Insects are the most speciose group of animals, but the phylogenetic relationships of many major lineages remain unresolved. We inferred the phylogeny of insects from 1478 protein-coding genes. Phylogenomic analyses of nucleotide and amino acid sequences, with site-specific nucleotide or domain-specific amino acid substitution models, produced statistically robust and congruent results resolving previously controversial phylogenetic relations hips. We dated the origin of insects to the Early Ordovician [~479 million years ago (Ma)], of insect flight to the Early Devonian (~406 Ma), of major extant lineages to the Mississippian (~345 Ma), and the major diversification of holometabolous insects to the Early Cretaceous. Our phylogenomic study provides a comprehensive reliable scaffold for future comparative analyses of evolutionary innovations among insects.
The advent in high-throughput-sequencing (HTS) technologies has revolutionized conventional biodiversity research by enabling parallel capture of DNA sequences possessing species-level diagnosis. However, polymerase chain reaction (PCR)-based implementation is biased by the efficiency of primer binding across lineages of organisms. A PCR-free HTS approach will alleviate this artefact and significantly improve upon the multi-locus method utilizing full mitogenomes. Here we developed a novel multiplex sequencing and assembly pipeline allowing for simultaneous acquisition of full mitogenomes from pooled animals without DNA enrichment or amplification. By concatenating assemblies from three de novo assemblers, we obtained high-quality mitogenomes for all 49 pooled taxa, with 36 species >15 kb and the remaining >10 kb, including 20 complete mitogenomes and nearly all protein coding genes (99.6%). The assembly quality was carefully validated with Sanger sequences, reference genomes and conservativeness of protein coding genes across taxa. The new method was effective even for closely related taxa, e.g. three Drosophila spp., demonstrating its broad utility for biodiversity research and mito-phylogenomics. Finally, the in silico simulation showed that by recruiting multiple mito-loci, taxon detection was improved at a fixed sequencing depth. Combined, these results demonstrate the plausibility of a multi-locus mito-metagenomics approach as the next phase of the current single-locus metabarcoding method.
To investigate the morphological and growth characteristics of rabbit keratocytes when cultured on decellularized cornea under simulate microgravity (SMG) rotary cell culture system (RCCS) and static culture or in plastic culture supplemented with small molecules of valproic acid (VPA) and vitamin C (VC). Bovine corneas were firstly decellularized with Triton X-100 and NH(4)OH and through short-term freezing process. Then cell count kit-8 (CCK-8) and flow cytometry were used to test the effects of VPA and VC on the proliferation, cell cycle and apoptosis of rabbit keratocytes. Hematoxylin-eosin (H&E) staining and scanning electron microscopy (SEM) imaging showed that cells were eliminated in the decellularized bovine corneas. The proliferation of cultured keratocytes was promoted by VPA and VC in the cell proliferation assay. VPA and VC moderately decreased the number of apoptotic cells and obviously promoted cell-cycle entrance of keratocytes. Rabbit keratocytes in plastic displayed spindle shape and rare interconnected with or without VPA and VC. Cells revealed dendritic morphology and reticular cellular connections when cultured on the carriers of decellularized corneas supplemented with VPA and VC even in the presence of 10% fetal bovine serum (FBS). When cultured in RCCS supplemented with VPA, VC and 10% FBS, keratocytes displayed round shape with many prominences and were more prone to grow into the pores of carriers with aggregation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis proved that the keratocytes cultured on decellularized bovine cornea under SMG with VPA and VC expressed keratocan and lumican. Keratocytes cultured on plastic expressed lumican but not keratocan. Immunofluorescence identification revealed that cells in all groups were positively immunostained for vimentin. Keratocytes on decellularized bovine cornea under SMG or in static culture were positively immunostained for keratocan and lumican. Thus, we reasonably made a conclusion that the combination of VPA, VC, RCCS and decellularized corneal carriers provide a good condition for keratocytes to well grow. Keratocytes can be manipulated to be aggregates or physiological morphological growth in vitro, which are important for the research of corneal stem cells and corneal tissue engineering.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a structurally endogenous peptide with many biological roles. Maxadilan, a 61-amino acid vasodilatory peptide, specifically activates the PACAP type I receptor (PAC1). Although PAC1 has been identified in embryonic stem cells, little is known about its presence or effects in human induced pluripotent stem (iPS) cells. In the present study, we investigated the expression of PAC1 in human iPS cells by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. To study the physiological effects mediated by PAC1, we evaluated the role of maxadilan in preventing apoptotic cell death induced by ultraviolet C (UVC). After exposure to UVC, the iPS cells showed a marked reduction in cell viability and a parallel increase of apoptotic cells, as demonstrated by WST-8 analysis, annexin V/propidium iodide (PI) analysis and the terminal transferase dUTP nick end labeling (TUNEL) assay. The addition of 30 nM of maxadilan dramatically increased iPS cell viability and reduced the percentage of apoptotic cells. The anti-apoptotic effects of maxadilan were correlated to the downregulation of caspase-3 and caspase-9. Concomitantly, immunofluorescence, western blot analysis, real-time quantitative polymerase chain reaction (RT-qPCR) analysis and in vitro differentiation results showed that maxadilan did not affect the pluripotent state of iPS cells. Moreover, karyotype analysis showed that maxadilan did not affect the karyotype of iPS cells. In summary, these results demonstrate that PAC1 is present in iPS cells and that maxadilan effectively protects iPS cells against UVC-induced apoptotic cell death while not affecting the pluripotent state or karyotype.
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