title: PeerJ description: Articles published in PeerJ link: https://peerj.com/articles/index.rss3?journal=peerj&page=926 creator: info@peerj.com PeerJ errorsTo: info@peerj.com PeerJ language: en title: Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells link: https://peerj.com/articles/11063 last-modified: 2021-04-15 description: BackgroundKRAS oncogenes harboring codon G12 and G13 substitutions are considered gatekeeper mutations which drive oncogenesis in many cancers. To date, there are still no target-specific vaccines or drugs available against this genotype, thus reinforcing the need towards the development of targeted therapies such as immunotoxins.MethodsThis study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells.ResultsThe VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells.DiscussionThese results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies. creator: Michelle Yee Mun Teo creator: Jeremy Jeack Ceen Ng creator: Jung Yin Fong creator: Jung Shan Hwang creator: Adelene Ai-Lian Song creator: Renee Lay Hong Lim creator: Lionel Lian Aun In uri: https://doi.org/10.7717/peerj.11063 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Teo et al. title: Bioinformatics analysis for the identification of differentially expressed genes and related signaling pathways in H. pylori-CagA transfected gastric cancer cells link: https://peerj.com/articles/11203 last-modified: 2021-04-15 description: AimHelicobacter pylori cytotoxin-associated protein A (CagA) is an important virulence factor known to induce gastric cancer development. However, the cause and the underlying molecular events of CagA induction remain unclear. Here, we applied integrated bioinformatics to identify the key genes involved in the process of CagA-induced gastric epithelial cell inflammation and can ceration to comprehend the potential molecular mechanisms involved.Materials and MethodsAGS cells were transected with pcDNA3.1 and pcDNA3.1::CagA for 24 h. The transfected cells were subjected to transcriptome sequencing to obtain the expressed genes. Differentially expressed genes (DEG) with adjusted P value < 0.05, — logFC —> 2 were screened, and the R package was applied for gene ontology (GO) enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The differential gene protein–protein interaction (PPI) network was constructed using the STRING Cytoscape application, which conducted visual analysis to create the key function networks and identify the key genes. Next, the Kaplan–Meier plotter survival analysis tool was employed to analyze the survival of the key genes derived from the PPI network. Further analysis of the key gene expressions in gastric cancer and normal tissues were performed based on The Cancer Genome Atlas (TCGA) database and RT-qPCR verification.ResultsAfter transfection of AGS cells, the cell morphology changes in a hummingbird shape and causes the level of CagA phosphorylation to increase. Transcriptomics identified 6882 DEG, of which 4052 were upregulated and 2830 were downregulated, among which q-value < 0.05, FC > 2, and FC under the condition of ≤2. Accordingly, 1062 DEG were screened, of which 594 were upregulated and 468 were downregulated. The DEG participated in a total of 151 biological processes, 56 cell components, and 40 molecular functions. The KEGG pathway analysis revealed that the DEG were involved in 21 pathways. The PPI network analysis revealed three highly interconnected clusters. In addition, 30 DEG with the highest degree were analyzed in the TCGA database. As a result, 12 DEG were found to be highly expressed in gastric cancer, while seven DEG were related to the poor prognosis of gastric cancer. RT-qPCR verification results showed that Helicobacter pylori CagA caused up-regulation of BPTF, caspase3, CDH1, CTNNB1, and POLR2A expression.ConclusionThe current comprehensive analysis provides new insights for exploring the effect of CagA in human gastric cancer, which could help us understand the molecular mechanism underlying the occurrence and development of gastric cancer caused by Helicobacter pylori. creator: Dingyu Chen creator: Chao Li creator: Yan Zhao creator: Jianjiang Zhou creator: Qinrong Wang creator: Yuan Xie uri: https://doi.org/10.7717/peerj.11203 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Chen et al. title: Identification and characterization of melon circular RNAs involved in powdery mildew responses through comparative transcriptome analysis link: https://peerj.com/articles/11216 last-modified: 2021-04-15 description: Circular RNAs (circRNAs) are a class of newly discovered non-coding RNAs that are typically derived from a genome’s exonic, intronic, and intergenic regions. Recent studies of circRNAs in animals and plants have shown that circRNAs are vital in response to various abiotic and biotic stresses. Powdery mildew disease (PM) is a serious fungal disease threatening the melon industry. We performed whole transcriptome sequencing using the leaves of a PM-resistant (M1) and a PM-susceptible (B29) melon to identify circRNAs and determine their molecular functions. A total of 303 circRNAs were identified and >50% circRNAs were derived from exonic regions. Expression levels were significantly altered in 17 and 23 circRNAs after PM infections in B29 and M1, respectively. Melon circRNAs may participate in the response to biotic stimuli, oxidation reduction, metabolic processes, and the regulation of gene expression based on the functional annotation of circRNA parental genes. Furthermore, 27 circRNAs were predicted to be potential targets or ‘sponges’ for 18 microRNAs (miRNAs). Our results are the first to identify and characterize circRNA functions in melon and may contribute to a better understanding of the role and regulatory mechanisms of circRNAs in resisting PM. creator: Jianlei Sun creator: Yumei Dong creator: Chongqi Wang creator: Shouhua Xiao creator: Zigao Jiao creator: Chao Gao uri: https://doi.org/10.7717/peerj.11216 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Sun et al. title: Ectomycorrhizal fungal communities associated with Larix gemelinii Rupr. in the Great Khingan Mountains, China link: https://peerj.com/articles/11230 last-modified: 2021-04-15 description: Larix gemelinii is an important tree species in the Great Khingan Mountains in Northeast China with a high economic and ecological value for its role in carbon sequestration and as a source of lumber and nuts. However, the ectomycorrhizal (EM) fungal diversity and community composition of this tree remain largely undefined. We examined EM fungal communities associated with L. gemelinii from three sites in the Great Khingan Mountains using Illumina Miseq to sequence the rDNA ITS2 region and evaluated the impact of spatial, soil, and climatic variables on the EM fungal community. A total of 122 EM fungal operational taxonomic units (OTUs) were identified from 21 pooled-root samples, and the dominant EM fungal lineages were /tricholoma, /tomentella-thelephora, /suillus-rhizopogon, and /piloderma. A high proportion of unique EM fungal OTUs were present; some abundant OTUs largely restricted to specific sites. EM fungal richness and community assembly were significantly correlated with spatial distance and climatic and soil variables, with mean annual temperature being the most important predictor for fungal richness and geographic distance as the largest determinant for community turnover. Our findings indicate that L. gemelinii has a rich and distinctive EM fungal community contributing to our understanding of the montane EM fungal community structure from the perspective of a single host plant that has not been previously reported. creator: Yonglong Wang creator: Yanling Zhao creator: Ying Xu creator: Jianjun Ma creator: Busayo Joshua Babalola creator: Yongjun Fan uri: https://doi.org/10.7717/peerj.11230 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Wang et al. title: Diversity patterns and drivers of soil microbial communities in urban and suburban park soils of Shanghai, China link: https://peerj.com/articles/11231 last-modified: 2021-04-15 description: BackgroundThe rapid expansion of urbanization leads to significant losses of soil ecological functions. Microbes directly participate in key soil processes and play crucial roles in maintaining soil functions. However, we still have a limited understanding of underlying mechanisms shaping microbial communities and the interactions among microbial taxa in park soils.MethodsIn this study, the community variations of bacteria and fungi in urban and suburban park soils were investigated in Shanghai, China. Real-time PCR and high-throughput Illumina sequencing were used to examine the microbial abundance and community composition, respectively.ResultsThe results showed that soil molecular biomass and fungal abundance in urban park soils were significantly higher than those in suburban park soils, while no significant difference was observed in the bacterial abundance between urban and suburban park soils. The alpha diversity of soil microbes in urban and suburban park soils was similar to each other, except for Chao1 index of fungal communities. The results of similarity analysis (ANOSIM) revealed remarkable differences in the composition of bacterial and fungal communities between urban and suburban park soils. Specifically, park soils in urban areas were enriched with the phyla Methylomirabilota and Verrucomicrobiota, while the relative abundance of Gemmatimonadota was higher in suburban park soils. Moreover, the fungal class Eurotiomycetes was also enriched in urban park soils. Compared with suburban park soils, nodes and average paths of the bacterial and fungal networks were higher in urban park soils, but the number of module hubs and connectors of the bacterial networks and negative interactions among bacterial taxa were lower. Compared with suburban park soils, Acidobacteriota bacterium and Mortierellomycota fungus played more important roles in the ecological networks of urban park soils. Soil available zinc (Zn), available nitrogen (N), pH, and total potassium (K) significantly affected fungal community composition in park soils in Shanghai. Soil available Zn was also the most important factor affecting the bacterial community composition in this study.ConclusionThere were significant differences in the soil molecular biomass, fungal abundance, and the community composition and co-occurrence relations of both soil bacterial and fungal communities between urban and suburban park soils. Soil available Zn played an important part in shaping the structures of both the bacterial and fungal communities in park soils in Shanghai. creator: Weiwei Zhang creator: Jigang Han creator: Haibing Wu creator: Qicheng Zhong creator: Wen Liu creator: Shanwen He creator: Lang Zhang uri: https://doi.org/10.7717/peerj.11231 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Zhang et al. title: Melatonin ameliorates myocardial injury by reducing apoptosis and autophagy of cardiomyocytes in a rat cardiopulmonary bypass model link: https://peerj.com/articles/11264 last-modified: 2021-04-15 description: BackgroundMyocardial injury is a frequent complication after cardiac surgery with cardiopulmonary bypass (CPB). This study aimed to test the hypothesis that melatonin could attenuate myocardial injury in a rat CPB model.MethodsEighteen male Sprague-Dawley rats were randomly divided into three groups, n = 6 for each group: the sham operation (SO) group, CPB group and melatonin group. Rats in the SO group underwent cannulation without CPB, rats in CPB group intraperitoneal injected an equal volume of vehicle daily for 7 days before being subjected to CPB and rats in melatonin group intraperitoneal injected 20 mg/kg of melatonin solution daily for 7 days before being subjected to CPB. After 120 min for CPB, the expression levels of plasma interleukin (IL) -6, IL-1β, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), creatine kinase (CK) -MB and cardiac troponin T (cTnT) were measured. Reactive oxygen species (ROS) were detected by dihydroethidium (DHE). Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Mitochondrial damage and autophagosomes were detected by electron microscopy. Apoptosis inducing factor (AIF) was detected by immunofluorescence. The expression of B cell lymphoma/leukemia2 associated X (Bax), B cell lymphoma/leukemia 2 (Bcl-2), cytochrome C (Cyto-C), cleaved caspase-9, AKT, p-AKT, signal transducer and activator of transcription 3 (STAT3), p-STAT3, LC3, P62, mechanistic target of rapamycin kinase (mTOR), p-mTOR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using western blotting.ResultsMelatonin significantly decreased the levels of IL-1β, IL-6, MDA, CK-MB and cTnT and increased the levels of SOD and GSH-Px, all of which were altered by CPB. Melatonin reduced cardiomyocyte superoxide production, the apoptosis index and autophagy in cardiomyocytes induced by CPB. The AKT, STAT3 and mTOR signaling pathways were activated by melatonin during CPB.ConclusionMelatonin may serve as a cardioprotective factor in CPB by inhibiting oxidative damage, apoptosis and autophagy. The AKT, STAT3 and mTOR signaling pathways were involved in this process. creator: Xiaolin Huang creator: Jian Hou creator: Suiqing Huang creator: Kangni Feng creator: Yuan Yue creator: Huayang Li creator: Shaojie Huang creator: Mengya Liang creator: Guangxian Chen creator: Zhongkai Wu uri: https://doi.org/10.7717/peerj.11264 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Huang et al. title: Explorations of avoidance and approach coping and perceived stress with a computer-based avatar task: detrimental effects of resignation and withdrawal link: https://peerj.com/articles/11265 last-modified: 2021-04-15 description: BackgroundIndividuals differ in how they react to stress or trauma through different coping styles in which they may deal directly with a stressor by adopting approach coping styles or disengage with a stressor by utilizing avoidant coping styles. Avoidant coping styles have been linked to adverse outcomes including psychological distress, anxiety disorders, and post-traumatic stress disorder (PTSD). Recently, avoidance coping styles as measured by a subset of items on the Brief COPE were found to have a weak positive relationship with performance on a computer-based avatar task which is related to avoidant personality temperaments. This avatar task was developed as an alternative for paper and pencil self-report inventories for measuring avoidant tendencies based on possible response biases of avoidant individuals. In the current study, avoidance and approach coping styles as measured by the Brief Approach/Avoidance Coping Questionnaire (BACQ) were compared to avoidant coping as measured by the Brief COPE and performance on the avatar task. In addition to approach and avoidance coping, the BACQ also measures active avoidance coping (i.e., diversion) and passive avoidance coping (i.e., resignation and withdrawal). The relationships between approach and avoidance coping and performance on the avatar task were also analyzed with the outcome of perceived stress as measured by the Perceived Stress Scale (PSS).MethodsOne hundred undergraduates voluntarily completed the BACQ, the Brief COPE, and the PSS. Participants also completed a computer-based task in which they guided an avatar through a series of social situations where they indicated how they would interact with or avoid interacting with strangers.ResultsApproach coping had a weak negative relationship to avoidance coping as measured by the BACQ and the Brief COPE. Performance on the avatar task had a moderate positive relationship with avoidance coping (diversion as well as resignation and withdrawal) as measured by the BACQ and a moderate negative relationship with approach coping as measured by the BACQ. A model including only approach, diversion, and resignation and withdrawal coping best predicted performance on the avatar task in a linear regression model. While resignation and withdrawal coping and diversion coping had moderate positive relationships to avatar task scores, only resignation and withdrawal had a strong positive relationship to perceived stress. A model than included only resignation and withdrawal coping best predicted perceived stress in a linear regression model. Overall, passive avoidant coping styles (i.e., resignation and withdrawal), but not active avoidant coping style (i.e., diversion), were related to perceived stress. These results support the continued study of multiple aspects of avoidant coping styles as well as the avatar task to increase our understanding of the maladaptive effects of excessive avoidance in the face of stress. creator: M. Todd Allen uri: https://doi.org/10.7717/peerj.11265 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Allen title: Conjuring up creativity: the effect of performing magic tricks on divergent thinking link: https://peerj.com/articles/11289 last-modified: 2021-04-15 description: Research suggests that learning to perform magic tricks can promote both physical and psychological wellbeing. The current study extended this work by examining the impact of learning magic tricks on divergent thinking. A group of 10- to 11-year-old children completed Guilford’s Alternate Uses Test both before and after participating in either a magic-based, or art-based, activity. As predicted, compared to the art-based activity, the magic-based activity resulted in a significantly greater increase in both AUT Fluency and AUT Originality scores. Rosenberg’s Self-Esteem Scale and Dweck’s Implicit Theories of Intelligence Scale for Children was also completed after each activity, and participants’ self-esteem scores were higher after the art-based activity than the magic-based activity. In an exploratory aspect of the study, the AUT was re-administered to both groups three weeks later, and yielded no significant differences. The practical and theoretical implications of these findings are discussed, along with recommendations for future research. creator: Richard Wiseman creator: Amy Wiles creator: Caroline Watt uri: https://doi.org/10.7717/peerj.11289 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Wiseman et al. title: Morphological grounds for the obligate aerial respiration of an aquatic snail: functional and evolutionary perspectives link: https://peerj.com/articles/10763 last-modified: 2021-04-14 description: The freshwater caenogastropod family Ampullariidae is emerging as a model for a variety of studies, among them, the evolution of terrestriality. A common character of the family is that all its members bear a lung while retaining the ancestral gill. This ensures that many ampullariids are able to inhabit poorly oxygenated waters, to bury in the mud during estivation, and to temporarily leave the water, in some species for oviposition. To these characters Pomacea canaliculata (Caenogastropoda, Ampullariidae) adds that is an obligate air-breather. In a recent paper, we showed the gill epithelium of P. canaliculata has a set of characteristics that suggest its role for oxygen uptake may be less significant than its role in ionic/osmotic regulation and immunity. We complement here our morphological investigation on the respiratory organs of P. canaliculata by studying the lung of this species at the anatomical (3D reconstructions of the blood system and nerve supply), histological and ultrastructural levels. The circulation of the gill and the lung are interconnected so that the effluence of blood from the gill goes to the lung where it completes oxygenation. Besides that, we found the lung cavity is lined by a pavement epithelium that encloses an anastomosing network of small blood spaces resting over a fibromuscular layer, which altogether form the respiratory lamina. The pavement cells form a blood-gas barrier that is 80–150 nm thick and thus fulfils the requirements for an efficient gas exchanger. Tufts of ciliary cells, together with some microvillar and secretory cells, are interspersed in the respiratory lamina. Rhogocytes, which have been proposed to partake in metal depuration and in the synthesis of hemocyanin in other gastropods, were found below the respiratory lamina, in close association with the storage cell tissue. In light of these findings, we discuss the functional role of the lung in P. canaliculata and compare it with that of other gastropods. Finally, we point to some similarities in the pattern of the evolution of air dependence in this family. creator: Cristian Rodriguez creator: Guido I. Prieto creator: Israel A. Vega creator: Alfredo Castro-Vazquez uri: https://doi.org/10.7717/peerj.10763 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Rodriguez et al. title: Unraveling the genetic diversity and structure of Quercus liaotungensis population through analysis of microsatellite markers link: https://peerj.com/articles/10922 last-modified: 2021-04-14 description: BackgroundQuercus liaotungensis Koidz. is an ecologically and economically important tree species widely distributed in Northern China. However, the effective assessment, utilization, and protection of Q. liaotungensis resources remain unexplored.MethodsIn total, 120 samples obtained from 12 Q. liaotungensis populations of Northern China were investigated for genetic diversity and structure using 19 simple sequence repeat (SSR) primer pairs.ResultsThe total number of alleles detected was 293, the average number of effective allele (Ne) was 6.084, the genetic differentiation coefficient (Fst) was 0.033, and the mean observed heterozygosity (Ho) and expected heterozygosity (He) were 0.690 and 0.801, respectively. Moreover, analysis of molecular variance (AMOVA) showed a 5.5% genetic variation among 12 Q. liaotungensis populations, indicating that a high level of genetic diversity and a low degree of genetic differentiation among Q. liaotungensis populations. STRUCTURE and cluster analysis divided the 12 Q. liaotungensis populations into the following three subpopulations: Bashang Plateau subpopulation (SH), Liaodong Peninsula subpopulation (NC), and Loess Plateau subpopulation (other 10 populations). The cluster analysis based on 19 climatic factors was consistent with the genetic structure. A positive correlation was found between genetic distance and geographical distance (r = 0.638, p = 0.028) by the Mantel test, and two boundaries were found among the 12 Q. liaotungensis populations by the Barrier analysis, indicating that Q. liaotungensis populations existed isolated by geographical distance and physical barrier.ConclusionThis study suggests that geographical isolation, physical barrier, climatic types, and natural hybridization promote the formation of genetic structures, which can contribute to future protection and genetic improvement of Q. liaotungensis. creator: Bin Guo creator: Xiangchun Hao creator: Lijun Han creator: Yu Zhai creator: Shuai Zhou creator: Si Chen creator: Da Ren creator: Xinmin An uri: https://doi.org/10.7717/peerj.10922 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Guo et al.