title: PeerJ description: Articles published in PeerJ link: https://peerj.com/articles/index.rss3?journal=peerj&page=367 creator: info@peerj.com PeerJ errorsTo: info@peerj.com PeerJ language: en title: Inhibition of non-small cell lung cancer (NSCLC) proliferation through targeting G6PD link: https://peerj.com/articles/16503 last-modified: 2023-12-05 description: BackgroundMounting evidence has linked cancer metabolic reprogramming with altered redox homeostasis. The pentose phosphate pathway (PPP) is one of the key metabolism-related pathways that has been enhanced to promote cancer growth. The glucose 6-phosphate dehydrogenase (G6PD) of this pathway generates reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is essential for controlling cellular redox homeostasis.ObjectiveThis research aimed to investigate the growth-promoting effects of G6PD in non-small cell lung cancer (NSCLC).MethodsClinical characteristics and G6PD expression levels in lung tissues of 64 patients diagnosed with lung cancer at the King Chulalongkorn Memorial Hospital (Bangkok, Thailand) during 2009-2014 were analyzed. G6PD activity in NSCLC cell lines, including NCI-H1975 and NCI-H292, was experimentally inhibited using DHEA and siG6PD to study cancer cell proliferation and migration.ResultsThe positive expression of G6PD in NSCLC tissues was detected by immunohistochemical staining and was found to be associated with squamous cells. G6PD expression levels and activity also coincided with the proliferation rate of NSCLC cell lines. Suppression of G6PD-induced apoptosis in NSCLC cell lines by increasing Bax/Bcl-2 ratio expression. The addition of D-(-)-ribose, which is an end-product of the PPP, increased the survival of G6PD-deficient NSCLC cell lines.ConclusionCollectively, these findings demonstrated that G6PD might play an important role in the carcinogenesis of NSCLC. Inhibition of G6PD might provide a therapeutic strategy for the treatment of NSCLC. creator: Makamas Chanda creator: Pornchai Anuntasomboon creator: Komkrit Ruangritchankul creator: Poonlarp Cheepsunthorn creator: Chalisa L. Cheepsunthorn uri: https://doi.org/10.7717/peerj.16503 license: https://creativecommons.org/licenses/by-nc/4.0 rights: ©2023 Chanda et al. title: Fascin actin-bundling protein 1 regulates non-small cell lung cancer progression by influencing the transcription and splicing of tumorigenesis-related genes link: https://peerj.com/articles/16526 last-modified: 2023-12-05 description: BackgroundHigh mortality rates are prevalent among patients with non-small-cell lung cancer (NSCLC), and effective therapeutic targets are key prognostic factors. Fascin actin-bundling protein 1 (FSCN1) promotes NSCLC; however, its role as an RNA-binding protein in NSCLC remains unexplored. Therefore, we aimed to explore FSCN1 expression and function in A549 cells.MethodWe screened for alternative-splicing events and differentially expressed genes (DEGs) after FSCN1 silence via RNA-sequencing (RNA-seq). FSCN1 immunoprecipitation followed by RNA-seq were used to identify target genes whose mRNA expression and pre-mRNA alternative-splicing levels might be influenced by FSCN1.ResultsSilencing FSCN1 in A549 cells affected malignant phenotypes; it inhibited proliferation, migration, and invasion, and promoted apoptosis. RNA-seq analysis revealed 2,851 DEGs and 3,057 alternatively spliced genes. Gene ontology-based functional enrichment analysis showed that downregulated DEGs and alternatively splicing genes were enriched for the cell-cycle. FSCN1 promoted the alternative splicing of cell-cycle-related mRNAs involved in tumorigenesis (i.e., BCCIP, DLGAP5, PRC1, RECQL5, WTAP, and SGO1). Combined analysis of FSCN1 RNA-binding targets and RNA-seq data suggested that FSCN1 might affect ACTG1, KRT7, and PDE3A expression by modulating the pre-mRNA alternative-splicing levels of NME4, NCOR2, and EEF1D, that were bound to long non-coding RNA transcripts (RNASNHG20, NEAT1, NSD2, and FTH1), which were highly abundant. Overall, extensive transcriptome analysis of gene alternative splicing and expression levels was performed in cells transfected with FSCN1 short-interfering RNA. Our data provide global insights into the regulatory mechanisms associated with the roles of FSCN1 and its target genes in lung cancer. creator: Qingchao Sun creator: Ruixue Liu creator: Haiping Zhang creator: Liang Zong creator: Xiaoliang Jing creator: Long Ma creator: Jie Li creator: Liwei Zhang uri: https://doi.org/10.7717/peerj.16526 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2023 Sun et al. title: Schrödinger’s yeast: the challenge of using transformation to compare fitness among Saccharomyces cerevisiae that differ in ploidy or zygosity link: https://peerj.com/articles/16547 last-modified: 2023-12-05 description: How the number of genome copies modifies the effect of random mutations remains poorly known. In yeast, researchers have investigated these effects for knock-out or other large-effect mutations, but have not accounted for differences at the mating-type locus. We set out to compare fitness differences among strains that differ in ploidy and/or zygosity using a panel of spontaneously arising mutations acquired in haploid yeast from a previous study. To ensure no genetic differences, even at the mating-type locus, we embarked on a series of transformations, which first sterilized and then temporarily introduced plasmid-borne mating types. Despite these attempts to equalize the haplotypes, fitness variation introduced during transformation swamped the differences among the original mutation-accumulation lines. While colony size looked normal, we observed a bi-modality in the maximum growth rate of our transformed yeast and determined that many of the slow growing lines were respiratory deficient (“petite”). Not previously reported, we found that yeast that were TID1/RDH54 knockouts were less likely to become petite. Even for lines with the same petite status, however, we found no correlation in fitness between the two replicate transformations performed. These results pose a challenge for any study using transformation to measure the fitness effect of genetic differences among strains. By attempting to hold haplotypes constant, we introduced more mutations that overwhelmed our ability to measure fitness differences between the genetic states. In this study, we transformed over one hundred different lines of yeast, using two independent transformations, and found that this common laboratory procedure can cause large changes to the microbe studied. Our study provides a cautionary tale of the need to use multiple transformants in fitness assays. creator: Linnea Sandell creator: Stephan G. König creator: Sarah P. Otto uri: https://doi.org/10.7717/peerj.16547 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2023 Sandell et al. title: pH-sensing G protein-coupled orphan receptor GPR68 is expressed in human cartilage and correlates with degradation of extracellular matrix during OA progression link: https://peerj.com/articles/16553 last-modified: 2023-12-05 description: BackgroundOsteoarthritis (OA) is a debilitating joints disease affecting millions of people worldwide. As OA progresses, chondrocytes experience heightened catabolic activity, often accompanied by alterations in the extracellular environment’s osmolarity and acidity. Nevertheless, the precise mechanism by which chondrocytes perceive and respond to acidic stress remains unknown. Recently, there has been growing interest in pH-sensing G protein-coupled receptors (GPCRs), such as GPR68, within musculoskeletal tissues. However, function of GPR68 in cartilage during OA progression remains unknown. This study aims to identify the role of GPR68 in regulation of catabolic gene expression utilizing an in vitro model that simulates catabolic processes in OA.MethodsWe examined the expression of GPCR by analyzing high throughput RNA-Seq data in human cartilage isolated from healthy donors and OA patients. De-identified and discarded OA cartilage was obtained from joint arthroplasty and chondrocytes were prepared by enzymatic digestion. Chondrocytes were treated with GPR68 agonist, Ogerin and then stimulated IL1β and RNA isolation was performed using Trizol method. Reverse transcription was done using the cDNA synthesis kit and the expression of GPR68 and OA related catabolic genes was quantified using SYBR® green assays.ResultsThe transcriptome analysis revealed that pH sensing GPCR were expressed in human cartilage with a notable increase in the expression of GPR68 in OA cartilage which suggest a potential role for GPR68 in the pathogenesis of OA. Immunohistochemical (IHC) and qPCR analyses in human cartilage representing various stages of OA indicated a progressive increase in GPR68 expression in cartilage associated with higher OA grades, underscoring a correlation between GPR68 expression and the severity of OA. Furthermore, IHC analysis of Gpr68 in murine cartilage subjected to surgically induced OA demonstrated elevated levels of GPR68 in knee cartilage and meniscus. Using IL1β stimulated in vitro model of OA catabolism, our qPCR analysis unveiled a time-dependent increase in GPR68 expression in response to IL1β stimulation, which correlates with the expression of matrix degrading proteases suggesting the role of GPR68 in chondrocytes catabolism and matrix degeneration. Using pharmacological activator of GPR68, our results further showed that GPR68 activation repressed the expression of MMPs in human chondrocytes.ConclusionsOur results demonstrated that GPR68 was robustly expressed in human cartilage and mice and its expression correlates with matrix degeneration and severity of OA progression in human and surgical model. GPR68 activation in human chondrocytes further repressed the expression of MMPs under OA pathological condition. These results identify GPR68 as a possible therapeutic target in the regulation of matrix degradation during OA. creator: Nazir M. Khan creator: Martha E. Diaz-Hernandez creator: William N. Martin creator: Bhakti Patel creator: Samir Chihab creator: Hicham Drissi uri: https://doi.org/10.7717/peerj.16553 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2023 Khan et al. title: miR-4270 suppresses hepatocellular carcinoma progression by inhibiting DNMT3A-mediated methylation of HGFAC promoter link: https://peerj.com/articles/16566 last-modified: 2023-12-05 description: BackgroundmiR-4270 is a regulatory factor has been linked with the progression of various cancers, such as nasopharyngeal carcinoma, hepatocellular carcinoma (HCC), and gastric cancer. However, the underlying mechanisms through which miR-4270 modulates HCC development are not fully understood.MethodsmiR-4270 expression levels were analyzed in various HCC cell lines and tissue samples. An online bioinformatics tool was then utilized to predict the miR-4270 target gene. The binding relationship between miR-4270 and its target gene DNMT3A was verified using dual-luciferase reporter and Ago2–RIP assays. Then, co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assays were conducted to investigate the association between DNMT3A and the hepatocyte growth factor activator (HGFAC) promoter region. To assess the methylation level of the HGFAC promoter, methylation-specific PCR (MSP) was employed. Furthermore, rescue analyses were carried out to evaluate the functional relevance of miR-4270 and HGFAC in the modulation of the malignant properties of HCC cells. Finally, HepG2 cells overexpressing miR-4270 were subcutaneously injected into nude mice to estimate the impact of miR-4270 on the xenograft tumor growth of HCC.ResultsA substantial miR-4270 downregulation was revealed in HCC patient samples and cell lines. miR-4270 upregulation suppressed both cell proliferation and invasion while promoting apoptosis. At the molecular level, miR-4270 was found to bind to the 3′untranslated region (3′UTR) of DNMT3A, thereby inhibiting DNMT3A-mediated methylation of the HGFAC promoter. Functional assays indicated that inhibition of miR-4270 stimulated HCC cell growth, an effect counteracted by overexpression of HGFAC. In vivo assays further verified that miR-4270 effectively suppressed the progression of HCC xenograft tumors.ConclusionsmiR-4270 was found to mitigate the malignant characteristics of HCC by inhibiting DNMT3A-mediated methylation of the HGFAC promoter, suggesting a potential therapeutic avenue for the management of HCC. creator: Qiang Zou creator: Shasha Cao uri: https://doi.org/10.7717/peerj.16566 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2023 Zou and Cao title: Acute heat priming promotes short-term climate resilience of early life stages in a model sea anemone link: https://peerj.com/articles/16574 last-modified: 2023-12-05 description: Across diverse taxa, sublethal exposure to abiotic stressors early in life can lead to benefits such as increased stress tolerance upon repeat exposure. This phenomenon, known as hormetic priming, is largely unexplored in early life stages of marine invertebrates, which are increasingly threatened by anthropogenic climate change. To investigate this phenomenon, larvae of the sea anemone and model marine invertebrate Nematostella vectensis were exposed to control (18 °C) or elevated (24 °C, 30 °C, 35 °C, or 39 °C) temperatures for 1 h at 3 days post-fertilization (DPF), followed by return to control temperatures (18 °C). The animals were then assessed for growth, development, metabolic rates, and heat tolerance at 4, 7, and 11 DPF. Priming at intermediately elevated temperatures (24 °C, 30 °C, or 35 °C) augmented growth and development compared to controls or priming at 39 °C. Indeed, priming at 39 °C hampered developmental progression, with around 40% of larvae still in the planula stage at 11 DPF, in contrast to 0% for all other groups. Total protein content, a proxy for biomass, and respiration rates were not significantly affected by priming, suggesting metabolic resilience. Heat tolerance was quantified with acute heat stress exposures, and was significantly higher for animals primed at intermediate temperatures (24 °C, 30 °C, or 35 °C) compared to controls or those primed at 39 °C at all time points. To investigate a possible molecular mechanism for the observed changes in heat tolerance, the expression of heat shock protein 70 (HSP70) was quantified at 11 DPF. Expression of HSP70 significantly increased with increasing priming temperature, with the presence of a doublet band for larvae primed at 39 °C, suggesting persistent negative effects of priming on protein homeostasis. Interestingly, primed larvae in a second cohort cultured to 6 weeks post-fertilization continued to display hormetic growth responses, whereas benefits for heat tolerance were lost; in contrast, negative effects of short-term exposure to extreme heat stress (39 °C) persisted. These results demonstrate that some dose-dependent effects of priming waned over time while others persisted, resulting in heterogeneity in organismal performance across ontogeny following priming. Overall, these findings suggest that heat priming may augment the climate resilience of marine invertebrate early life stages via the modulation of key developmental and physiological phenotypes, while also affirming the need to limit further anthropogenic ocean warming. creator: Benjamin H. Glass creator: Katelyn G. Jones creator: Angela C. Ye creator: Anna G. Dworetzky creator: Katie L. Barott uri: https://doi.org/10.7717/peerj.16574 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2023 Glass et al. title: How does abstract and concrete garbage classification signage influence waste sorting behavior? link: https://peerj.com/articles/16597 last-modified: 2023-12-05 description: Despite the issuance of standardized garbage classification signage, the rate of garbage classification in China remains low. We conducted a pair of laboratory experiments to explore the cognitive processing differences between abstract (including recyclables, hazardous garbage, and food signs) and concrete (including paper, plastic, glass, metal, textiles, batteries, household chemicals, tubes, and food signs) classification signs. We tested a nudging strategy to enhance garbage classification behavior. In Experiment 1, we divided garbage classification signs into two conditions: an abstract condition (comprising abstract signs) and a concrete condition (comprising concrete signs). The Go/No Go task was used to simulate garbage classification behavior. Participants were instructed to press a key when the garbage stimulus matched the classification signs (Go condition) and to refrain from pressing the key when there was a mismatch (No Go condition). The results showed that responses under the concrete condition were expedited compared to those under the abstract condition. This suggests that concrete signage requires less cognitive exertion, thereby enhancing the efficiency of waste classification. In Experiment 2, we optimized the existing bin signage, which predominantly featured abstract signs (traditional condition), and transformed it into a bin signage that emphasized concrete classification signs. These concrete signs were strategically positioned on the upper part of the bins to draw attention (nudging condition). The results suggested that the nudging condition required fewer cognitive resources than the traditional condition, which in turn increased the efficiency of processing garbage classification. This study not only validates the effects of concreteness in garbage classification but also provides effective nudge strategies to complement existing garbage classification management policy tools in a realistic Chinese context. creator: Gai Cao creator: Rong Cao creator: Peng Liu uri: https://doi.org/10.7717/peerj.16597 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2023 Cao et al. title: Restudy of shoulder motion in the theropod dinosaur Mononykus olecranus (Alvarezsauridae) link: https://peerj.com/articles/16605 last-modified: 2023-12-05 description: BackgroundRange of motion in the forelimb of the Upper Cretaceous theropod dinosaur Mononykus olecranus, a member of the family Alvarezsauridae, has previously been investigated. However, the method used to investigate range of motion at the shoulder in M. olecranus did not follow the standardized procedure used in subsequent studies. The latter procedure yields more reliable results, and its standardization provides that its results are directly comparable to the results of similar studies in other species. I therefore reinvestigated the range of motion at the shoulder in M. olecranus, using the latter procedure.MethodsCasts of the left scapula and coracoid of M. olecranus were posed on a horizontal surface, supported from beneath with modeling clay, with the medial surface of the scapula facing toward the horizontal surface. A cast of the left humerus was posed at the limits of motion through the transverse and parasagittal planes. Photos of the poses in orthal views were superimposed and used to measure range of motion, which was measured as the angle between lines drawn down the long axis of the humerus in each position.ResultsThrough the transverse plane, the humerus of M. olecranus could be elevated to a subhorizontal position and depressed to a subvertical position. It could move through the parasagittal plane from a subvertical position at full protraction to a position above the horizontal at full retraction. These results correct the previous mischaracterization of shoulder motion in M. olecranus as restricted to a small arc with the arms held in a permanent sprawl. The range of humeral motion in M. olecranus is much greater than that found by the previous method and allowed the animal to tuck its arms in at the sides, in addition to allowing them to sprawl so as to orient the palm downward. The wide range of humeral motion allowed M. olecranus to forage for insects by employing hook-and-pull digging at surfaces with a wider range of orientations than the previous study showed to be possible. creator: Philip J. Senter uri: https://doi.org/10.7717/peerj.16605 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2023 Senter title: taxalogue: a toolkit to create comprehensive CO1 reference databases link: https://peerj.com/articles/16253 last-modified: 2023-12-04 description: BackgroundTaxonomic identification through DNA barcodes gained considerable traction through the invention of next-generation sequencing and DNA metabarcoding. Metabarcoding allows for the simultaneous identification of thousands of organisms from bulk samples with high taxonomic resolution. However, reliable identifications can only be achieved with comprehensive and curated reference databases. Therefore, custom reference databases are often created to meet the needs of specific research questions. Due to taxonomic inconsistencies, formatting issues, and technical difficulties, building a custom reference database requires tremendous effort. Here, we present taxalogue, an easy-to-use software for creating comprehensive and customized reference databases that provide clean and taxonomically harmonized records. In combination with extensive geographical filtering options, taxalogue opens up new possibilities for generating and testing evolutionary hypotheses.Methodstaxalogue collects DNA sequences from several online sources and combines them into a reference database. Taxonomic incongruencies between the different data sources can be harmonized according to available taxonomies. Dereplication and various filtering options are available regarding sequence quality or metadata information. taxalogue is implemented in the open-source Ruby programming language, and the source code is available at https://github.com/nwnoll/taxalogue. We benchmark four reference databases by sequence identity against eight queries from different localities and trapping devices. Subsamples from each reference database were used to compare how well another one is covered.Resultstaxalogue produces reference databases with the best coverage at high identities for most tested queries, enabling more accurate, reliable predictions with higher certainty than the other benchmarked reference databases. Additionally, the performance of taxalogue is more consistent while providing good coverage for a variety of habitats, regions, and sampling methods. taxalogue simplifies the creation of reference databases and makes the process reproducible and transparent. Multiple available output formats for commonly used downstream applications facilitate the easy adoption of taxalogue in many different software pipelines. The resulting reference databases improve the taxonomic classification accuracy through high coverage of the query sequences at high identities. creator: Niklas W. Noll creator: Christoph Scherber creator: Livia Schäffler uri: https://doi.org/10.7717/peerj.16253 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2023 Noll et al. title: Margelopsid species search taxonomic home within Corymorphidae and Boreohydridae link: https://peerj.com/articles/16265 last-modified: 2023-12-04 description: Planktonic lifestyle of polyps in representatives of Margelopsidae are very different from all other species in the hydrozoan clade Aplanulata. Their evolutionary origin and phylogenetic position have been the subject of significant speculation. A recent molecular study based only on COI data placed Margelopsidae as a sister group to all Aplanulata, an unexpected result because margelopsid morphology suggests affiliation with Tubulariidae or Corymorphidae. Here we used multigene analyses, including nuclear (18S rRNA and 28S rRNA) and mitochondrial (16S rRNA and COI) markers of the hydroid stage of the margelopsid species Margelopsis haeckelii and the medusa stage of Margelopsis hartlaubii to resolve their phylogenetic position with respect to other hydrozoans. Our data provide strong evidence that M. haeckelii, the type species of Margelopsis, is a member of the family Corymorphidae. In contrast, M. hartlaubii is sister to Plotocnide borealis, a member of Boreohydridae. These results call into question the validity of the genus Margelopsis and the family Margelopsidae. The systematic position of M. haeckelii is discussed in light of the phylogeny of Corymorphidae. creator: Daria Kupaeva creator: Tatiana Lebedeva creator: Zachariah Kobrinsky creator: Daniel Vanwalleghem creator: Andrey Prudkovsky creator: Stanislav Kremnyov uri: https://doi.org/10.7717/peerj.16265 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2023 Kupaeva et al.