title: PeerJ description: Articles published in PeerJ link: https://peerj.com/articles/index.rss3?journal=peerj&page=992 creator: info@peerj.com PeerJ errorsTo: info@peerj.com PeerJ language: en title: A new caimanine alligatorid from the Middle Eocene of Southwest Texas and implications for spatial and temporal shifts in Paleogene crocodyliform diversity link: https://peerj.com/articles/10665 last-modified: 2021-01-15 description: Dramatic early Cenozoic climatic shifts resulted in faunal reorganization on a global scale. Among vertebrates, multiple groups of mammals (e.g., adapiform and omomyiform primates, mesonychids, taeniodonts, dichobunid artiodactyls) are well known from the Western Interior of North America in the warm, greenhouse conditions of the early Eocene, but a dramatic drop in the diversity of these groups, along with the introduction of more dry-tolerant taxa, occurred near the Eocene–Oligocene boundary. Crocodyliforms underwent a striking loss of diversity at this time as well. Pre-Uintan crocodyliform assemblages in the central Western Interior are characterized by multiple taxa, whereas Chadronian assemblages are depauperate with only Alligator prenasalis previously known. Crocodyliform diversity through the intervening Uintan and Duchesnean is not well understood. The middle Eocene Devil’s Graveyard Formation (DGF) of southwest Texas provides new data from southern latitudes during that crucial period. A new specimen from the middle member of the DGF (late Uintan–Duchesnean) is the most complete cranial material of an alligatorid known from Paleogene deposits outside the Western Interior. We identify this specimen as a caimanine based on notched descending laminae of the pterygoids posterior to the choanae and long descending processes of the exoccipitals that are in contact with the basioccipital tubera. Unlike Eocaiman cavernensis, the anterior palatine process is rounded rather than quadrangular. The relationships and age of this new taxon support the hypothesis that the modern distribution of caimanines represents a contraction of a more expansive early Cenozoic distribution. We hypothesize that the range of caimanines tracked shifting warm, humid climatic conditions that contracted latitudinally toward the hothouse-icehouse transition later in the Eocene. creator: Michelle R. Stocker creator: Christopher A. Brochu creator: E. Christopher Kirk uri: https://doi.org/10.7717/peerj.10665 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Stocker et al. title: RNA sequencing analysis of FGF2-responsive transcriptome in skin fibroblasts link: https://peerj.com/articles/10671 last-modified: 2021-01-15 description: BackgroundFibroblast growth factor 2 (FGF2) is a highly pleiotropic cytokine with antifibrotic activity in wound healing. During the process of wound healing and fibrosis, fibroblasts are the key players. Although accumulating evidence has suggested the antagonistic effects of FGF2 in the activation process of fibroblasts, the mechanisms by which FGF2 hinders the fibroblast activation remains incompletely understood. This study aimed to identify the key genes and their regulatory networks in skin fibroblasts treated with FGF2.MethodsRNA-seq was performed to identify the differentially expressed mRNA (DEGs) and lncRNA between FGF2-treated fibroblasts and control. DEGs were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Furthermore, the networks between mRNAs and lncRNAs were constructed by Pearson correlation analysis and the networkanalyst website. Finally, hub genes were validated by real time-PCR.ResultsBetween FGF2-treated fibroblasts and control fibroblasts, a total of 1475 DEGs was obtained. These DEGs were mainly enriched in functions such as the ECM organization, cell adhesion, and cell migration. They were mainly involved in ECM-receptor interaction, PI3K-Akt signaling, and the Hippo pathway. The hub DEGs included COL3A1, COL4A1, LOX, PDGFA, TGFBI, and ITGA10. Subsequent real-time PCR, as well as bioinformatics analysis, consistently demonstrated that the expression of ITGA10 was significantly upregulated while the other five DEGs (COL3A1, COL4A1, LOX, PDGFA, TGFBI) were downregulated in FGF2-treated fibroblasts. Meanwhile, 213 differentially expressed lncRNAs were identified and three key lncRNAs (HOXA-AS2, H19, and SNHG8) were highlighted in FGF2-treated fibroblasts.ConclusionThe current study comprehensively analyzed the FGF2-responsive transcriptional profile and provided candidate mechanisms that may account for FGF2-mediated wound healing. creator: Baojin Wu creator: Xinjie Tang creator: Zhaoping Zhou creator: Honglin Ke creator: Shao Tang creator: Ronghu Ke uri: https://doi.org/10.7717/peerj.10671 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Wu et al. title: KEYLINK: towards a more integrative soil representation for inclusion in ecosystem scale models—II: model description, implementation and testing link: https://peerj.com/articles/10707 last-modified: 2021-01-15 description: New knowledge on soil structure highlights its importance for hydrology and soil organic matter (SOM) stabilization, which however remains neglected in many wide used models. We present here a new model, KEYLINK, in which soil structure is integrated with the existing concepts on SOM pools, and elements from food web models, that is, those from direct trophic interactions among soil organisms. KEYLINK is, therefore, an attempt to integrate soil functional diversity and food webs in predictions of soil carbon (C) and soil water balances. We present a selection of equations that can be used for most models as well as basic parameter intervals, for example, key pools, functional groups’ biomasses and growth rates. Parameter distributions can be determined with Bayesian calibration, and here an example is presented for food web growth rate parameters for a pine forest in Belgium. We show how these added equations can improve the functioning of the model in describing known phenomena. For this, five test cases are given as simulation examples: changing the input litter quality (recalcitrance and carbon to nitrogen ratio), excluding predators, increasing pH and changing initial soil porosity. These results overall show how KEYLINK is able to simulate the known effects of these parameters and can simulate the linked effects of biopore formation, hydrology and aggregation on soil functioning. Furthermore, the results show an important trophic cascade effect of predation on the complete C cycle with repercussions on the soil structure as ecosystem engineers are predated, and on SOM turnover when predation on fungivore and bacterivore populations are reduced. In summary, KEYLINK shows how soil functional diversity and trophic organization and their role in C and water cycling in soils should be considered in order to improve our predictions on C sequestration and C emissions from soils. creator: Omar Flores creator: Gaby Deckmyn creator: Jorge Curiel Yuste creator: Mathieu Javaux creator: Alexei Uvarov creator: Sietse van der Linde creator: Bruno De Vos creator: Harry Vereecken creator: Juan Jiménez creator: Olga Vinduskova creator: Andrea Schnepf uri: https://doi.org/10.7717/peerj.10707 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Flores et al. title: Effective ribosomal RNA depletion for single-cell total RNA-seq by scDASH link: https://peerj.com/articles/10717 last-modified: 2021-01-15 description: 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. creator: Danson S.C. Loi creator: Lei Yu creator: Angela R. Wu uri: https://doi.org/10.7717/peerj.10717 license: https://creativecommons.org/licenses/by-nc/4.0 rights: ©2021 Loi et al. title: A preliminary comparative study of percutaneous CT-guided cryoablation with surgical resection for osteoid osteoma link: https://peerj.com/articles/10724 last-modified: 2021-01-15 description: BackgroundThe 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).MethodsFifteen 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.ResultsThe 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.ConclusionCryoablation is a minimally invasive, safe, and effective treatment strategy for osteoid osteoma, and is fully comparable to surgical resection. creator: Liangliang Meng creator: Xiao Zhang creator: Ruijiang Xu creator: Bin Wu creator: Xiaobo Zhang creator: Yingtian Wei creator: Jing Li creator: Husheng Shan creator: Yueyong Xiao uri: https://doi.org/10.7717/peerj.10724 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Meng et al. title: Overexpressed PLAU and its potential prognostic value in head and neck squamous cell carcinoma link: https://peerj.com/articles/10746 last-modified: 2021-01-15 description: BackgroundMetastasis 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.MethodsPLAU 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.ResultsPLAU 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.ConclusionsPLAU 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. creator: Zhexuan Li creator: Changhan Chen creator: Juncheng Wang creator: Ming Wei creator: Guancheng Liu creator: Yuexiang Qin creator: Li She creator: Yong Liu creator: Donghai Huang creator: Yongquan Tian creator: Gangcai Zhu creator: Xin Zhang uri: https://doi.org/10.7717/peerj.10746 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Li et al. title: A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)—patterns of nucleotide misincorporation link: https://peerj.com/articles/10420 last-modified: 2021-01-14 description: 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. creator: Jacopo D’Ercole creator: Sean W.J. Prosser creator: Paul D.N. Hebert uri: https://doi.org/10.7717/peerj.10420 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 D’Ercole et al. title: Identification of a novel association for the WWOX/HIF1A axis with gestational diabetes mellitus (GDM) link: https://peerj.com/articles/10604 last-modified: 2021-01-14 description: BackgroundAlthough 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.MethodsLeukocytes 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).ResultsGDM 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.ConclusionsThe 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. creator: Izabela Baryla creator: Elzbieta Pluciennik creator: Katarzyna Kośla creator: Marzena Wojcik creator: Andrzej Zieleniak creator: Monika Zurawska-Klis creator: Katarzyna Cypryk creator: Lucyna Alicja Wozniak creator: Andrzej K Bednarek uri: https://doi.org/10.7717/peerj.10604 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Baryla et al. title: Tumor microenvironment related novel signature predict lung adenocarcinoma survival link: https://peerj.com/articles/10628 last-modified: 2021-01-14 description: BackgroundLung 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.MethodThe 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.ResultA 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.ConclusionA 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. creator: Juan Chen creator: Rui Zhou uri: https://doi.org/10.7717/peerj.10628 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Chen and Zhou title: Comparative analysis and implications of the chloroplast genomes of three thistles (Carduus L., Asteraceae) link: https://peerj.com/articles/10687 last-modified: 2021-01-14 description: BackgroundCarduus, 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.MethodsWe 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.ResultsThe 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. creator: Joonhyung Jung creator: Hoang Dang Khoa Do creator: JongYoung Hyun creator: Changkyun Kim creator: Joo-Hwan Kim uri: https://doi.org/10.7717/peerj.10687 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Jung et al.