The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus.

The SARS-CoV-2 pandemic is currently causing an unprecedented global health emergency since its emergence in December 2019. In December 2021, the FDA granted emergency use authorization to nirmatrelvir, a SARS-CoV-2 main protease inhibitor, for treating infected patients. This peptidomimetic is designed with a nitrile warhead, which forms a covalent bond to the viral protease. Herein, we investigate nirmatrelvir analogs with different warheads and their inhibitory activities. In addition, antiviral activities against human alphacoronavirus 229E was also investigated along with a cell-based assay. We discovered that the hydroxymethylketone and ketobenzothiazole warheads were equipotent to the nitrile warhead, suggesting that these analogs can also be used for treating coronavirus infections.

Hepatocellular carcinoma (HCC) is now the major malignant disease with high morbidity and mortality, which seriously endangers human lives and health. Alpha fetoprotein (AFP) assay is a commonly used serological biomarker for clinical diagnosis of HCC, but it lacks specificity. Analysis of its isoform AFP-L3, especially the AFP-L3 ratio in total AFP (AFP-L3%), can significantly improve the specificity for HCC identification. Herein, an electrochemical approach has been first proposed for simple, accurate, and fast determination of AFP-L3% in clinical samples. On the basis of two independent electrochemical signals generated from the synthesized nanoparticles, 4-mercaptophenylboronic acid (MPA)-functionalized copper nanoparticles (MPA-CuNPs) and the Lens culinaris agglutinin (LCA)-functionalized silver nanoparticles (LCA-AgNPs), simultaneous quantification of the AFP-L3 and total AFP in serum sample has been achieved, thus achieving directly the electrochemical assay of AFP-L3%. To be noted, both the assay time and the assay procedure have been significantly compressed when compared to that of available techniques in clinical use. Therefore, with the integration of electrochemical techniques, this new approach for AFP-L3% analysis would be promising for the accurate diagnosis of HCC.

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A cold-shock protein, RbfA (ribosome-binding factor A), is essential for cell growth at low temperature. In an rbfA-deletion strain, 30S and 50S ribosomal subunits increase relative to 70S monosomes with concomitant accumulation of a precursor 16S rRNA (17S rRNA). Recently, we have reported that overexpression of Era, an essential GTP-binding protein, suppresses not only the cold-sensitive cell growth but also defective ribosome biogenesis in the rbfA-deletion strain. Here, in order to elucidate how RbfA and Era functionally overlap, we characterized a cold-sensitive Era mutant (a point mutation at the Glu-200 to Lys; E200K) which shows a similar phenotype as the rbfA-deletion strain; accumulation of free ribosome subunits and 17S rRNA. To examine the effect of E200K in the rbfA-deletion strain, we constructed an E200K-inducible expression system. Interestingly, unlike wild-type Era, overexpression of Era(E200K) protein in the rbfA-deletion strain severely inhibited cell growth even at permissive temperature with further concomitant reduction of 16S rRNA. Purified Era(E200K) protein binds to 30S ribosomal subunits in a nucleotide-dependent manner like wild-type Era and retains both GTPase and autophosphorylation activities. Furthermore, we isolated spontaneous revertants of the E200K mutant. These revertants partially suppressed the accumulation of 17S rRNA. All the spontaneous mutations were found to result in higher Era(E200K) expression. These results suggest that the Era(E200K) protein has an impaired function in ribosome biogenesis without losing its ribosome binding activity. The severe growth defect caused by E200K in the rbfA-deletion strain may be due to competition between intrinsic wild-type Era and overexpressed Era(E200K) for binding to 30S ribosomal subunits. We propose that Era and RbfA have an overlapping function that is essential for ribosome biogenesis, and that RbfA becomes dispensable only at high temperatures because Era can complement its function only at higher temperatures.

The naturally fermented yak yogurt of pastoralists in the Tibetan Plateau, China, because of its unique geographical environment and the unique lifestyle of Tibetan pastoralists, is very different from other kinds of sour milk, and the microorganisms it contains are special. Lactococcus lactis subsp. lactis HFY14 (LLSL-HFY14) is a new lactic acid bacterium isolated from naturally fermented yak yogurt. The purpose of this study was to study the inhibitory effect of the bacterium on constipation. Methods: Constipation was induced in ICR mice with diphenoxylate, and the constipated mice were treated with LLSL-HFY14. The weight and feces of the mice were visually detected. Colonic tissues were observed on hematoxylin and eosin-stained sections. Serum indices were detected with kits. mRNA expression in the colon was determined by quantitative polymerase chain reaction assay. Results: Constipation caused weight loss, the number of defecation granules, defecation weight, fecal water content decreased, and the first black stool excretion time increased. LLSL-HFY14 alleviated these symptoms, and the effects were similar to those of lactulose (drug). The pathological examination revealed that constipation caused pathological changes in the colon, and LLSL-HFY14 effectively alleviated the disease. LLSL-HFY14 increased serum levels of motilin, gastrin, endothelin, substance P, acetylcholinesterase, and vasoactive intestinal peptide (VIP) and decreased serum levels of somatostatin in constipated mice. In addition, LLSL-HFY14 upregulated VIP, cAMP, protein kinase A, and aquaporin 3 expression in colonic tissues of constipated mice in a dose-dependent manner. Conclusion: LLSL-HFY14 inhibited constipation, similar to lactulose, and has the potential to become a biological agent.

In this article, we analyze and report cases of three patients who were admitted to Renmin Hospital, Wuhan University, China, for treating COVID-19 pneumonia in February 2020 and were unresponsive to initial treatment of steroids. They were then received titrated steroids treatment based on the assessment of computed tomography (CT) images augmented and analyzed with the artificial intelligence (AI) tool and output. Three patients were finally recovered and discharged. The result indicated that sufficient steroids may be effective in treating the COVID-19 patients after frequent evaluation and timely adjustment according to the disease severity assessed based on the quantitative analysis of the images of serial CT scans.