Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines
Background Biobanking of cell lines is a promising tool of support for wildlife conservation. In particular, the ability to preserve fibroblast cell lines derived from collared peccaries is of significance as these wild mammals are unique to the Americas and play a large role in maintaining the ecosystem. We identified collared peccary fibroblasts by immunofluorescence and evaluated their morphology, growth and adherence capacity. Further, we monitored the viability and metabolic activity of the fibroblasts to determine the effects of passage number and cryopreservation on establishment of cell lines. Methods Skin biopsies were collected from the peripheral ear region from five adult animals in captivity. Initially, cells were isolated from fragments and cultured in the Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 2% antibiotic–antimycotic solution under a controlled atmosphere (38.5 °C, 5% CO2). We evaluated the maintenance of primary cells for morphology, adherence capacity of explants, explants in subconfluence, cell growth and absence of contamination. Moreover, we identified the fibroblast cells by immunofluorescence. Additionally, to evaluate the influence of the number of passages (first, third and tenth passage) and cryopreservation on establishment of cell lines, fibroblasts were analysed for the viability, metabolic activity, population doubling time (PDT), levels of reactive oxygen species (ROS), and mitochondrial membrane potential (ΔΨm). Results All explants (20/20) adhered to the dish in 2.4 days ± 0.5 with growth around the explants in 4.6 days ± 0.7, and subconfluence was observed within 7.8 days ± 1.0. Moreover, by morphology and immunocytochemistry analyses, cells were identified as fibroblasts which presented oval nuclei, a fusiform shape and positive vimentin staining. No contamination was observed after culture without antibiotics and antifungals for 30 days. While there was no difference observed for cell viability after the passages (first vs. third: P = 0.98; first vs. tenth: P = 0.76; third vs. tenth: P = 0.85), metabolic activity was found to be reduced in the tenth passage (23.2 ± 12.1%) when compared to that in the first and third passage (100.0 ± 24.4%, P = 0.006). Moreover, the cryopreservation did not influence the viability (P = 0.11), metabolic activity (P = 0.77), or PDT (P = 0.11). Nevertheless, a greater ΔΨm (P = 0.0001) was observed for the cryopreserved cells (2.12 ± 0.14) when compared to that in the non-cryopreserved cells (1.00 ± 0.05). Additionally, the cryopreserved cells showed greater levels of intracellular ROS after thawing (1.69 ± 0.38 vs. 1.00 ± 0.22, P = 0.04). Conclusions This study is the first report on isolation, characterization and cryopreservation of fibroblasts from collared peccaries. We showed that adherent cultures were efficient for obtaining fibroblasts, which can be used as donor cells for nuclei for species cloning and other applications.
Borges et al. (2020) Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines. PeerJ 8:e9136 https://doi.org/10.7717/peerj.9136
Identification of DEGs and transcription factors involved in H. pylori-associated inflammation and their relevance with gastric cancer
Background Previous studies have indicated that chronic inflammation linked to H. pylori infection is the leading causes for gastric cancer (GC). However, the exact mechanism is not entirely clear until now. Purpose To identify the key molecules and TFs involved in H. pylori infection and to provide new insights into H. pylori-associated carcinogenesis and lay the groundwork for the prevention of GC. Results GO and KEGG analysis revealed that the DEGs of Hp+-NAG were mainly associated with the immune response, chemokine activity, extracellular region and rheumatoid arthritis pathway. The DEGs of Hp+-AG-IM were related to the apical plasma membrane, intestinal cholesterol absorption, transporter activity and fat digestion and absorption pathway. In Hp+-NAG network, the expression of TNF, CXCL8, MMP9, CXCL9, CXCL1, CCL20, CTLA4, CXCL2, C3, SAA1 and FOXP3, JUN had statistical significance between normal and cancer in TCGA database. In Hp+-AG-IM network the expression of APOA4, GCG, CYP3A4, XPNPEP2 and FOXP3, JUN were statistically different in the comparison of normal and cancer in TCGA database. FOXP3 were negatively associated with overall survival, and the association for JUN was positive. Conclusion The current study identified key DEGs and their transcriptional regulatory networks involved in H. pylori-associated NAG, AG-IM and GC and found that patients with higher expressed FOXP3 or lower expressed JUN had shorter overall survival time. Our study provided new directions for inflammation-associated oncogenic transformation involved in H. pylori infection.
Yin et al. (2020) Identification of DEGs and transcription factors involved in H. pylori-associated inflammation and their relevance with gastric cancer. PeerJ 8:e9223 https://doi.org/10.7717/peerj.9223
Incorporating reef fish avoidance behavior improves accuracy of species distribution models
Species distribution models (SDMs) are used to interpret and map fish distributions based on habitat variables and other drivers. Reef fish avoidance behavior has been shown to vary in the presence of divers and is primarily driven by spearfishing pressure. Diver avoidance behavior or fish wariness may spatially influence counts and other descriptive measures of fish assemblages. Because fish assemblage metrics are response variables for SDMs, measures of fish wariness may be useful as predictors in SDMs of fishes targeted by spearfishing. We used a diver operated stereo-video system to conduct fish surveys and record minimum approach distance (MAD) of targeted reef fishes inside and outside of two marine reserves on the island of Oʻahu in the main Hawaiian Islands. By comparing MAD between sites and management types we tested the assumption that it provides a proxy for fish wariness related to spearfishing pressure. We then compared the accuracy of SDMs which included MAD as a predictor with SDMs that did not. Individual measures of MAD differed between sites though not management types. When included as a predictor, MAD averaged at the transect level greatly improved the accuracy of SDMs of targeted fish biomass.
Spatial and Geographic Information Science
Stamoulis et al. (2020) Incorporating reef fish avoidance behavior improves accuracy of species distribution models. PeerJ 8:e9246 https://doi.org/10.7717/peerj.9246
Addressing initialisation uncertainty for end-to-end ecosystem models: application to the Chatham Rise Atlantis model
Ecosystem models require the specification of initial conditions, and these initial conditions have some level of uncertainty. It is important to allow for uncertainty when presenting model results, because it reduces the risk of errant or non-representative results. It is crucial that model results are presented as an envelope of what is likely, rather than presenting only one instance. We perturbed the initial conditions of the Chatham Rise Atlantis model and analysed the effect of this uncertainty on the model’s dynamics by comparing the model outputs resulting from many initial condition perturbations. At the species group level, we found some species groups were more sensitive than others, with lower trophic level species groups generally more sensitive to perturbations of the initial conditions. We recommend testing for robust system dynamics by assessing the consistency of ecosystem indicators in response to fishing pressure under perturbed initial conditions. In any set of scenarios explored using complex end-to-end ecosystem models, we recommend that associated uncertainty analysis be included with perturbations of the initial conditions.
McGregor et al. (2020) Addressing initialisation uncertainty for end-to-end ecosystem models: application to the Chatham Rise Atlantis model. PeerJ 8:e9254 https://doi.org/10.7717/peerj.9254
Identification of gene expression and DNA methylation of SERPINA5 and TIMP1 as novel prognostic markers in lower-grade gliomas
Background Lower-grade gliomas (LGGs) is characteristic with great difference in prognosis. Due to limited prognostic biomarkers, it is urgent to identify more molecular markers to provide a more objective and accurate tumor classification system for LGGs. Methods In the current study, we performed an integrated analysis of gene expression data and genome-wide methylation data to determine novel prognostic genes and methylation sites in LGGs. Results To determine genes that differentially expressed between 44 short-term survivors (<2 years) and 48 long-term survivors (≥2 years), we searched LGGs TCGA RNA-seq dataset and identified 106 differentially expressed genes. SERPINA5 and TIMP1 were selected for further study. Kaplan–Meier plots showed that SERPINA5 and TIMP1 expression were significantly correlated with overall survival (OS) and relapse-free survival (RFS) in TCGA LGGs patients. We next validated the correlation between the candidate genes expression and clinical outcome in CGGA LGGs patients. Multivariate analysis showed that TIMP1 mRNA expression had a significant prognostic value independent of other variables (HR = 4.825, 95% CI = 1.370–17.000, P = 0.014). Then, differential methylation sites were identified from differentially candidate gene expression groups, and all four methylation sites were significantly negatively correlated with gene expression (spearman r < − 0.5, P < 0.0001). Moreover, hyper-methylation of four methylation sites indicated better OS (P < 0.05), and three of them also shown statistical significantly association with better RFS, except for SERPINA5 cg15509705 (P = 0.0762). Conclusion Taken together, these findings indicated that the gene expression and methylation of SERPINA5 and TIMP1 may serve as prognostic predictors in LGGs and may help to precise the current histology-based tumors classification system and to provide better stratification for future clinical trials.
Zeng et al. (2020) Identification of gene expression and DNA methylation of SERPINA5 and TIMP1 as novel prognostic markers in lower-grade gliomas. PeerJ 8:e9262 https://doi.org/10.7717/peerj.9262
Identification of differentially expressed methylated genes in melanoma versus nevi using bioinformatics methods
Background Melanoma is a highly invasive malignant skin tumor. While melanoma may share some similarities with that of melanocytic nevi, there also exist a number of distinct differences between these conditions. An analysis of these differences may provide a means to more effectively evaluate the etiology and pathogenesis of melanoma. In particular, differences in aberrant methylation expression may prove to represent a critical distinction. Methods Data from gene expression datasets (GSE3189 and GSE46517) and gene methylation datasets (GSE86355 and GSE120878) were downloaded from the GEO database. GEO2R was used to obtain differentially expressed genes (DEGs) and differentially methylation genes (DMGs). Function and pathway enrichment of selected genes were performed using the DAVID database. A protein-protein interaction (PPI) network was constructed by STRING while its visualization was achieved with use of cytoscape. Primary melanoma samples from TCGA were used to identify significant survival genes. Results There was a total of 199 genes in the hypermethylation-low expression group, while 136 genes in the hypomethylation-high expression group were identified. The former were enriched in the biological processes of transcription regulation, RNA metabolism and regulation of cell proliferation. The later were highly involved in cell cycle regulation. 13 genes were screened out after survival analysis and included: ISG20, DTL, TRPV2, PLOD3, KIF3C, DLGAP4, PI4K2A, WIPI1, SHANK2, SLC16A10, GSTA4O, LFML2A and TMEM47. Conclusion These findings reveal some of the methylated differentially expressed genes and pathways that exist between melonoma and melanocytic nevi. Moreover, we have identified some critical genes that may help to improve the diagnosis and treatment of melanoma.
He et al. (2020) Identification of differentially expressed methylated genes in melanoma versus nevi using bioinformatics methods. PeerJ 8:e9273 https://doi.org/10.7717/peerj.9273
The linkages of plant, litter and soil C:N:P stoichiometry and nutrient stock in different secondary mixed forest types in the Qinling Mountains, China
Background Carbon (C), nitrogen (N) and phosphorus (P) stoichiometric ratios are important indicators of ecosystem function and productivity. However, few studies have assessed the nutrient relationship between plant, litter and soil, and the nutrient stock in different secondary mixed forest types. Methods We investigated the C, N and P concentrations and stoichiometric ratios in trees, understory plants, litter and soil layers in three different secondary mixed forest types (broadleaf mixed forests (BM), broadleaf-conifer mixed forests (BCM) and coniferous mixed forests (CM)) in the Qinling Mountains. Results The results showed that significant differences in C:N:P stoichiometry were detected in multiple organs in the vegetation layers in the different forest types. Trees, shrubs and herbs all allocated more N and P in leaves and had a higher N:P ratio in leaves than in other organs. The C concentrations, C:N ratios and C:P ratios of all tree organs showed a decreasing order: BM < BCM < CM, while the N and P concentrations showed an increasing order: BM > BCM > CM. For litter and soil, BM had generally higher N and P concentrations than those of BCM and CM. The highest N and P stock was in tree branches-not in the stem, which had the highest biomass (except for P in CM). Compared with other forest types, CM stored more nutrients in the labile litter layer, while BM stored more nutrients in the stable soil layer. The net ecosystem nutrient element stock in BM was generally higher than that in BCM and CM. The C, N and P concentrations and stoichiometry in the plant organs, litter and soil were significantly correlated. Conclusion Our findings demonstrate that nutrient concentrations in plant organs, litter and soil are tightly linked in secondary mixed forests.
Pang et al. (2020) The linkages of plant, litter and soil C:N:P stoichiometry and nutrient stock in different secondary mixed forest types in the Qinling Mountains, China. PeerJ 8:e9274 https://doi.org/10.7717/peerj.9274
Association of carotid intima-media thickness with the risk of sudden sensorineural hearing loss
Cardiovascular factors are associated with the pathophysiological features and risk of sudden sensorineural hearing loss (SSNHL). However, little is known about the link between carotid intima-media thickness (IMT), SSNHL risk, and their respective treatment outcomes. In this study, we retrospectively reviewed 47 SSNHL cases and 33 control subjects from a single medical center and compared their demographic data and clinical characteristics, including their carotid IMT and audiological data. Of the 80 enrolled subjects, the proportion of those with high carotid IMT was greater in the SSNHL group (53.2%) than in the control group (21.2%), with an odds ratio (OR) of 4.22 (95% confidence interval (CI) [1.53–11.61], P = 0.004). Notably, high carotid IMT was more common in female SSNHL patients than females in the control group (54.2% vs. 12.5%; OR, 8.27 (95% CI [1.53–44.62]), P = 0.008), particularly in female patients ≥50 years of age (75% vs. 25%; OR, 9.0 (95% CI [1.27–63.9]), P = 0.032). The multivariate regression analyses showed the association between high carotid IMT and SSNHL with an adjusted OR of 4.655 (95% CI [1.348–16.076], P = 0.015), particularly in female SSNHL patients (adjusted OR, 9.818 (95% CI [1.064–90.587], P = 0.044). The carotid IMT was not associated with the treatment outcomes of SSNHL. Our results indicate that early-stage atherosclerosis may be associated with SSNHL, particularly in female patients more than 50 years old.
Ho et al. (2020) Association of carotid intima-media thickness with the risk of sudden sensorineural hearing loss. PeerJ 8:e9276 https://doi.org/10.7717/peerj.9276
Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2
Background Highly sensitive real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-CoV-2. However, they are costly. Loop-mediated isothermal amplification (LAMP) assay has emerged as a novel alternative isothermal amplification method for the detection of nucleic acid. Methods A rapid, sensitive and specific real-time reverse transcription LAMP (RT-LAMP) assay was developed for SARS-CoV-2 detection. Results This assay detected one copy/reaction of SARS-CoV-2 RNA in 30 min. Both the clinical sensitivity and specificity of this assay were 100%. The RT-LAMP showed comparable performance with RT-qPCR. Combining simplicity and cost-effectiveness, this assay is therefore recommended for use in resource resource-limited settings.
Lau et al. (2020) Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2. PeerJ 8:e9278 https://doi.org/10.7717/peerj.9278
Ginsenoside panaxatriol reverses TNBC paclitaxel resistance by inhibiting the IRAK1/NF-κB and ERK pathways
Background Paclitaxel (PTX) resistance is a major obstacle in the treatment of triple-negative breast cancer (TNBC). Previously, we have reported that interleukin-1 receptor-associated kinase 1 (IRAK1) and its downstream pathways are associated with PTX resistance in TNBC cells. In this study, we sought to investigate the combination treatment of ginsenoside panaxatriol (GPT), one of the main active components in Panax ginseng, with PTX on viability and apoptosis of TNBC PTX resistant cells, and explore the role of IRAK1 mediated signaling pathways in the therapeutic effects. Methods CellTiter-Glo and colony formation assays were used to assess cell viability. Flow cytometry was used to analyze subG1 and apoptosis. Western blot was used to detect expressions of proteins involved in apoptosis and the IRAK1/NF-κB and ERK pathways. The mRNA expression of inflammatory cytokines, S100A7/8/9 and cancer stem cell (CSC)-related genes were examined by qPCR. Stem cells were identified by tumor sphere assay. Cell invasion ability was examined by transwell assay. Results We show that GPT inhibits MDA-MB-231 PTX resistant (MB231-PR) cell viability in a dose-dependent manner. When combined with PTX, GPT synergistically causes more cell death, induces subG1 accumulation and cell apoptosis. Besides, up-regulation of BAX/BCL-2 ratio, and down-regulation of MCL-1 are also observed. Moreover, this combination inhibits IRAK1, NF-κB and ERK1/2 activation, and leads to down-regulation of inflammatory cytokines (IL6, IL8, CXCL1, CCL2), S100A7/9 and CSC-related genes (OCT4, SOX2, NANOG, ALDH1, CD44) expression. In addition, the combination treatment suppresses MB231-PR cell invasion ability, and impairs tumor sphere growth both in MB231-PR and SUM159 PTX resistant (SUM159-PR) cells. Conclusion Our study demonstrates that GPT can resensitize TNBC PTX resistant cells to PTX by inhibiting the IRAK1/NF-κB and ERK pathways and reducing stem cell characteristics.
Wang et al. (2020) Ginsenoside panaxatriol reverses TNBC paclitaxel resistance by inhibiting the IRAK1/NF-κB and ERK pathways. PeerJ 8:e9281 https://doi.org/10.7717/peerj.9281