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To elucidate the application value of insect endogenous protease and its inhibitor genes in pest control, we analyzed in detail the transcriptome sequence of the Asian corn borer, Ostrinia furnacalis. We obtained 12 protease genes and 11 protease inhibitor genes, and comprehensively analyzed of their spatiotemporal expression by qRT-PCR. In which, a previous unstudied serine protease inhibitor gene attracted our attention. It belongs to the canonical serine proteinase inhibitor family, a trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitor, but its TIL domain lacks two cysteine residues, and it was named as ACB-TIL. Its expression level is relatively very low in the absence of pathogen stimulation, and can be up-regulated expression induced by Gram-negative bacteria (Escherichia coli), virus (BmNPV), and dsRNA (dsEGFP), but cannot be induced by fungus spores (Metarrhizium anisopliae). Prokaryotic expressed ACB-TIL protein can significantly inhibit the melanization in vitro. Injecting this protein into insect body can inhibit the production of antimicrobial peptides of attacin, lebocin and gloverin. Inhibition of ACB-TIL by RNAi can cause the responses of other immune-, protease- and inhibitor-related genes. ACB-TIL is primarily involved in Asian corn borer humoral immunity in responses to Gram-negative bacteria and viruses. This gene can be a potential target for pest control since this will mainly affect insect immune response.

Optic atrophy1 (OPA1) is crucial for inner mitochondrial membrane (IMM) fusion and essential for maintaining crista structure and mitochondrial morphology. Optic atrophy and hearing impairment are the most prevalent clinical features associated with mutations in the OPA1 gene, but the function of OPA1 in hearing is still unknown. In this study, we examined the ability of Opa1 to protect against cisplatin-induced cochlear cell death in vitro and in vivo. Our results revealed that knockdown of Opa1 affects mitochondrial function in HEI-OC1 and Neuro 2a cells, as evidenced by an elevated reactive oxygen species (ROS) level and reduced mitochondrial membrane potential. The dysfunctional mitochondria release cytochrome c, which triggers apoptosis. Opa1 expression was found to be significantly reduced after cell exposed to cisplatin in HEI-OC1 and Neuro 2a cells. Loss of Opa1 aggravated the apoptosis and mitochondrial dysfunction induced by cisplatin treatment, whereas overexpression of Opa1 alleviated cisplatin-induced cochlear cell death in vitro and in explant. Our results demonstrate that overexpression of Opa1 prevented cisplatin-induced ototoxicity, suggesting that Opa1 may play a vital role in ototoxicity and/or mitochondria-associated cochlear damage.

Double-stranded RNA (dsRNA)-induced genes are usually related to RNA interference (RNAi) mechanisms and are involved in immune-related pathways. In a previous study, we found a lepidopteran-specific nuclease gene REase that was up-regulated by dsRNA and that affected RNAi efficiency in Asian corn borer (Ostrinia furnacalis). In this study, to verify the function of REase, the homologous gene HaREase in cotton bollworm (Helicoverpa armigera) was knocked out using CRISPR/Cas9 system. We found that the midgut epithelium structure was apparently not affected in the ΔHaREase mutant [Knock out (KO)]. Transcript sequencing results showed that most of the known insect immune-related genes were up-regulated in KO. When second instar larvae were fed artificial diet with Cry1Ac, a protoxin from Bacillus thuringiensis (Bt), in sublethal doses (2.5 or 4 μg/g), the growth rate of KO was repressed significantly. The dsRNA stability was also enhanced in midgut extraction of KO; however, RNAi efficiency was not obviously improved compared with the wild type (WT). The KO and WT were injected with dsEGFP (Enhanced green fluorescent protein) and subjected to transcriptome sequencing. The results showed that the expression levels of 14 nuclease genes were enhanced in KO after the dsRNA treatment. These findings revealed that HaREase expression level was not only related with dsRNA stability, but also with Bt resistance in cotton bollworm. When HaREase was knocked out, other immune- or nuclease-related genes were enhanced significantly. These results remind us that insect immune system is complex and pest control for cotton bollworm is an arduous task.

Exogenous dsRNA enters the insect body and can induce the RNAi effect only when it is cleaved into siRNA. However, what kinds of base composition are easier to cut and what kinds of siRNA will be produced in vivo is largely unknown. In this study, we found that dsRNA processing into siRNA has sequence preference and regularity in insects. We injected 0.04 mg/g dsRNA into Asian corn borers or cotton bollworms according to their body weight, and then the siRNAs produced in vivo were analyzed by RNA-Seq. We discovered that a large number of siRNAs were produced with GGU nucleotide residues at the 5′- and 3′-ends and produced a siRNA peak on the sequence. Once the GGU site is mutated, the number of siRNAs will decrease significantly and the siRNA peak will also lost. However, in the red flour beetle, a member of Coleoptera, dsRNA was cut at more diverse sites, such as AAG, GUG, and GUU; more importantly, these enzyme restriction sites have a high conservation base of A/U. Our discovery regarding dsRNA in vivo cleavage preference and regularity will help us understand the RNAi mechanism and its application.

Abstract Background Kangaroo mother care (KMC) can have a positive effect on the mental well-being of a mother. However, there are specific challenges associated with the process that may contribute to increased anxiety for the mother. By integrating nurse-assisted mindfulness training alongside KMC guidance, nurses may effectively alleviate maternal stress to a greater extent. Methods A single-centre randomized controlled trial was conducted to investigate the effects of KMC combined with nurse-assisted mindfulness training. The study included preterm infants with a gestational age of less than 32 weeks or a birth weight of less than 1500 g and their mothers, who were randomly divided into two groups. The intervention group consisted of mothers who received KMC combined with nurse-assisted mindfulness training for 14 days. The control group comprised mothers who received only KMC for 14 days. Data from both groups were collected and compared for analysis. Results Forty-seven infants and their mothers were included in the intervention group, whereas 44 pairs were included in the control group. After the intervention, the parental stressor scale scores for the neonatal intensive care unit (PSS: NICU) (3), PSS: NICU (4), and Hospital Anxiety and Depression Scale (HADS) scores for the intervention group were lower than those for the control group, whereas the Five Facet Mindfulness Questionnaire (FFMQ) (1), FFMQ (4), and FFMQ (5) scores for the intervention group were higher. The degree of change in the PSS: NICU and HADS scores was inversely correlated with the degree of change in the FFMQ score. The breast milk feed rate and weight gain rate were greater in the intervention group than in the control group. No adverse reactions were observed in either group. Conclusions Kangaroo mother care combined with nurse-assisted mindfulness training is an acceptable, feasible, and effective procedure for reducing anxiety in mothers of preterm infants in the NICU, with potential benefits for the short-term prognosis of these infants. Trial registration Chinese Clinical Trial Registry, ChiCTR1900023697, registered on June 8, 2019, retrospectively registered.