No abstract

MicroRNAs (miRNAs) are non-coding but functional RNA molecules of 21–25 nucleotides in length. MiRNAs play significant regulatory roles in diverse plant biological processes. In order to decipher the relationship between nbe-miR1919c-5p and the accumulations of tobacco curly shoot virus (TbCSV) and its betasatellite (TbCSB) DNAs, as well as viral symptom development, we investigated the function of nbe-miR1919c-5p during TbCSV and TbCSB co-infection in plants using a PVX-and a TRV-based short tandem target mimic (STTM) technology. Suppression of nbe-miR1919c-5p expression using these two technologies enhanced TbCSV and TbCSB co-infection-induced leaf curling symptoms in Nicotiana benthamiana plants. Furthermore, suppression of nbe-miR1919c-5p expression enhanced TbCSV and TbCSB DNA accumulations in the infected plants. Our results can advance our knowledge on the nbe-miR1919c-5p function during TbCSV and TbCSB co-infection.

Background: Aldrichina grahami (Diptera: Calliphoridae) is a forensically important fly, which has been widely applied to practical legal investigations. Unlike other necrophagous flies, A. grahami exhibits cold tolerance which helps to maintain its activity during low-temperature months, when other species are usually not active. Hence, A. grahami is considered an important forensic insect especially in cold seasons. In this study, we aim to explore the molecular mechanisms of cold tolerance of A. grahami through transcriptome. Results: We collected eggs and larvae (first-instar, second-instar and third-instar) at three different temperatures (4°C, 12°C and 20°C) and performed RNA-seq analyses. The differentially expressed genes (DEGs) associated with the cold-tolerance were screened out. The Venn analysis of DEGs from egg to third-instar larvae at three different temperatures showed there were 9 common genes. Candidate biological processes and genes were identified which refer to growth, and development of different temperatures, especially the chitin and cuticle metabolic process. The series-clusters showed crucial and unique trends when the temperature changed. Moreover, by comparing the results of growth and developmental transcriptomes from different temperatures, we found that DEGs belonging to the family of larval cuticle proteins (LCP), pupal cuticle protein (CUP), and heat shock proteins (HSP) have certain differences. Conclusions: This study identified functional genes and showed differences in the expression pattern of diverse temperatures. The DEGs series-clusters with increasing or decreasing trends were analyzed which may play an important role in cold-tolerance. Moreover, the findings in LCP, CUP and HSP showed more possible modulations in a cold environment. This work will provide valuable information for the future investigation of the molecular mechanism of cold tolerance in A. grahami.

The minimum postmortem interval (PMImin) could be evaluated from the developmental stage of forensically important insects colonize a corpse, such as blow flies (Diptera: Calliphoridae). Unlike larvae, the developmental stage of which is well established according to their morphology, estimating the age of pupae is proven to be challenging. Recently, several studies reported the regulation of special genes during the development of blow fly pupae. However, gene regulation in Aldrichina grahami during the intrapuparial period remains to be studied. Therefore, we set out to investigate the mRNA levels of heat shock protein 23 (Hsp23), heat shock protein 24 (Hsp24), and 1_16 during the metamorphosis of A. grahami pupae. First, we examined seven candidate reference genes (ribosomal protein 49 (RP49), 18S ribosomal RNA (18S rRNA), 28S ribosomal RNA (28S rRNA), beta-tubulin at 56D (β-tubulin), Ribosomal protein L23 (RPL23), glutathione S-transferase (GST1), and Actin. Three widely used algorithms (NormFinder, BestKeeper, and geNorm) were applied to evaluate the mRNA levels of reference gene candidates in puparium at three stable temperatures (15, 22, and 27°C). Next, mRNA expression of Hsp23, Hsp24, and 1_16 during A. grahami metamorphosis was examined. We demonstrated that mRNA expression levels of Hsp23, Hsp24, and 1_16 showed time-specific regulation. In summary, our study identified three gene markers for the intrapuparial period of A. grahami and might provide a potential application in PMImin estimation.

The prevalent use of electronic cigarettes (e-cigarettes) has increased exponentially in recent years, especially in youth who are attracted to flavored e-cigarettes. Indeed, e-cigarette or vaping product use-associated lung injury (EVALI) cases started to emerge in the United States in August 2019, resulting in 2807 hospitalized cases and 68 deaths as of 18 February 2020. In the present study, we investigated, for the first time, whether flavored and nicotine containing e-cigarettes induce endothelial dysfunction to result in impaired angiogenesis and wound healing particularly under diabetic condition. Nicotine containing e-cigarettes with various contents of nicotine (0, 1.2%, 2.4%), and flavored e-cigarettes of classic tobacco, mint, menthol, and vanilla or fruit from BLU (nicotine 2.4%) or JUUL (nicotine 3%), were used to treat endothelial cells in vitro and streptozotocin-induced diabetic mice in vivo. Endothelial cell superoxide production, determined by dihydroethidium (DHE) fluorescent imaging and electron spin resonance (ESR), was markedly increased by exposure to e-cigarette extract (e-CSE) in a nicotine-content dependent manner, while nitric oxide (NO) bioavailability detected by DAF-FM fluorescent imaging was substantially decreased. All of the different flavored e-cigarettes examined also showed significant effects in increasing superoxide production while diminishing NO bioavailability. Endothelial cell apoptosis evaluated by caspase 3 activity was markedly increased by exposure to e-CSE prepared from flavored and nicotine containing e-cigarettes. Endothelial monolayer wound assays revealed that nicotine-containing and flavored e-cigarettes induced impaired angiogenic wound repair of endothelial cell monolayers. Furthermore, vascular endothelial growth factor (VEGF) stimulated wound healing in diabetic mice was impaired by exposure to e-CSEs prepared from nicotine-containing and flavored e-cigarettes. Taken together, our data demonstrate for the first time that flavored and nicotine-containing e-cigarettes induce endothelial dysfunction through excessive ROS production, resulting in decreased NO bioavailability, increased endothelial cell apoptosis, and impairment in angiogenesis and wound healing, especially under diabetic condition. These responses of endothelial dysfunction likely underlie harmful effects of e-cigarettes in endothelial-rich organs, such as heart and lungs. These data also indicate that rigorous regulation on e-cigarette use should be enforced in diabetic and/or surgical patients to avoid severe consequences from impaired angiogenesis/wound healing.

Background Blowflies (Diptera: Calliphoridae) are the most commonly found entomological evidence in forensic investigations. Distinguished from other blowflies, Aldrichina grahami has some unique biological characteristics and is a species of forensic importance. Its development rate, pattern, and life cycle can provide valuable information for the estimation of the minimum postmortem interval. Findings Herein we provide a chromosome-level genome assembly of A. grahami that was generated by Pacific BioSciences sequencing platform and chromosome conformation capture (Hi-C) technology. A total of 50.15 Gb clean reads of the A. grahami genome were generated. FALCON and Wtdbg were used to construct the genome of A. grahami, resulting in an assembly of 600 Mb and 1,604 contigs with an N50 size of 1.93 Mb. We predicted 12,823 protein-coding genes, 99.8% of which was functionally annotated on the basis of the de novo genome (SRA: PRJNA513084) and transcriptome (SRA: SRX5207346) of A. grahami. According to the co-analysis with 11 other insect species, clustering and phylogenetic reconstruction of gene families were performed. Using Hi-C sequencing, a chromosome-level assembly of 6 chromosomes was generated with scaffold N50 of 104.7 Mb. Of these scaffolds, 96.4% were anchored to the total A. grahami genome contig bases. Conclusions The present study provides a robust genome reference for A. grahami that supplements vital genetic information for nonhuman forensic genomics and facilitates the future research of A. grahami and other necrophagous blowfly species used in forensic medicine.

Tobacco curly shoot virus (TbCSV) belongs to the genus Begomovirus of the family Geminiviridae, and causes leaf curling and curly shoot symptoms in tobacco and tomato crops. MicroRNAs (miRNAs) are pivotal modulators of plant development and host-virus interactions. However, the relationship between TbCSV infection and miRNAs accumulation has not been well investigated. The present study was conducted to analyze different expressions of miRNAs in Nicotiana benthamiana in response to the infection of TbCSV via small RNAs sequencing. The results showed that 15 up-regulated miRNAs and 12 down-regulated miRNAs were differentially expressed in TbCSV infected N. benthamiana, and nbe-miR167b-3p was down-regulated. To decipher the relationship between nbe-miR167b-3p expression and the accumulations of TbCSV DNA, pCVA mediation of miRNA overexpression and PVX based short tandem target mimic (STTM) were used in this study. It was found that overexpression of nbe-miR167b-3p attenuated leaf curling symptom of TbCSV and decreased viral DNA accumulation, but suppression of nbe-miR167b-3p expression enhanced the symptoms and accumulation of TbCSV. PRCP, the target gene of nbe-miR167b-3p, was silenced in plants using VIGS and this weakened the viral symptoms and DNA accumulation of TbCSV in the plants. Overall, this study clarified the effect of nbe-miR167b-3p on plant defense during TbCSV infection, and provided a framework to reveal the molecular mechanisms of miRNAs between plants and viruses.

Background: The excessive and frequent intake of refined sugar leads to caries. However, the relationship between the amount of sugar intake and the risk of caries is not always consistent. Oral microbial profile and function may impact the link between them. This study aims to identify the plaque microbiota characteristics of caries subjects with low (CL) and high (CH) sugar consumption, and of caries-free subjects with low (FL) and high sugar (FH) consumption. Methods: A total of 40 adolescents were enrolled in the study, and supragingival plaque samples were collected and subjected to metagenomic analyses. The caries status, sugar consumption, and oral-health behaviors of the subjects were recorded. Results: The results indicate that the CL group showed a higher abundance of several cariogenic microorganisms Lactobacillus, A. gerencseriae, A. dentails, S. mutans, C. albicans, S. wiggsiae and P. acidifaciens. C. gingivalis, and P. gingivalis, which were enriched in the FH group. In terms of gene function, the phosphotransferase sugar uptake system, phosphotransferase system, and several two-component responses–regulator pairs were enriched in the CL group. Conclusion: Overall, our data suggest the existence of an increased cariogenic microbial community and sugar catabolism potential in the CL group, and a healthy microbial community in the FH group, which had self-stabilizing functional potential.