CRISPR/Cas9-mediated disruption of orf76 as an antiviral therapy against BmNPV in the transgenic silkworm.

Viral diseases pose a significant threat to livestock husbandry and plant cultivation. CRISPR/Cas9-mediated targeted editing of viral genes offers a promising approach to antiviral therapy. The silkworm, Bombyx mori, is an economically important insect susceptible to infection by B. mori nucleopolyhedrovirus (BmNPV), and viral outbreaks cause severe economic losses to the sericulture industry. Here, we identified BmNPV orf76 as a viral late gene that is highly similar to Autographa californica multiple nucleopolyhedrovirus Ac93. The deletion of orf76 abolished BmNPV proliferation and hindered the production of infectious budded viruses. We generated a transgenic line, Cas9(+)/sgorf76(+), that did not affect the growth or development of the silkworm and demonstrated that the transgenic line Cas9(+)/sgorf76(+) efficiently cleaved orf76 at the sgorf76 site, resulting in large deletions at 120 h post-infection, with no observed off-target effects. Survival analyses revealed that the transgenic line Cas9(+)/sgorf76(+) exhibited significantly higher survival rates than the control lines Cas9(-)/sgorf76(-), regardless of the BmNPV inoculation dose. Additionally, the number of BmNPV DNA copies and the expression levels of viral genes were markedly inhibited in the transgenic line Cas9(+)/sgorf76(+) compared with the control line Cas9(-)/sgorf76(-). The results provide a promising target for Cas9-mediated antiviral therapy against BmNPV, and the findings provide new insights for baculovirus gene function studies and lepidopteran pest control.

Antibacterial activity of lysozyme after association with carboxymethyl konjac glucomannan.

The unique high isoelectric point of lysozyme (LYZ) restricts its application in composite antibacterial coating due to the unfavorable liability to electrostatic interaction with other components. In this work, the antibacterial activity of a dispersible LYZ-carboxymethyl konjac glucomannan (CMKGM) polyelectrolyte complex was evaluated. Kinetic analysis revealed that, compared with free LYZ, the complexed enzyme exhibited decreased affinity (Km) but markedly increased Vmax against Micrococcus lysodeikticus, and QCM and dynamic light scattering analysis confirmed that the complex could bind with the substrate but in a much lower ratio. The complexation with CMKGM did not alter the antibacterial spectrum of LYZ, and the complex exerted antibacterial function by delaying the logarithmic growth phase and impairing the cell integrity of Staphylococcus aureus. Since the LYZ-CMKGM complex is dispersible in water and could be assembled easily, it has great potential as an edible coating in food preservation.

New stilbenoligan and flavonoid from the roots of Caragana stenophylla Pojark. and their anti-inflammatory activity.

A phytochemical investigation on the 80% ethanol extract of the roots of Caragana stenophylla Pojark. resulted in the isolation of 20 compounds, including two new ones, named kompasinol P (2) and 3,5,7,2',3'-pentahydroxy-4'-methoxyisoflavanone (3). Among them, a pair of enantiomers, (7S, 8 R, 7'R, 8'S)-kompasinol A (1a) and (7 R, 8S, 7'S, 8'R)-kompasinol A (1b), were successfully separated by the chiral-phase HPLC resolution for the first time. The absolute configurations of 1a and 1b were determined by the experimental and calculated electronic circular dichroism (ECD) data. 15 isolates were evaluated for their anti-inflammatory activity via inhibiting the production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. Compounds 1a/1b, 6, 7, 9, 10, 12, 14, and 16-18 showed moderate inhibition with IC50 values ranging from 11.45 to 68.54 µM.

[Chemical constituents from roots of Caragana stenophylla and their anti-tumor activities].

Fourteen compounds were isolated from the 80% ethanol extract of Caragana stenophylla root, by using a combination of various chromatographic approaches, including silica gel sephadex LH-20 column chromatography, and preparative HPLC. On the basis of their physical and chemical properties and spectroscopic data, their structures were elucidated as 2-(4-hydroxy-3-methoxy lphenyl)-3-methoxyl benzofuran-6-ol (1), mucodianin C (2), isopterofuran (3), formononetin (4), afromosin (5), calycosin (6), acacetin (7), 3-O-methylkaempferol (8), liquiritigenin (9), isoliquiritigenin (10), variabilin (11), resveratrol (12), zhebeiresinol (13), and 2, 3-dicarboxy-6, 7-dihydroxy-1-(3', 4'-dihydroxy)-phenyl-1, 2-dihydronaphthalen (14). Compound 1 is a new benzofuran derivative, named as mucodianin S; compounds 2, 3, 11, 13, 14 were isolated from the genus Caragana for the first time, and compounds 4-10 were firstly isolated from Caragana stenophylla. MTT assay was used to determine their cytotoxicity of the isolated compounds against human tumor cell lines, and 2 showed cytotoxicity against human hepato cellular cancer (HepG2) and human cervical (HeLa) lines, with IC50 values of (16.18±0.95), (3.75±0.08) µmol•L ⁻¹, respectively.