Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice
Background Gene doping is the misuse of genome editing and gene therapy technologies for the purpose of manipulating specific genes or gene functions in order to improve athletic performance. However, a non-invasive detection method for gene doping using recombinant adenoviral (rAdV) vectors containing human follistatin (hFST) genes (rAdV<hFST>) has not yet been developed. Therefore, the aim of this study was to develop a method to detect gene doping using rAdV<hFST>. Methods First, we generated rAdV<hFST> and evaluated the overexpression of the hFST gene, FST protein, and muscle protein synthesis signaling using cell lines. Next, rAdV<hFST> was injected intravenously or intramuscularly into mice, and whole blood was collected, and hFST and cytomegalovirus promoter (CMVp) gene fragments were detected using TaqMan-quantitative polymerase chain reaction (qPCR). Finally, to confirm the specificity of the primers and the TaqMan probes, samples from each experiment were pooled, amplified using TaqMan-qPCR, and sequenced using the Sanger sequencing. Results The expression of hFST and FST proteins and muscle protein synthesis signaling significantly increased in C2C12 cells. In long-term, transgene fragments could be detected until 4 days after intravenous injection and 3 days after intramuscular injection. Finally, the Sanger sequencing confirmed that the primers and TaqMan probe specifically amplified the gene sequence of interest. Conclusions These results indicate the possibility of detecting gene doping using rAdV<hFST> using TaqMan-qPCR in blood samples. This study may contribute to the development of detection methods for gene doping using rAdV<hFST>.
Yanazawa et al. (2021) Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice. PeerJ 9:e12285 https://doi.org/10.7717/peerj.12285
Comparative transcriptome analyses of different Salvia miltiorrhiza varieties during the accumulation of tanshinones
Salvia miltiorrhiza (Labiatae) is an important medicinal plant in traditional Chinese medicine. Tanshinones are one of the main active components of S. miltiorrhiza. It has been found that the intraspecific variation of S. miltiorrhiza is relatively large and the content of tanshinones in its roots of different varieties is also relatively different. To investigate the molecular mechanisms that responsible for the differences among these varieties, the tanshinones content was determined and comparative transcriptomics analysis was carried out during the tanshinones accumulation stage. A total of 52,216 unigenes were obtained from the transcriptome by RNA sequencing among which 23,369 genes were differentially expressed among different varieties, and 2,016 genes including 18 diterpenoid biosynthesis-related genes were differentially expressed during the tanshinones accumulation stage. Functional categorization of the differentially expressed genes (DEGs) among these varieties revealed that the pathway related to photosynthesis, oxidative phosphorylation, secondary metabolite biosynthesis, diterpenoid biosynthesis, terpenoid backbone biosynthesis, sesquiterpenoid and triterpenoid biosynthesis are the most differentially regulated processes in these varieties. The six tanshinone components in these varieties showed different dynamic changes in tanshinone accumulation stage. In addition, combined with the analysis of the dynamic changes, 277 DEGs (including one dehydrogenase, three CYP450 and 24 transcription factors belonging to 12 transcription factor families) related to the accumulation of tanshinones components were obtained. Furthermore, the KEGG pathway enrichment analysis of these 277 DEGs suggested that there might be an interconnection between the primary metabolic processes, signaling processes and the accumulation of tanshinones components. This study expands the vision of intraspecific variation and gene regulation mechanism of secondary metabolite biosynthesis pathways in medicinal plants from the “omics” perspective.
Zhou et al. (2021) Comparative transcriptome analyses of different Salvia miltiorrhiza varieties during the accumulation of tanshinones. PeerJ 9:e12300 https://doi.org/10.7717/peerj.12300
Functional analysis of a dihydroflavonol 4-reductase gene in Ophiorrhiza japonica (OjDFR1) reveals its role in the regulation of anthocyanin
Dihydroflavonol 4-reductase (DFR), a key regulatory enzyme, participated in the biosynthesis of anthocyanins, proanthocyanidins and other flavonoids that essential for plant survival and human health. However, the role of this enzyme in Ophiorrhiza japonica is still unknown. Here, three putative DFR-like genes were firstly isolated from O. japonica. Phylogenetic analysis indicated that OjDFR1 was classified into DFR subgroup, while the rest two were clustered into other NADPH-dependent reductases. Then, functions of the three genes were further characterized. Expression analysis showed that OjDFR1 transcripts had strong correlations with the accumulation pattern of anthocyanin during the flower developmental, whereas other two were not, this suggested the potential roles of OjDFR1 in anthocyanin biosynthesis. Subsequently, all three clones were functionally expressed in Escherichia coli, but confirming that only OjDFR1 encode active DFR proteins that catalyzed the reduction of dihydroflavonols to leucoanthocyanidin. Consistant with the biochemical assay results, overexpressing OjDFR1 in Arabidopsis tt3-1 mutant successfully restored the deficiency of anthocyanin and proanthocyanidin, hinting its function as DFR in planta. Additionally, heterologous expression of OjDFR1 in transgenic tobacco contributed to darker flower color via up-regulating the expressions of endogenous NtANS and NtUFGT, which suggested that OjDFR1 was involved in flower color development. In summary, this study validates the functions of OjDFR1 and expands our understanding of anthocyanin biosynthesis in O. japonica.
Sun et al. (2021) Functional analysis of a dihydroflavonol 4-reductase gene in Ophiorrhiza japonica (OjDFR1) reveals its role in the regulation of anthocyanin. PeerJ 9:e12323 https://doi.org/10.7717/peerj.12323
DNA barcode reference library construction and genetic diversity and structure analysis of Amomum villosum Lour. (Zingiberaceae) populations in Guangdong Province
Background Amomum villosum Lour. is the plant that produces the famous traditional Chinese medicine Amomi Fructus. Frequent habitat destruction seriously threatens A. villosum germplasm resources. Genetic diversity is very important to the optimization of germplasm resources and population protection, but the range of inherited traits within A. villosum is unclear. In this study, we analyzed the genetic diversity and genetic structures of A. villosum populations in Guangdong and constructed a local reference DNA barcode library as a resource for conservation efforts. Methods DNA barcoding and Inter-Simple Sequence Repeat (ISSR) markers were used to investigate the population genetics of A. villosum. Five universal DNA barcodes were amplified and used in the construction of a DNA barcode reference library. Parameters including percentage of polymorphic sites (PPB), number of alleles (Na), effective number of alleles (Ne), Nei’s gene diversity index (H), and Shannon’s polymorphism information index (I) were calculated for the assessment of genetic diversity. Genetic structure was revealed by measuring Nei’s gene differentiation coefficient (Gst), total population genetic diversity (Ht), intra-group genetic diversity (Hs), and gene flow (Nm). Analysis of molecular variance (AMOVA), Mantel tests, unweighted pair-group method with arithmetic mean (UPGMA) dendrogram, and principal co-ordinates (PCoA) analysis were used to elucidate the genetic differentiation and relationship among populations. Results A total of 531 sequences were obtained from the five DNA barcodes with no variable sites from any of the barcode sequences. A total of 66 ISSR bands were generated from A. villosum populations using the selected six ISSR primers; 56 bands, 84.85% for all the seven A. villosum populations were polymorphic. The A. villosum populations showed high genetic diversity (H = 0.3281, I = 0.4895), whereas the gene flow was weak (Nm = 0.6143). Gst (0.4487) and AMOVA analysis indicated that there is obvious genetic differentiation amongA. villosum populations and more genetic variations existed within each population. The genetic relationship of each population was relatively close as the genetic distances were between 0.0844 and 0.3347.
Gong et al. (2021) DNA barcode reference library construction and genetic diversity and structure analysis of Amomum villosum Lour. (Zingiberaceae) populations in Guangdong Province. PeerJ 9:e12325 https://doi.org/10.7717/peerj.12325
Transcriptomic analysis reveals candidate genes for male sterility in Prunus sibirica
Background The phenomenon of male sterility widely occurs in Prunus sibirica and has a serious negative impact on yield. We identified the key stage and cause of male sterility and found differentially expressed genes related to male sterility in Prunus sibirica, and we analyzed the expression pattern of these genes. This work aimed to provide valuable reference and theoretical basis for the study of reproductive development and the mechanisms of male sterility in Prunus sibirica. Method The microstructures of male sterile flower buds and male fertile flower buds were observed by paraffin section. Transcriptome sequencing was used to screen genes related to male sterility in Prunus sibirica. Quantitative real-time PCR analysis was performed to verify the transcriptome data. Results Anther development was divided into the sporogenous cell stage, tetrad stage, microspore stage, and pollen maturity stage. Compared with male fertile flower buds, in the microspore stage, the pollen sac wall tissue in the male sterile flower buds showed no signs of degeneration. In the pollen maturity stage, the tapetum and middle layer were not fully degraded, and anther development stopped. Therefore, the microspore stage was the key stage for anther abortion , and the pollen maturity stage was the post stage for anther abortion. A total of 4,108 differentially expressed genes were identified by transcriptome analysis. Among them, 1,899 were up-regulated, and 2,209 were down-regulated in the transcriptome of male sterile flower buds. We found that “protein kinase activity”, “apoptosis process”, “calcium binding”, “cell death”, “cytochrome c oxidase activity”, “aspartate peptidase activity”, “cysteine peptidase activity” and other biological pathways such as “starch and sucrose metabolism” and “proteasome” were closely related to male sterility in Prunus sibirica. A total of 331 key genes were preliminarily screened. Conclusion The occurrence of male sterility in Prunus sibirica involved many biological processes and metabolic pathways. According to the results of microstructure observations, related physiological indexes determination and transcriptome analysis, we reveal that the occurrence of male sterility in Prunus sibirica may be caused by abnormal metabolic processes such as the release of cytochrome c in the male sterile flower buds, the imbalance of the antioxidant system being destroyed, and the inability of macromolecular substances such as starch to be converted into soluble small molecules at the correct stage of reproductive development, resulting in energy loss. As a result, the tapetum cannot be fully degraded, thereby blocking anther development, which eventually led to the formation of male sterility.
Chen et al. (2021) Transcriptomic analysis reveals candidate genes for male sterility in Prunus sibirica. PeerJ 9:e12349 https://doi.org/10.7717/peerj.12349
Effect of SARS-CoV-2 infection on host competing endogenous RNA and miRNA network
Competing endogenous RNAs (ceRNA) play a crucial role in cell functions. Computational methods that provide large-scale analysis of the interactions between miRNAs and their competitive targets can contribute to the understanding of ceRNA regulations and critical regulatory functions. Recent reports showed that viral RNAs can compete with host RNAs against host miRNAs. Regarding SARS-CoV-2 RNA, no comprehensive study had been reported about its competition with cellular ceRNAs. In this study, for the first time, we used the ceRNAnetsim package to assess ceRNA network effects per individual cell and competitive behavior of SARS-CoV-2 RNA in the infected cells using single-cell sequencing data. Our computations identified 195 genes and 29 miRNAs which vary in competitive behavior specifically in presence of SARS-CoV-2 RNA. We also investigated 18 genes that are affected by genes that lost perturbation ability in presence of SARS-CoV-2 RNA in the human miRNA:ceRNA network. These transcripts have associations with COVID-19-related symptoms as well as many dysfunctions such as metabolic diseases, carcinomas, heart failure. Our results showed that the effects of the SARS-CoV-2 genome on host ceRNA interactions and consequent dysfunctions can be explained by competition among various miRNA targets. Our perturbation ability perspective has the potential to reveal yet to be discovered SARS-CoV-2 induced effects invisible to conventional approaches.
Ari Yuka et al. (2021) Effect of SARS-CoV-2 infection on host competing endogenous RNA and miRNA network. PeerJ 9:e12370 https://doi.org/10.7717/peerj.12370
In silico evaluation of molecular virus–virus interactions taking place between Cotton leaf curl Kokhran virus- Burewala strain and Tomato leaf curl New Delhi virus
Background Cotton leaf curl disease (CLCuD) is a disease of cotton caused by begomoviruses, leading to a drastic loss in the annual yield of the crop. Pakistan has suffered two epidemics of this disease leading to the loss of billions in annual exports. The speculation that a third epidemic of CLCuD may result as consequence of the frequent occurrence of Tomato leaf curl New Delhi virus (ToLCNDV) and Cotton leaf curl Kokhran Virus-Burewala Strain (CLCuKoV-Bu) in CLCuD infected samples, demand that the interactions taking between the two viruses be properly evaluated. This study is designed to assess virus-virus interactions at the molecular level and determine the type of co-infection taking place. Methods Based on the amino acid sequences of the gene products of both CLCuKoV-Bu and ToLCNDV, protein structures were generated using different software, i.e., MODELLER, I-TASSER, QUARKS, LOMETS and RAPTORX. A consensus model for each protein was selected after model quality assessment using ERRAT, QMEANDisCo, PROCHECK Z-Score and Ramachandran plot analysis. The active and passive residues in the protein structures were identified using the CPORT server. Protein–Protein Docking was done using the HADDOCK webserver, and 169 Protein–Protein Interaction (PPIs) were performed between the proteins of the two viruses. The docked complexes were submitted to the PRODIGY server to identify the interacting residues between the complexes. The strongest interactions were determined based on the HADDOCK Score, Desolvation energy, Van der Waals Energy, Restraint Violation Energy, Electrostatic Energy, Buried Surface Area and Restraint Violation Energy, Binding Affinity and Dissociation constant (Kd). A total of 50 ns Molecular Dynamic simulations were performed on complexes that exhibited the strongest affinity in order to validate the stability of the complexes, and to remove any steric hindrances that may exist within the structures. Results Our results indicate significant interactions taking place between the proteins of the two viruses. Out of all the interactions, the strongest were observed between the Replication Initiation protein (Rep) of CLCuKoV-Bu with the Movement protein (MP), Nuclear Shuttle Protein (NSP) of ToLCNDV (DNA-B), while the weakest were seen between the Replication Enhancer protein (REn) of CLCuKoV-Bu with the REn protein of ToLCNDV. The residues identified to be taking a part in interaction belonged to domains having a pivotal role in the viral life cycle and pathogenicity. It maybe deduced that the two viruses exhibit antagonistic behavior towards each other, and the type of infection may be categorised as a type of Super Infection Exclusion (SIE) or homologous interference. However, further experimentation, in the form of transient expression analysis, is needed to confirm the nature of these interactions and increase our understanding of the direct interactions taking place between two viruses.
Ali et al. (2021) In silico evaluation of molecular virus–virus interactions taking place between Cotton leaf curl Kokhran virus- Burewala strain and Tomato leaf curl New Delhi virus. PeerJ 9:e12018 https://doi.org/10.7717/peerj.12018
New phiocricetomyine rodents (Hystricognathi) from the Jebel Qatrani Formation, Fayum Depression, Egypt
Background The rich rodent assemblages from the Eocene–Oligocene deposits of the Jebel Qatrani Formation (Fayum Depression, Egypt) have important implications for our understanding of the origin and paleobiogeography of Hystricognathi, a diverse clade that is now represented by the Afro-Asiatic Hystricidae, New World Caviomorpha, and African Phiomorpha. Methods Here we present previously undescribed material of the enigmatic hystricognath clade Phiocricetomyinae, from two stratigraphic levels in the lower sequence of the Jebel Qatrani Formation—a new genus and species (Qatranimys safroutus) from the latest Eocene Locality 41 (~34 Ma, the oldest and most productive quarry in the formation) and additional material of Talahphiomys lavocati from that species’ type locality, early Oligocene Quarry E (~31–33.2 Ma). Results The multiple specimens of Qatranimys safroutus from L-41 document almost the entire lower and upper dentition, as well as mandibular fragments and the first cranial remains known for a derived phiocricetomyine. Specimens from Quarry E allow us to expand comparisons with specimens from Libya (late Eocene of Dur at-Talah and early Oligocene of Zallah Oasis) that have been placed in T. lavocati, and we show that the Dur at-Talah and Zallah specimens do not pertain to this species. These observations leave the Fayum Quarry E as the only locality where T. lavocati occurs.
Al-Ashqar et al. (2021) New phiocricetomyine rodents (Hystricognathi) from the Jebel Qatrani Formation, Fayum Depression, Egypt. PeerJ 9:e12074 https://doi.org/10.7717/peerj.12074
Visual assessment of dynamic knee joint alignment in patients with patellofemoral pain: an agreement study
Background Assessment of knee kinematics plays an important role in the clinical examination of patients with patellofemoral pain (PFP). There is evidence that visual assessments are reliable in healthy subjects, but there is a lack of evidence in injured populations. The purpose of this study was to examine the intra- and interrater agreement in the visual assessment of dynamic knee joint alignment in patients with PFP. Methods The study was a cross-sectional agreement study. Sixty participants (42 females) were included. We assessed the intra- and interrater agreement of two functional tests: The single leg squat (SLS) and the forward lunge (FL). One investigator scored the movement according to preset criteria while video recording the movement for retest. Moreover, the performance was scored by another investigator using the video recording. Agreement was assessed using weighted kappa statistics. Results The intrarater agreement ranged from moderate to good (Kappa 0.58 (FL) to 0.70 (SLS)) whereas the interrater agreement ranged from fair to moderate (Kappa 0.22 (SLS) to 0.50 (FL)). Conclusion The agreement within raters was better than between raters, which suggests that assessments should preferably be performed by the same tester in research and in a clinical setting, e.g., to evaluate any treatment effect. We promote FL as a reliable clinical tool for evaluating dynamic knee alignment, since it shows equally good intra- and interrater agreement, and it is an inexpensive and easy method to use.
Hansen et al. (2021) Visual assessment of dynamic knee joint alignment in patients with patellofemoral pain: an agreement study. PeerJ 9:e12203 https://doi.org/10.7717/peerj.12203
Upright walking has driven unique vascular specialization of the hominin ilium
Background A novel physis in hominins modulates broadening and shortening of the ilium. We report analysis of a vascular canal system whose origin may be associated with this physis and which appears to be also unique to hominins. Its presence is potentially identifiable in the fossil record by its association with a highly enlarged foramen that is consistently present in modern humans and hominin fossils. Methods We measured the diameter of this foramen in humans, fossil hominins, and African great apes and corrected for body size. Results The mean relative human foramen diameter is significantly greater than those of either Pan or Gorilla. Moreover, eight of the nine values of the Cohen’s d for these differences in ratios are highly significant and support the ordering of magnitudes: Pan < Gorilla < Homo. The relative foramen diameter of A.L. 288-1 is above the 75th percentile of all other hominoids and at the high end of humans. The foramen is also present in ARA-VP-6/500. Conclusions We posit that the presence and significant enlargement of this foramen in fossils can reasonably serve as an indicator that its anterior inferior iliac spine emerged via the unique hominin physis. The foramen can therefore serve as an indicator of hominin iliac ontogenetic specialization for bipedality in fossil taxa.
Zirkle et al. (2021) Upright walking has driven unique vascular specialization of the hominin ilium. PeerJ 9:e12240 https://doi.org/10.7717/peerj.12240