Targeted DNA N6-methyladenine editing by dCas9 fused to METTL4 in the lepidopteran model insect Bombyx mori.

We have established a novel CRISPR-dCas9-METTL4 epigenome editing tool that can methylate target regions to achieve site-specific DNA 6mA methylation in both hypermethylated and hypomethylated genes. Targeted methylation on genes by dCas9-METTL4 results in misexpression, allowing for the functional investigation of target genes of interest in silkworm.

CRISPR-Cas9-Mediated Mutation of Methyltransferase METTL4 Results in Embryonic Defects in Silkworm Bombyx mori.

DNA N6-methyladenine (6mA) has recently been found to play regulatory roles in gene expression that links to various biological processes in eukaryotic species. The functional identification of 6mA methyltransferase will be important for understanding the underlying molecular mechanism of epigenetic 6mA methylation. It has been reported that the methyltransferase METTL4 can catalyze the methylation of 6mA; however, the function of METTL4 remains largely unknown. In this study, we aim to investigate the role of the Bombyx mori homolog METTL4 (BmMETTL4) in silkworm, a lepidopteran model insect. By using CRISPR-Cas9 system, we somatically mutated BmMETTL4 in silkworm individuates and found that disruption of BmMETTL4 caused the developmental defect of late silkworm embryo and subsequent lethality. We performed RNA-Seq and identified that there were 3192 differentially expressed genes in BmMETTL4 mutant including 1743 up-regulated and 1449 down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that genes involved in molecular structure, chitin binding, and serine hydrolase activity were significantly affected by BmMETTL4 mutation. We further found that the expression of cuticular protein genes and collagens were clearly decreased while collagenases were highly increased, which had great contributions to the abnormal embryo and decreased hatchability of silkworm. Taken together, these results demonstrated a critical role of 6mA methyltransferase BmMETTL4 in regulating embryonic development of silkworm.

Effects of Cardiac Rehabilitation on Cardiac Function and Quality of Life in Patients with Ischemic Nonobstructive Coronary Artery Disease and Diabetes Mellitus.

Objective: To investigate the effect of cardiac rehabilitation on the quality of life in patients with ischemic and nonobstructive coronary artery disease complicated with diabetes mellitus. Methods: From January 2020 to June 2021, 100 patients with ischemic nonobstructive coronary heart disease complicated with diabetes were randomly divided into the control group (n = 50). The routine drug therapy observation group (50 cases) was treated with routine drugs combined with cardiac rehabilitation at 6 months for 1 course. The curative effect, cardiac function, 6 min walking distance (6MWD), cardiopulmonary exercise test (CPFT) index, SF-36 Health Status Survey Scale, and quality of life score were compared between the two groups. Results: The total effective rate of the observation group was significantly higher than that of the control group (P < 0.05). After treatment, there was no significant change in cardiac function and 6MWD in the control group. The left ventricular ejection fraction and 6MWD in the observation group were significantly higher/longer than those before treatment and the control group (all P < 0.05). After treatment, the indexes of CPET in the two groups were improved in varying degrees. The forced vital capacity and oxygen uptake of anaerobic valve in the observation group were significantly higher than those in the control group (all P < 0.05). After treatment, the scores of the SF-36 in both groups were improved in varying degrees. The physiological function, general health, energy, mental health, and total scores of the observation group were significantly higher than those of the control group (all P < 0.05). Conclusion: Cardiac rehabilitation can significantly improve the cardiorespiratory function, exercise ability, and quality of life in patients with ischemic and nonobstructive coronary artery disease complicated with diabetes mellitus.

SUMOylation of Translationally Regulated Tumor Protein Modulates Its Immune Function.

Translationally controlled tumor protein (TCTP) is a highly conserved protein possessing numerous biological functions and molecular interactions, ranging from cell growth to immune responses. However, the molecular mechanism by which TCTP regulates immune function is largely unknown. Here, we found that knockdown of Bombyx mori translationally controlled tumor protein (BmTCTP) led to the increased susceptibility of silkworm cells to virus infection, whereas overexpression of BmTCTP significantly decreased the virus replication. We further demonstrated that BmTCTP could be modified by SUMOylation molecular BmSMT3 at the lysine 164 via the conjugating enzyme BmUBC9, and the stable SUMOylation of BmTCTP by expressing BmTCTP-BmSMT3 fusion protein exhibited strong antiviral activity, which confirmed that the SUMOylation of BmTCTP would contribute to its immune responses. Further work indicated that BmTCTP is able to physically interact with interleukin enhancer binding factor (ILF), one immune molecular, involved in antivirus, and also induce the expression of BmILF in response to virus infection, which in turn enhanced antiviral activity of BmTCTP. Altogether, our present study has provided a novel insight into defending against virus via BmTCTP SUMOylation signaling pathway and interacting with key immune molecular in silkworm.

Bioengineered silkworm model for expressing human neurotrophin-4 with potential biomedical application.

Neurotrophin-4 (NT-4) is a neurotrophic factor that plays important roles in maintaining nerve cell survival, regulating neuronal differentiation and apoptosis, and promoting nerve injury repair. However, the source of sufficient NT-4 protein and efficient delivery of NT-4 remain a challenge. This study aims to express an activated human NT-4 protein in a large scale by genetically engineering silk gland bioreactor of silkworm as a host. We showed that the expression of human NT-4-functionalized silk material could promote proliferation of mouse HT22 cells when compared to the natural silk protein, and no obvious cytotoxicity was observed under the conditions of different silk materials. Importantly, this functional silk material was able to induce the potential differentiation of HT22 cells, promote peripheral neural cell migration and neurite outgrowth of chicken embryo dorsal root ganglion (DRG). All these results demonstrated a high bioactivity of human NT-4 protein produced in silk gland. Therefore, based on the silkworm model, the further fabrication of different silk materials-carrying active NT-4 protein with good mechanical properties and great biocompatibility will give promising applications in tissue engineering and neurons regeneration.

Function of Polyamines in Regulating Cell Cycle Progression of Cultured Silkworm Cells.

BACKGROUND: Putrescine, spermidine, and spermine are polyamines that are ubiquitously distributed in prokaryotic and eukaryotic cells, which play important roles in cell proliferation and differentiation. METHODS: We investigated the expression profiles of polyamine pathway genes by qRT-PCR in different tissues of the lepidopteran silkworm. The polyamine levels in cultured silkworm cells were measured by HPLC. Spermidine and polyamine biosynthetic inhibitors were used for treating the cultured silkworm cells in order to clarify their effects on cell cycle progression. RESULTS: We identified the anabolic and catabolic enzymes that are involved in the polyamine biosynthetic pathway in silkworm. Transcriptional expression showed at least seven genes that were expressed in different silkworm tissues. Treatments of the cultured silkworm cells with spermidine or inhibitor mixtures of DFMO and MGBG induced or inhibited the expression of cell cycle-related genes, respectively, and thus led to changed progression of the cell cycle. CONCLUSIONS: The present study is the first to identify the polyamine pathway genes and to demonstrate the roles of polyamines on cell cycle progression via regulation of the expression of cell cycle genes in silkworm.