Proteomic and metabolomic analysis of Nicotiana benthamiana under dark stress.

Exposure to extended periods of darkness is a common source of abiotic stress that significantly affects plant growth and development. To understand how Nicotiana benthamiana responds to dark stress, the proteomes and metabolomes of leaves treated with darkness were studied. In total, 5763 proteins and 165 primary metabolites were identified following dark treatment. Additionally, the expression of autophagy-related gene (ATG) proteins was transiently upregulated. Weighted gene coexpression network analysis (WGCNA) was utilized to find the protein modules associated with the response to dark stress. A total of four coexpression modules were obtained. The results indicated that heat-shock protein (HSP70), SnRK1-interacting protein 1, 2A phosphatase-associated protein of 46 kDa (Tap46), and glutamate dehydrogenase (GDH) might play crucial roles in N. benthamiana's response to dark stress. Furthermore, a protein-protein interaction (PPI) network was constructed and top-degreed proteins were predicted to identify potential key factors in the response to dark stress. These proteins include isopropylmalate isomerase (IPMI), eukaryotic elongation factor 5A (ELF5A), and ribosomal protein 5A (RPS5A). Finally, metabolic analysis suggested that some amino acids and sugars were involved in the dark-responsive pathways. Thus, these results provide a new avenue for understanding the defensive mechanism against dark stress at the protein and metabolic levels in N. benthamiana.

Isolation of a Novel Bat Rhabdovirus with Evidence of Human Exposure in China.

Bats are well-recognized reservoirs of zoonotic viruses. Several spillover events from bats to humans have been reported, causing severe epidemic or endemic diseases including severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome-CoV (MERS-CoV), henipaviruses, and filoviruses. In this study, a novel rhabdovirus species, provisionally named Rhinolophus rhabdovirus DPuer (DPRV), was identified from the horseshoe bat (Rhinolophus affinis) in Yunnan province, China, using next-generation sequencing. DPRV shedding in the spleen, liver, lung, and intestinal contents of wild bats with high viral loads was detected by real-time quantitative PCR, indicating that DPRV has tropism for multiple host tissues. Furthermore, DPRV can replicate in vitro in multiple mammalian cell lines, including BHK-21, A549, and MA104 cells, with the highest efficiency in hamster kidney cell line BHK-21, suggesting infectivity of DPRV in these cell line-derived hosts. Ultrastructure analysis revealed a characteristic bullet-shaped morphology and tightly clustered distribution of DPRV particles in the intracellular space. DPRV replicated efficiently in suckling mouse brains and caused death of suckling mice; death rates increased with passaging of DPRV in suckling mice. Moreover, 421 serum samples were collected from individuals who lived near the bat collection site and had fever symptoms within 1 year. DPRV-specific antibodies were detected in 20 (4.75%) human serum samples by indirect immunofluorescence assay. Furthermore, 10 (2.38%) serum samples were DPRV positive according to plaque reduction neutralization assay, which revealed potential transmission of DPRV from bats to humans and highlighted the potential public health risk. Potential vector association with DPRV was not found with negative viral RNA in bloodsucking arthropods. IMPORTANCE We identified a novel rhabdovirus from the horseshoe bat (Rhinolophus thomasi) in China with probable infectivity in humans. DPRV was isolated in vitro from several mammalian cell lines, indicating wide host tropism, excluding bats, of DPRV. DPRV replicated in the brains of suckling mice, and the death rate of suckling mice increased with passaging of DPRV in vivo. Serological tests indicated the possible infectivity of DPRV in humans and the potential transmission to humans. The present findings provide preliminary evidence for the potential risk of DPRV to public health. Additional studies with active surveillance are needed to address interspecies transmission and determine the pathogenicity of DPRV in humans.

MAGI-2 downregulation: a potential predictor of tumor progression and early recurrence in Han Chinese patients with prostate cancer.

Membrane-associated guanylate kinase (MAGUK) family protein MAGUK invert 2 (MAGI-2) has been demonstrated to be involved in the tumorigenic mechanism of prostate cancer. The objective of this study was to investigate the expression of MAGI-2 at mRNA and protein levels. The prognostic value of MAGI-2 in Han Chinese patients with prostate cancer was also investigated. The expression data of MAGI-2 were assessed through database retrieval, analysis of sequencing data from our group, and tissue immunohistochemistry using digital scoring system (H-score). The clinical, pathological, and follow-up data were collected. The expression of MAGI-2 in prostate tumor tissues and prostate normal tissues was evaluated and compared. MAGI-2 expression was associated with clinical parameters including tumor stage, lymph node status, Gleason score, PSA level, and biochemical recurrence of prostate cancer. The relative expression of MAGI-2 mRNA was lower in the tumor tissue in The Cancer Genome Atlas (TCGA) database and sequencing data (P < 0.001). There was no difference in MAGI-2 protein expression between tumor and normal tissues in tissue microarray (TMA) results. MAGI-2 expression was associated with pathological tumor stage (P = 0.02), Gleason score (P = 0.05), and preoperation prostate-specific antigen (PSA; P = 0.04). A positive correlation was identified between MAGI-2 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expressions through the analysis of TCGA and TMA data (P < 0.0001). Patients with higher MAGI-2 expression had longer biochemical recurrence-free survival in the univariate analysis (P = 0.005), which indicates an optimal prognostic value of MAGI-2 in Han Chinese patients with prostate cancer. In conclusion, MAGI-2 expression gradually decreases with tumor progression, and can be used as a predictor of tumor recurrence in Chinese patients.

Detection and characterization of a novel hepacivirus in long-tailed ground squirrels (Spermophilus undulatus) in China.

Rodent populations are known to be reservoirs of viruses with the potential to infect humans. However, a large number of such viruses remain undiscovered. In this study, we investigated the shedding of unknown viruses in long-tailed ground squirrel (Spermophilus undulatus) feces by high-throughput sequencing. A novel and highly divergent virus related to members of the genus Hepacivirus was identified in ground squirrel liver. This virus, tentatively named RHV-GS2015, was found to have a genome organization that is typical of hepaciviruses, including a long open reading frame encoding a polyprotein of 2763 aa. Sequence alignment of RHV-GS2015 with the most closely related hepaciviruses yielded p-distances of the NS3 and NS5B regions of 0.546 and 0.476, respectively, supporting the conclusion that RHV-GS2015 is a member of a new hepacivirus species, which we propose to be named "Hepacivirus P". Phylogenetic analysis of the NS3 and NS5B regions indicated that RHV-GS2015 shares common ancestry with other rodent hepaciviruses (species Hepacivirus E, and species Hepacivirus F), Norway rat hepacivirus 1 (species Hepacivirus G), and Norway rat hepacivirus 2 (species Hepacivirus H). A phylogenetic tree including the seven previously identified rodent hepaciviruses revealed extreme genetic heterogeneity among these viruses. RHV-GS2015 was detected in 7 out of 12 ground squirrel pools and was present in liver, lung, and spleen tissues. Furthermore, livers showed extremely high viral loads of RHV-GS2015, ranging from 2.5 × 106 to 2.0 × 108 copies/g. It is reasonable to assume that this novel virus is hepatotropic, like hepatitis C virus. The discovery of RHV-GS2015 extends our knowledge of the genetic diversity and host range of hepaciviruses, helping to elucidate their origins and evolution.

Identification of chilling-responsive transcripts in peanut (Arachis hypogaea L. )

To isolate differentially expressed peanut genes responsive to chilling, a suppression subtractive hybridization (SSH) cDNA library was constructed for a chilling tolerant peanut cultivar A4 with mRNAs extracted from the seeds imbibed at 2ºC and 15ºC, respectively, for 24 hrs. A total of 466 cDNA clones were sequenced, from which 193 unique transcripts (73 contigs and 120 singlets) were assembled. Of these unique transcripts, 132 (68.4 percent) were significantly similar to the sequences in GenBank non-redundant (nr) protein database, which belonged to diverse functional categories including metabolism, signal transduction, stress response, cell defense and transcriptional regulation. The remaining 61 (31.6 percent) showed no similarity to either hypothetical or known proteins. Six differentially expressed transcripts were further confirmed with real-time quantitative PCR (RT-qPCR).

Large-scale identification of odorant-binding proteins and chemosensory proteins from expressed sequence tags in insects.

BACKGROUND: Insect odorant binding proteins (OBPs) and chemosensory proteins (CSPs) play an important role in chemical communication of insects. Gene discovery of these proteins is a time-consuming task. In recent years, expressed sequence tags (ESTs) of many insect species have accumulated, thus providing a useful resource for gene discovery. RESULTS: We have developed a computational pipeline to identify OBP and CSP genes from insect ESTs. In total, 752,841 insect ESTs were examined from 54 species covering eight Orders of Insecta. From these ESTs, 142 OBPs and 177 CSPs were identified, of which 117 OBPs and 129 CSPs are new. The complete open reading frames (ORFs) of 88 OBPs and 123 CSPs were obtained by electronic elongation. We randomly chose 26 OBPs from eight species of insects, and 21 CSPs from four species for RT-PCR validation. Twenty two OBPs and 16 CSPs were confirmed by RT-PCR, proving the efficiency and reliability of the algorithm. Together with all family members obtained from the NCBI (OBPs) or the UniProtKB (CSPs), 850 OBPs and 237 CSPs were analyzed for their structural characteristics and evolutionary relationship. CONCLUSIONS: A large number of new OBPs and CSPs were found, providing the basis for deeper understanding of these proteins. In addition, the conserved motif and evolutionary analysis provide some new insights into the evolution of insect OBPs and CSPs. Motif pattern fine-tune the functions of OBPs and CSPs, leading to the minor difference in binding sex pheromone or plant volatiles in different insect Orders.