Effective ribosomal RNA depletion for single-cell total RNA-seq by scDASH
A decade since its invention, single-cell RNA sequencing (scRNA-seq) has become a mainstay technology for profiling transcriptional heterogeneity in individual cells. Yet, most existing scRNA-seq methods capture only polyadenylated mRNA to avoid the cost of sequencing non-messenger transcripts, such as ribosomal RNA (rRNA), that are usually not of-interest. Hence, there are not very many protocols that enable single-cell analysis of total RNA. We adapted a method called DASH (Depletion of Abundant Sequences by Hybridisation) to make it suitable for depleting rRNA sequences from single-cell total RNA-seq libraries. Our analyses show that our single-cell DASH (scDASH) method can effectively deplete rRNAs from sequencing libraries with minimal off-target non-specificity. Importantly, as a result of depleting the rRNA, the rest of the transcriptome is significantly enriched for detection.
Loi et al. (2021) Effective ribosomal RNA depletion for single-cell total RNA-seq by scDASH. PeerJ 9:e10717 https://doi.org/10.7717/peerj.10717
A preliminary comparative study of percutaneous CT-guided cryoablation with surgical resection for osteoid osteoma
Background The traditional treatment for osteoid osteoma is the nidus’ surgical resection, which was difficult to eradicate with more invasive and complications because of osteosclerosis surrounding the nidus. This study aimed to analyze the efficacy and safety of percutaneous CT-guided cryoablation of osteoid osteoma at different sites (especially refractory sites such as the spine). Methods Fifteen patients with osteoid osteoma who underwent cryoablation at our institution were analyzed retrospectively on their imaging data and clinical visual analog scale (VAS) pain scores before and after the procedure. Fifty-three patients underwent surgical resection during the period were also included in this study as a control group. Treatment efficacy was assessed primarily by comparing the differences in VAS scores at different time points in each group of patients by paired-sample t-test. Differences in length of hospital stay and complications between the two groups were also compared. Results The technical success rate was 100% in both the cryoablation and surgical resection group. Cryoablation had a significantly shorter hospitalization time than surgery (p = 0.001). Clinically, the post-operative VAS scores were all significantly improved compared to the pre-operative period, and the clinical cure was achieved in both groups. Surgical operations had more complications than cryoablation, although there was no significant difference. In the group of cryoablation, only one patient had mild numbness of the left lower extremity, which relieved itself; two patients had mild post-operative pain. No patients in the cryoablation group experienced recurrence during the follow-up period. In the surgery group, three of the patients experienced massive bleeding (>500 ml), and two underwent transfusion therapy. Only one patient in the surgical resection group experienced a recurrence at 29 months postoperatively and underwent a second resection. All patients had local scars on the skin after surgical resection. Conclusion Cryoablation is a minimally invasive, safe, and effective treatment strategy for osteoid osteoma, and is fully comparable to surgical resection.
Meng et al. (2021) A preliminary comparative study of percutaneous CT-guided cryoablation with surgical resection for osteoid osteoma. PeerJ 9:e10724 https://doi.org/10.7717/peerj.10724
Overexpressed PLAU and its potential prognostic value in head and neck squamous cell carcinoma
Background Metastasis is a major event for survival and prognosis in patients with head and neck squamous cell carcinomas (HNSCC). A primary cause of metastasis is the proteolytic degradation of the extracellular matrix (ECM). The plasminogen activator urokinase (PLAU) is involved in the transformation of plasminogen to plasmin leading to hydrolyzation of ECM-related proteins. However, the role of PLAU expression in HNSCC is unclear and the worth being investigated. Methods PLAU expression profiles and clinical parameters from multiple HNSCC datasets were used to investigate the relationship of PLAU expression and HNSCC survival. GO and PPI network were established on PLAU-related downstream molecular. The stroma score was deconvoluted for analysis of PLAU’s association with the immune environment. ROC analysis was applied to show the performance of PLAU in predicting HNSCC prognosis. Results PLAU mRNA was significantly elevated, as opposed to its methylation, in HNSCC tumor samples over normal specimens (all p < 0.01). Univariate and multivariate cox analysis showed PLAU could be an independent indicator for HNSCC prognosis. Combining with neck lymph node status, the AUC of PLAU in predicting 5-years overall survival reached to 0.862. GO enrichment analysis showed the major biological process (extracellular matrix organization and the P13K-Akt signaling pathway) may involve to the possible mechanism of PLAU’s function on HNSCC prognosis. Furthermore, PLAU expression was positively correlated with stroma cell score, M1 type macrophages, and negatively associated with CD4 + T cell, Tregs cell, and follicular helper T cell. Conclusions PLAU might be an independent biomarker for predicting outcomes of HNSCC patients. The elevated expression of PLAU was associated with HPV positivity and neck node status. The PI3K-Akt pathway and aberrant proportions of immune cells might underly the mechanism of PLAU’s oncogene role in HNSCC.
Li et al. (2021) Overexpressed PLAU and its potential prognostic value in head and neck squamous cell carcinoma. PeerJ 9:e10746 https://doi.org/10.7717/peerj.10746
A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)—patterns of nucleotide misincorporation
Natural history collections are a valuable resource for molecular taxonomic studies and for examining patterns of evolutionary diversification, particularly in the case of rare or extinct species. However, the recovery of sequence information is often complicated by DNA degradation. This article describes use of the Sequel platform (Pacific Biosciences) to recover the 658 bp barcode region of the mitochondrial cytochrome c oxidase I (COI) gene from 380 butterflies with an average age of 50 years. Nested multiplex PCR was employed for library preparation to facilitate sequence recovery from extracts with low concentrations of highly degraded DNA. By employing circular consensus sequencing (CCS) of short amplicons (circa 150 bp), full-length barcodes could be assembled without a reference sequence, an important advance from earlier protocols which required reference sequences to guide contig assembly. The Sequel protocol recovered COI sequences (499 bp on average) from 318 of 380 specimens (84%), much higher than for Sanger sequencing (26%). Because each read derives from a single molecule, it was also possible to quantify the incidence of substitutions arising from DNA damage. In agreement with past work on sequence changes induced by DNA degradation, the transition C/G → T/A was the most prevalent category of change, but its rate of occurrence (4.58E−4) was so low that it did not impede the recovery of reliable sequences. Because the current protocol recovers COI sequence from most museum specimens, and because sequence fidelity is unaffected by nucleotide misincorporations, large-scale sequence characterization of museum specimens is feasible.
D’Ercole et al. (2021) A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)—patterns of nucleotide misincorporation. PeerJ 9:e10420 https://doi.org/10.7717/peerj.10420
Identification of a novel association for the WWOX/HIF1A axis with gestational diabetes mellitus (GDM)
Background Although the WW-domain-containing oxidoreductase (WWOX)/Hypoxia-inducible factor 1 (HIF1) pathway is a well-known regulator of cellular glucose and energy metabolism in pathophysiological processes, its role in gestational diabetes mellitus (GDM), remains elusive. We undertook this study to determine the effect of WWOX/HIF1A signaling on the expression of glucose metabolism genes in GDM patients. Methods Leukocytes were obtained from 135 pregnant women with (n = 98) or without (n = 37) GDM and, in turn, 3 months (n = 8) and 1 year (n = 12) postpartum. Quantitative RT-PCR was performed to determine gene expression profiles of the WWOX/HIF1A-related genes, including those involved in glucose transport (SLC2A1, SLC2A4), glycolytic pathway (HK2, PKM2, PFK, LDHA), Wnt pathway (DVL2, CTNNB1), and inflammatory response (NFKB1). Results GDM patients displayed a significant downregulation of WWOX with simultaneous upregulation of HIF1A which resulted in approximately six times reduction in WWOX/HIF1A ratio. As a consequence, HIF1A induced genes (SLC2A1, HK2, PFK, PKM) were found to be overexpressed in GDM compared to normal pregnancy and negative correlate with WWOX/HIF1A ratio. The postpartum WWOX expression was higher than during GDM, but its level was comparable to that observed in normal pregnancy. Conclusions The obtained results suggest a significant contribution of the WWOX gene to glucose metabolism in patients with gestational diabetes. Decreased WWOX expression in GDM compared to normal pregnancy, and in particular reduction of WWOX/HIF1A ratio, indicate that WWOX modulates HIF1α activity in normal tissues as described in the tumor. The effect of HIF1α excessive activation is to increase the expression of genes encoding proteins directly involved in the glycolysis which may lead to pathological changes in glucose metabolism observed in gestational diabetes.
Baryla et al. (2021) Identification of a novel association for the WWOX/HIF1A axis with gestational diabetes mellitus (GDM). PeerJ 9:e10604 https://doi.org/10.7717/peerj.10604
Tumor microenvironment related novel signature predict lung adenocarcinoma survival
Background Lung adenocarcinoma (LUAD) is the most common histological type of lung cancers, which is the primary cause of cancer‐related mortality worldwide. Growing evidence has suggested that tumor microenvironment (TME) plays a pivotal role in tumorigenesis and progression. Hence, we investigate the correlation of TME related genes with LUAD prognosis. Method The information of LUAD gene expression data was obtained from The Cancer Genome Atlas (TCGA). According to their immune/stromal scores calculated by the ESTIMATE algorithm, differentially expressed genes (DEGs) were identified. Then, we performed univariate Cox regression analysis on DEGs to obtain genes that are apparently bound up with LUAD survival (SurGenes). Functional annotation and protein-protein interaction (PPI) was also conducted on SurGenes. By validating the SurGenes with data sets of lung cancer from the Gene Expression Omnibus (GEO), 106 TME related SurGenes were generated. Further, intersection analysis was executed between the 106 TME related SurGenes and hub genes from PPI network, PTPRC and CD19 were obtained. Gene Set Enrichment Analysis and CIBERSORT analysis were performed on PTPRC and CD19. Based on the TCGA LUAD dataset, we conducted factor analysis and Step-wise multivariate Cox regression analysis for 106 TME related SurGenes to construct the prognostic model for LUAD survival prediction. The LUAD dataset in GEO (GSE68465) was used as the testing dataset to confirm the prognostic model. Multivariate Cox regression analysis was used between risk score from the prognostic model and clinical parameters. Result A total of 106 TME related genes were collected in our research totally, which were markedly correlated with the overall survival (OS) of LUAD patient. Bioinformatics analysis suggest them mainly concentrated on immune response, cell adhesion, and extracellular matrix. More importantly, among 106 TME related SurGenes, PTPRC and CD19 were highly interconnected nodes among PPI network and correlated with immune activity, exhibiting significant prognostic potential. The prognostic model was a weighted linear combination of the 106 genes, by which the low-OS LUAD samples could be separated from the high-OS samples with success. This model was also able to rebustly predict the situation of survival (training set: p-value < 0.0001, area under the curve (AUC) = 0.649; testing set: p-value = 0.0009, AUC = 0.617). By combining with clinical parameters, the prognostic model was optimized. The AUC achieved 0.716 for 3 year and 0.699 for 5 year. Conclusion A series of TME-related prognostic genes were acquired in this research, which could reflect immune disorders within TME, and PTPRC and CD19 show the potential to be an indicator for LUAD prognosis and tumor microenvironment modulation. The prognostic model constructed base on those prognostic genes presented a high predictive ability, and may have clinical implications in the overall survival prediction of LUAD.
Chen et al. (2021) Tumor microenvironment related novel signature predict lung adenocarcinoma survival. PeerJ 9:e10628 https://doi.org/10.7717/peerj.10628
Comparative analysis and implications of the chloroplast genomes of three thistles (Carduus L., Asteraceae)
Background Carduus, commonly known as plumeless thistles, is a genus in the Asteraceae family that exhibits both medicinal value and invasive tendencies. However, the genomic data of Carduus (i.e., complete chloroplast genomes) have not been sequenced. Methods We sequenced and assembled the chloroplast genome (cpDNA) sequences of three Carduus species using the Illumina Miseq sequencing system and Geneious Prime. Phylogenetic relationships between Carduus and related taxa were reconstructed using Maximum Likelihood and Bayesian Inference analyses. In addition, we used a single nucleotide polymorphism (SNP) in the protein coding region of the matK gene to develop molecular markers to distinguish C. crispus from C. acanthoides and C. tenuiflorus. Results The cpDNA sequences of C. crispus, C. acanthoides, and C. tenuiflorus ranged from 152,342 bp to 152,617 bp in length. Comparative genomic analysis revealed high conservation in terms of gene content (including 80 protein-coding, 30 tRNA, and four rRNA genes) and gene order within the three focal species and members of subfamily Carduoideae. Despite their high similarity, the three species differed with respect to the number and content of repeats in the chloroplast genome. Additionally, eight hotspot regions, including psbI-trnS_GCU, trnE_UUC-rpoB, trnR_UCU-trnG_UCC, psbC-trnS_UGA, trnT_UGU-trnL_UAA, psbT-psbN, petD-rpoA, and rpl16-rps3, were identified in the study species. Phylogenetic analyses inferred from 78 protein-coding and non-coding regions indicated that Carduus is polyphyletic, suggesting the need for additional studies to reconstruct relationships between thistles and related taxa. Based on a SNP in matK, we successfully developed a molecular marker and protocol for distinguishing C. crispus from the other two focal species. Our study provides preliminary chloroplast genome data for further studies on plastid genome evolution, phylogeny, and development of species-level markers in Carduus.
Jung et al. (2021) Comparative analysis and implications of the chloroplast genomes of three thistles (Carduus L., Asteraceae). PeerJ 9:e10687 https://doi.org/10.7717/peerj.10687
Taphonomic patterns mimic biologic structures: diagenetic Liesegang rings in Mesozoic coleoids and coprolites
Because of physiology of coleoids, their fossils preserve soft-tissue-remains more often than other cephalopods. Sometimes, the phosphatized soft-tissues, particularly parts of the muscular mantle, display dark circular patterns. Here, we showcase that these patterns, here documented for fossil coleoids from the Jurassic of Germany and the Cretaceous of Lebanon, superficially resemble chromatophores (which enable living coleoids to alter their coloration). We examined and chemically analyzed the circular structures in these specimens, describe them, and discuss their genesis. Based on their structure and color, we visually differentiate between three types of circles. By comparison with similar structures, we suggest that these structures are not biogenic but Liesegang rings, which formed due to reaction-diffusion processes very soon after death.
Klug et al. (2021) Taphonomic patterns mimic biologic structures: diagenetic Liesegang rings in Mesozoic coleoids and coprolites. PeerJ 9:e10703 https://doi.org/10.7717/peerj.10703
Genome-wide miRNA expression profiling in potato (Solanum tuberosum L.) reveals TOR-dependent post-transcriptional gene regulatory networks in diverse metabolic pathway
Target of rapamycin (TOR) operates as a hub of the signal transduction that integrates nutrient and energy signaling to promote cell proliferation and growth through mediating the transcriptional and post- transcriptional regulator networks in all eukaryotic species. MicroRNAs (miRNAs) are widespread classes of small, single-stranded, non-coding endogenous RNAs and are widely found in eukaryotes, which play a vital role in regulating gene expression by degrading targeted mRNAs or translational repression at post-transcriptional level. Recent studies found that there were necessarily close connections between miRNA and TOR pathways in mammals. However, there is little information about the interplay between the miRNA and TOR in plants. Thus, the aim of this study was to identify potential TOR-miRNA-mRNA regulatory networks in TOR signaling through global mRNA and microRNA expression profiling in potato. Based on the previous high-throughput transcriptome sequencing and filtering, a total of 2,899 genes were significantly differentially expressed in potato under TOR inhibitors treatment. Pathway analysis revealed that these genes were significantly enriched in multiple metabolic processes. Similarly, in the present study, suppression of TOR resulted in 41 miRNAs up-regulated and 45 down-regulated, revealing that TOR plays a crucial role in the regulation of miRNA regulatory network. Furthermore, integrated mRNA and miRNA expression profiling uncovered that these miRNAs participated in large-scale metabolic process in the TOR signal pathway in potato, such as regulation of autophagy and ubiquitination, and biosynthesis of secondary metabolites. Overall, the results shed new insight into TOR related post-transcriptional gene regulatory networks in potato and suggesting TOR-miRNA-targeting genes relevant networks as a potential genetic resource for potato improvement.
Deng et al. (2021) Genome-wide miRNA expression profiling in potato (Solanum tuberosum L.) reveals TOR-dependent post-transcriptional gene regulatory networks in diverse metabolic pathway. PeerJ 9:e10704 https://doi.org/10.7717/peerj.10704
Blood markers of endothelial dysfunction and their correlation to cerebrovascular reactivity in patients with chronic hepatitis C infection
Although liver cirrhosis and hepatocellular carcinoma are major consequences of hepatitis C (HCV), there has been an increasing number of studies examining extrahepatic manifestations, especially those caused by systemic chronic inflammation and metabolic complications that might predispose HCV patients to atherosclerosis and ischemic cerebrovascular disease (CVD). The aim of our study was to assess E-selectin, VCAM-1, ICAM-1 and VEGF-A serum levels in patients with chronic HCV infection and to correlate them with cerebrovascular reactivity. A blood sample was taken from eighteen patients with chronic hepatitis C infection and from the same number of healthy blood donors in the control group. The aim was to analyse markers of endothelial dysfunction and to correlate them with cerebrovascular reactivity expressed as breath-holding index (BHI) determined using transcranial color Doppler. The obtained results revealed significant differences between the groups in all endothelial markers except for the E selectin. While the ICAM-1 and sVCAM-1 were significantly increased in the hepatitis group, VEGF-A was significantly decreased. A significant reduction of 0.5 (95% CI 0.2, 0.8) in the mean BHI was found in the hepatitis group (mean BHI 0.64) compared to controls (mean BHI 1.10). No significant association between the BHI and any of the endothelial markers was found in the control group, while in the hepatitis group, the scatter plot of ICAM-1 vs BHI suggested that the association might be present. In conclusion, the results of this study confirm an association between a chronic HCV infection and altered cerebrovascular reactivity as well as higher levels of markers of endothelial activation (ICAM-1, VCAM-1) as possible indicators of an increased CVD risk.
Pavicic Ivelja et al. (2021) Blood markers of endothelial dysfunction and their correlation to cerebrovascular reactivity in patients with chronic hepatitis C infection. PeerJ 9:e10723 https://doi.org/10.7717/peerj.10723