrimidine scaffold was generated via a three-component one-pot reaction from iodochromone, alkyne, and an amidine through a Sonogashira coupling, condensation, and cycloaddition. This combinatorial synthetic approach provides an efficient, easy construction of a diversified heterocyclic compounds library.

Responses in the morphology, physiology and biochemistry of Taxus chinensis var. mairei grown under supplementary UV-B radiation.

The effects of supplemental UV-B radiation on Taxus chinensis var. mairei were studied. Leaf traits, gas exchange parameters and the concentrations of photosynthetic pigments, cellular defense system products, secondary metabolites and ultrastructure were determined. UV-B radiation significantly decreased leaf area (p<0.05). Leaf number, secondary branch number, leaf weight per plant and leaf moisture all increased dramatically (p<0.05). Neither the leaf weight nor the specific leaf weight (SLW) exhibited significant differences between ambient and enhanced UV-B radiation. Gas exchange parameters were all dramatically reduced by enhanced UV-B radiation (p<0.05). The contents of chlorophyll and the chlorophyll a/b ratio were not distinctly affected by UV-B radiation, while carotenoids content significantly decreased (p<0.05). Supplemental UV-B treatment induced significant flavonoid accumulation (p<0.05), which was able to protect plant from radiation damage. Meanwhile, the appendage content, abaxial stomatal density, papilla density and particulate matter content in substomatic chambers increased noticeably by supplemental UV-B radiation, whereas the aperture size of single stomata was diminished. The number and area of plastoglobuli were apparently reduced by UV-B radiation, but stroma and grana lamellae were not destroyed. Our results demonstrated that T. chinensis var. mairei can activate several defense mechanisms against oxidative stress injury caused by supplemental UV-B radiation.