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Background: In vitro fertilization (IVF) and embryo transfer (ET) are widely used in reproductive biology. Despite the transfer of high-quality blastocysts, the implantation rate of IVF-derived blastocysts remains low after ET., Methods: This article provides a comprehensive review of current research on embryo implantation regulators and their application to improve the implantation potential of IVF-derived blastocysts., Main Findings: The in vivo mouse model revealed selective proteolysis immediately after expression in activated blastocysts, that is, degradation of ERα expression in activated blastocysts regulated by the ubiquitin-proteasome pathway, followed by completion of blastocyst implantation. Treatment of blastocysts to induce appropriate protein expression during in vitro culture prior to ET is a useful approach for improving implantation rates. This approach showed that combined treatment with PRL, EGF, and 4-OH-E 2 (PEC) improved the blastocyst implantation rates. Furthermore, arginine and leucine drive reactive oxygen species (ROS)-mediated integrin α5β1 expression and promote blastocyst implantation., Conclusion: Findings based on analysis of molecular and cellular regulators are useful for improving the implantation potential of IVF-derived blastocysts. These approaches may help to elucidate the mechanisms underlying the completion of the blastocyst implantation, although further investigation is required to improve the success of implantation and pregnancy., Competing Interests: Author H.M. was supported by JSPS KAKENHI (Grant Nussmbers 25 450 390, 18K05936, 21K05903, and 24K09098), and author H.M. has a patent for quality of embryos., (© 2025 The Author(s). Reproductive Medicine and Biology published by John Wiley & Sons Australia, Ltd on behalf of Japan Society for Reproductive Medicine.)

The cellular and molecular mechanisms underlying lymphocyte development are diverse among teleost species. Although recent scRNA-seq analyses of zebrafish hematopoietic cells have advanced our understanding of teleost hematopoiesis, comparative studies using another genetic model, medaka, which is evolutionarily distant among teleosts, is useful for understanding commonality and species-specificity in teleosts. In order to gain insight into how different molecular and cellular mechanisms of lymphocyte development in medaka and zebrafish, we established a recombination activating gene 1 ( rag1 ) mutant medaka, which exhibited defects in V(D)J rearrangement of lymphocyte antigen receptor genes, accordingly lacking mature B and T cells. scRNA-seq analysis of wild type and rag1 mutant lymphocytes in the thymus and kidney characterized the developing stages of T and B cells, and found that most developed cd4 + cd8 - and cd4 - cd8 + single-positive (SP) T-cell populations are absent in the thymus, and identified lymphoid progenitor cells already committed to the T lineage in kidney, implying unique features of medaka lymphocyte development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Sakaguchi, Matsuda and Iwanami.)

In cattle, maternal metabolic health has been suggested to influence oocyte and embryo quality. Here, we examined whether maternal liver abnormalities affected in vitro oocyte maturation by screening meiotic maturation, spindle morphology, actin filaments, and lysosomes. In oocytes from the abnormal liver group, the maturation rate (80.2%) was significantly lower compared to a control group with healthy livers (90.8%; P < 0.05). Mean spindle area in oocytes of the abnormal group (50.4 ± 3.4 μm 2 ) was significantly larger than in the control (40.8 ± 1.6 μm 2 ; P < 0.05). Likewise, mean spindle width in the abnormal group (8.8 ± 0.3 μm) was significantly larger than in the control group (7.8 ± 0.2 μm; P < 0.05). The proportion of cells with correctly aligned chromosomes in the abnormal group (48.0%) was significantly lower than in the control (78.3%; P < 0.05). The number of cortical actin filaments in mature oocytes of the abnormal group (299.3 ± 3.7) was significantly lower than in the control (314.7 ± 3.2; P < 0.05). The number of lysosomes in mature oocytes of the abnormal group (1363.6 ± 39.0) was significantly higher than in the control (1123.4 ± 26.3; P < 0.05). In conclusion, our findings indicate that the quality of in vitro matured oocytes is lower in cattle with liver abnormalities than in healthy cattle.

Ribonucleic acid (RNA) extraction is the first critical step in gene expression analysis. In this chapter, we describe a high-throughput RNA extraction method using guanidine thiocyanate and isopropyl alcohol (HighGI). The use of carboxyl-coated paramagnetic beads, instead of silica membrane columns, enables semi-automation using a liquid handling system and high-throughput RNA extraction for large-scale transcriptome studies. Homemade mixes of paramagnetic beads and buffers make HighGI inexpensive. In addition, HighGI-extracted RNA retains low molecular weight RNA molecules less than 200 bp, which is typically lost in commercial column-based kits., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Neprilysin is a skin wrinkle-inducing membrane bound elastase that is expressed abundantly in UV-exposed and in aged dermal fibroblasts. The overexpression of neprilysin is closely associated with enhanced epithelial-mesenchymal cytokine interactions mainly via interleukin (IL)-1α, which has the distinct potential to stimulate the expression of neprilysin by human dermal fibroblasts (HDFs). The over-expression of neprilysin also accelerates the formation of wrinkles, accompanied by disruptions of the three-dimensional architecture of dermal elastic fibers that are responsible for the loss of skin elasticity. Because the signaling pathway(s) that lead to the IL-1α-stimulated expression of neprilysin in HDFs remain unclear, we characterized the signaling pathway involved, including their related transcription factors, in IL-1α-treated HDFs. Since qRT-PCR analysis revealed that the mRNA expression level of neprilysin is stimulated to a stronger extent in adult HDFs (aHDFs) by IL-1α than in neonatal HDFs, we used aHDFs for the signaling analysis. Western blotting analysis of the phosphorylation of signaling factors revealed that IL-1α significantly stimulated the phosphorylation of ERK1/2, RSK, JNK, p38, MSK1, NFkB, c-Jun, ATF-2, CREB, and STAT3. Analysis using various signaling inhibitors demonstrated that inhibiting ERK and JNK but not p38, MSK1, NFkB, or STAT3 significantly abrogated the IL-1α stimulated expression of neprilysin at the mRNA, protein, and enzyme activity levels. Furthermore, silencing c-Fos significantly down-regulated the IL-1α-increased expression of neprilysin at the protein and enzyme activity levels. These findings strongly suggest that the IL-1α-stimulated expression of neprilysin in aHDFs is mediated via the intracellular signaling axis of ERK/JNK/c-Jun/c-Fos/AP-1., (© 2024 The Author(s). The Journal of Dermatology published by John Wiley & Sons Australia, Ltd on behalf of Japanese Dermatological Association.)

The evolution of green plants from aquatic to terrestrial environments is thought to have been facilitated by the acquisition of gametangia, specialized multicellular organs housing gametes. Antheridia and archegonia, responsible for producing and protecting sperm and egg cells, undergo formative cell divisions to produce a cell to differentiate into germ cell lineages and the other cell to give rise to surrounding structures. However, the genes governing this process remain unidentified. We isolated genes expressed during gametangia development from previously established gene-trap lines of Physcomitrium patens and characterized their function during gametangia formation. We identified P. patens LATERAL SUPPRESSOR 1 (PpLAS1) from the gene-trap library, encoding a GRAS transcription factor. The double-deletion mutant with its paralog PpLAS2 failed to form inner cells in both gametangia. PpLASs are expressed in cells undergoing formative cell division, and introducing PpLAS1 into the double-deletion mutant successfully rescued the phenotype. These findings underscore the pivotal role of PpLASs in regulating formative cell divisions, ensuring the separation of reproductive cell lineages from surrounding cells in antheridia and archegonia. Furthermore, they suggest a link between PpLASs and the evolutionary origin of male and female gametangia in the common ancestor of land plants., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

A curcumin-derived chemosensor 4,4'-((1E,3Z,5Z,6E)-3,5-bis((2hydroxyphenyl)imino) hepta-1,6-diene-1,7-diyl)bis(2-methoxyphenol) (HIBMP) was developed from curcumin and o-aminophenol using Schiff base condensation method. HIBMP selectively recognizes Cu (II) ion (Cu (II)) relative to other tested metal ions. Selective binding of Cu (II) ion turns off the fluorescent property of HIBMP and shows no interference with other metal ions. Moreover, the favourable binding of Cu (II) with HIBMP was noticed by a good linearity between the fluorescence intensity and concentration of Cu (II) with a range of 0-15.6 × 10 -7  M, and the limit of detection was determined to be as low as 30.1 nM. Furthermore a 1:1 stoichiometry was identified from the results of Job's plot and HR-MS. Density functional theory (DFT) calculation results expressed that the intramolecular charge transfer (ICT) during chelation is responsible for quenching of fluorescence. Besides, the fluorescence life time measurement and ethylenediaminetetraacetic acid (EDTA) titration method revealed the stability and reversibility of the sensor. Practical use of the sensor was achieved through the quantitative analysis of spiked Cu (II) ion in real water samples with good recoveries, and biological experiments exposed that the sensor was less toxic and could be applied in fluorescence imaging of Cu (II) in living cells., (© 2024 John Wiley & Sons Ltd.)

A spatiotemporal understanding of gene function requires the precise control of gene expression in each cell. Here, we use an infrared laser-evoked gene operator (IR-LEGO) system to induce gene expression at the single-cell level in the moss Physcomitrium patens by heating a living cell with an IR laser and thereby activating the heat shock response. We identify the laser irradiation conditions that provide higher inducibility with lower invasiveness by changing the laser power and irradiation duration. Furthermore, we quantitatively characterize the induction profile of the heat shock response using a heat-induced fluorescence reporter system after the IR laser irradiation of single cells under different conditions. Our data indicate that IR laser irradiation with long duration leads to higher inducibility according to increase in the laser power but not vice versa, and that the higher laser power even without conferring apparent damage to the cells decelerates and/or delayed gene induction. We define the temporal shift in expression as a function of onset and duration according to laser power and irradiation duration. This study contributes to the versatile application of IR-LEGO in plants and improves our understanding of heat shock-induced gene expression., (© 2024. The Author(s).)

The green alga Pediastrum duplex forms colonies through asexual reproduction and has a unique life cycle. To elucidate the mechanisms that regulate the asexual reproductive cycle in P. duplex, we analyzed the effects of light on the processes and gene expression involved in each step of the asexual reproductive cycle, revealing light irradiation to be essential for increasing the number of colonies. Among the processes in the asexual reproductive cycle, the transition from cell hypertrophy to zoospore formation could proceed even in the dark if glucose was added to the medium. Transcriptome analysis revealed that the expression of different groups of genes was significantly promoted or suppressed before and after the number of colonies increased. Our findings indicate that the asexual reproductive cycle of P. duplex includes a process promoted by photosynthesis. This study enhances our understanding of the growth characteristics of P. duplex and other microalgae., (© 2024. The Author(s) under exclusive licence to The Botanical Society of Japan.)

A comprehensive LC-MS study examined the venom components of the solitary scoliid wasp Scolia oculata . Online mass fingerprinting showed that crude venom contains 25 small molecules (amino acids, biogenic amines, and nucleosides/nucleotides) and 45 peptides with MW 400-2700. The small molecules were identified by elemental composition analysis, and peptide sequences were determined by ESI-MS/MS and MALDI-TOF/TOF MS analyses. As major peptide components, a known peptide, β-scoliidine (DYVTVKGFSPLRKA), and three new peptides, γ-scoliidine (YVTVKGFSPLR), δ-scoliidine (YVTVKGFSPLREP) and ε-scoliidine (DYVTVKGFSPLREP) were identified, all of which are closely homologous to each other. Once the neuroprotective effects of β-scoliidine have already been described, the other three new scoliidine peptides were analyzed against oxidative stress-induced toxicity in PC12 neuronal cells by mitochondrial metabolism assay, and the structure-activity relationship was evaluated. Interestingly, pre-treatment with ε-scoliidine increased the mitochondrial metabolism of PC12 cells (106 ± 3.6%; p = 0.007) exposed to H 2 O 2 -induced oxidative stress in contrast to γ- and δ-scoliidines (77.6 ± 4.8 and 68.5 ± 4.1%, respectively) in compared to cells treated only H 2 O 2 (75.8 ± 2.4%). These new peptides were also analyzed for enzyme inhibitor/substrate assays with angiotensin-converting enzyme (ACE), neprilysin (NEP), and acetylcholinesterase (AChE). In these assays, only δ- and ε-scoliidines increased the AChE activity (128.7 ± 3.8%; p = 0.01; and 116.8 ± 3.8% p = 0.03; respectively) in relation to basal activity (100.1 ± 1.6%). In addition, the four peptides were analyzed through in silico analysis, and none of them demonstrated possible hemolytic and toxic activities. In our study, the comprehensive LC-MS and MS/MS analyses of Scolia oculate venom identified four major peptide components of the venom β-, γ-, δ- and ε-scoliidines, and small differences in their primary structures are important to their neuroprotective properties.

In the long history of human relations with flowering cherry trees in Japan, 'Somei-Yoshino' occupies an exceptional position among a variety of flowering trees: it is a self-incompatible interspecific hybrid but has been enthusiastically planted by grafting throughout Japan, due most likely to its flamboyant appearance upon full bloom. Thus, 'Somei-Yoshino' gives us a rare opportunity to trace and investigate the occurrence and distribution of somatic mutations within a single plant species through analysis of the genomes of the clonally propagated trees grown under a variety of geographical and artificial environments. In the studies presented here, a total of 46 samples of 'Somei-Yoshino' trees were collected and their genomes were analysed. We identified 684 single nucleotide mutations, of which 71 were present in more than two samples. Clustering analysis of the mutations indicated that the 46 samples were classified into eight groups, four of which included 36 of the 46 samples analysed. Interestingly, all the four tree samples collected in Ueno Park of Tokyo were members of the four groups mentioned above. Based on comparative analysis of their mutations, one of the four trees growing in Ueno Park was concluded to be the closest to the original ancestor. We propose that somatic mutations may be used as tracers to establish the ancestral relationship amongst clonally propagated individuals., (© The Author(s) 2024. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

This study investigated the antistress effects of yuzu synthetic fragrances by employing three experiments on humans and mice using two yuzu synthetic fragrances and five single compounds. We prepared two synthetic fragrances based on the component analysis of two natural yuzu essential oils extracted by cold-pressed and steam-distilled extraction methods. Chromogranin A (CgA) and heart rate (HR) were used as stress indices in human experiments. Immobility time during the forced swim test was used as a stress index in mice experiments. We analyzed brain mechanisms by measuring the expression of neurotrophic factors, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) in the mice experiments. Synthetic yuzu fragrance mimicked steam-distilled oil (SD) significantly reduced participants' heart rate in experiment 1. In the forced swim test conducted in experiment 2, SD significantly reduced immobility time, and increased the expression of neurotrophic factors BDNF, NGF, and NT-3 in the hippocampus of mice. In experiment 3, focusing on single compounds, terpinen-4-ol significantly reduced immobility time in the forced swim test. These findings indicate that inhalation of SD and terpinen-4-ol has antistress effects. Terpinen-4-ol is a strong candidate for further investigation as a potential stress-reducing agent.

Complete sequences of RNA1 and RNA2 of tulip streak virus (TuSV) were already reported, but other segments were not yet. In this study, we reported RNA3 and RNA4 of TuSV, which shared around 69% nucleotide identity with those of closely related virus, suggesting that these are additional RNA segments., Competing Interests: The authors declare no conflict of interest.

This study evaluates the structural stability of large underground spaces in seismic conditions, represented by the Oya underground stone mining plant. By directly monitoring the seismic response of the underground mining site, significant earthquake activities at the plate boundaries of the Tokyo region and Ibaraki Prefecture offshore area were observed. Additionally, through an in-depth analysis of seismic records from different locations within the underground structure, the dynamic characteristics and motion patterns of the Oya underground stone mining plant were revealed, revealing its movement trajectory during earthquakes. Additionally, this study innovatively applied seismic waves measured at the original site as input parameters and artificially generated seismic waves based on their response spectra. A numerical analysis was performed after ensuring the model's high correlation with the original site was met. The findings demonstrate that the results of both parameter input methods are confirmable and valuable. Under severe seismic conditions, instability was observed in some regions of the underground mining site. The study also discusses the location and damage mechanisms of the mining site's structure under seismic effects, providing valuable insights for the safety assessment of similar large underground spaces and proposing new approaches for selecting input parameters in seismic analysis., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: CHENG CHUANTAO reports financial support was provided by 10.13039/501100002241Japan Science and Technology Agency. Takafumi SEIKI reports financial support was provided by Chuden Engineering Consultants Co. Ltd, Japan. Takafumi SEIKI reports financial support was provided by Utsunomiya City Hall for Oya area development. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

JH and ecdysone signaling regulate insect metamorphosis through the master transcription factors, Krüppel homolog 1 (kr-h1), Broad-Complex (BR-C), and E93. Ecdysone signaling activates successively expressed ecdysone responsive transcription factors (ERTFs), and the interaction between ERTFs determines the expression profiles of ERTFs themselves. Through the construction of expressed sequence tag (EST) database of Bombyx mori from many tissues, the existence of a large number of cuticular protein (CP) genes was identified in wing disc cDNA library of the 3 days after the start of wandering (W3). From the genomic analysis, 12 types of CP clusters of CP genes were identified. DNA sequences of CP genes revealed the duplication of CP genes, which suggests to reflect the insect evolution. These CP genes responded to ecdysone and ecdysone pulse; therefore, CP genes were applied for the analysis of transcriptional regulation by ERTF. The binding sites of ERTF have been reported to exist upstream of CP genes in several insects, and the activation of CP genes occurred by the binding of ERTFs. Through the analysis, the following were speculated; the successive appearance of ERTFs and the activation of target genes resulted in the successively produced CPs and cuticular layer. The sequence of the ERTF and CP gene expression was the same at larval to pupal and pupal to adult transformation. The involvement of several ERTFs in one CP gene expression was also clarified; BmorCPG12 belongs to group showing expression peak at W3 and was regulated by two ERTFs; BHR3 and ßFTZ-F1, BmorCPH2 belongs to group showing expression peak at P0 and was regulated by two ERTFs; ßFTZ-F1 and E74A. The involvement of BHR39 as a negative regulator of CP gene expression was found. Larval, pupal, and adult cuticular layers were supposed to be constructed by the combination of different and similar types of CPs, through the expressed timing of CP genes., (© 2024 Wiley Periodicals LLC.)

Global warming has led to the expansion of arid lands and more frequent droughts, which are the largest cause of global food production losses. In our previous study, we developed TaPYLox wheat overexpressing the plant hormone abscisic acid (ABA) receptor, which is important for the drought stress response in plants. TaPYLox showed resistance to drought stress and acquired water-saving traits that enable efficient grain production with less water use. In this study, we used TaPYLox to identify ABA-dependent and -independent metabolites in response to drought stress. We compared the variation of metabolites in wheat under well-watered, ABA treatment, and drought stress conditions using the ABA-sensitive TaPYLox line and control lines. The results showed that tagatose and L-serine were ABA-dependently regulated metabolites, because their stress-induced accumulation was increased by ABA treatment in TaPYLox. In contrast, L-valine, L-leucine, and DL-isoleucine, which are classified as branched chain amino acids, were not increased by ABA treatment in TaPYLox, suggesting that they are metabolites regulated in an ABA-independent manner. Interestingly, the accumulation of L-valine, L-leucine, and DL-isoleucine was suppressed in drought-tolerant TaPYLox under drought stress, suggesting that drought-tolerant wheat might be low in these amino acids. 3-dehydroshikimic acid and α-ketoglutaric acid were decreased by drought stress in an ABA-independent manner. In this study, we have succeeded in identifying metabolites that are regulated by drought stress in an ABA-dependent and -independent manner. The findings of this study should be useful for future breeding of drought-tolerant wheat., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Weng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: This study examined the immediate effects of oculomotor and bimanual coordination exercises, as well as a combination of the two, on stability of balance in athletes., Patients and Methods: Changes in center-of-gravity sway were measured in 30 college student athletes before and after the following three exercise conditions: 1) oculomotor exercises, 2) bimanual coordination exercises, and 3) a combination of oculomotor and bimanual coordination exercises (1+2). The order of these exercises was counterbalanced., Results: The combination of exercises (condition 3) reduced large swaying during balancing and immediately increased postural stability. Moreover, the oculomotor and bimanual coordination exercises (conditions 1 and 2) immediately reduced large sway during balancing when performed independently. Thus, the present study revealed that the combination of oculomotor and bimanual coordination exercises immediately reduced accidental swaying during balancing and also improved postural stability., Conclusion: This combination could be effective as an immediate balance adjustment method for athletes., Competing Interests: The authors have no conflicts of interest directly relevant to the content of this article., (© 2024 Matsuura et al.)

α,α-trehalose is a well-known sugar that plays a key role in establishing tolerance to environmental stresses in many organisms, except unicellular eukaryotes. However, almost nothing is known about α,β-trehalose, including their synthesis, function, and even presence in living organisms. In this study, we identified α,β-trehalose in the resting cyst, a dormancy cell form characterized by extreme tolerance to environmental stresses, of the ciliated protist Colpoda cucullus, using high-performance liquid chromatography (HPLC), and a proton nuclear magnetic resonance ( 1 H NMR). Gene expression analysis revealed that the expression of trehalose-6-phosphate synthase (TPS), glycosyltransferase (GT), alpha-amylase (AMY), and trehalose transporter 1 (TRET1), were up-regulated in encystment, while the expression of α-glucosidase 2 (AG2) and trehalase (TREH) was up-regulated in excystment. These results suggest that α,β-trehalose is synthesized during encystment process, while and contributes to extreme tolerances to environmental stressors, stored carbohydrates, and energy reserve during resting cyst and/or during excystment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The dorsal and anal fins can vary widely in position and length along the anterior-posterior axis in teleost fishes. However, the molecular mechanisms underlying the diversification of these fins remain unknown. Here, we used genetic approaches in zebrafish and medaka, in which the relative positions of the dorsal and anal fins are opposite, to demonstrate the crucial role of hox genes in the patterning of the teleost posterior body, including the dorsal and anal fins. By the CRISPR-Cas9-induced frameshift mutations and positional cloning of spontaneous dorsalfinless medaka, we show that various hox mutants exhibit the absence of dorsal or anal fins, or a stepwise posterior extension of these fins, with vertebral abnormalities. Our results indicate that multiple hox genes, primarily from hoxc -related clusters, encompass the regions responsible for the dorsal and anal fin formation along the anterior-posterior axis. These results further suggest that shifts in the anterior boundaries of hox expression which vary among fish species, lead to diversification in the position and size of the dorsal and anal fins, similar to how modulations in Hox expression can alter the number of anatomically distinct vertebrae in tetrapods. Furthermore, we show that hox genes responsible for dorsal fin formation are different between zebrafish and medaka. Our results suggest that a novel mechanism has occurred during teleost evolution, in which the gene network responsible for fin formation might have switched to the regulation downstream of other hox genes, leading to the remarkable diversity in the dorsal fin position., Competing Interests: Competing interests statement:The authors declare no competing interest.

Gene doping is prohibited in horse sports and can involve the administration of exogenous genes, called transgenes, to postnatal animals. Quantitative polymerase chain reaction (qPCR) methods have been developed to detect gene doping; however, these generally require DNA extraction from the plasma prior to qPCR. In this study, we developed two methods, direct droplet digital PCR (ddPCR) and nested ddPCR, to detect the equine erythropoietin (EPO) transgene without DNA extraction. Direct ddPCR used pretreated plasma and PCR to detect the EPO transgene spiked at 10 copies/μL. Nested ddPCR utilised pre-amplification using nontreated plasma, purification of PCR products and PCR to detect the EPO transgene spiked at 1 copy/μL in plasma. These methods successfully detected the EPO transgene after intramuscular injection into horses. Since each method has different detection sensitivity, the combined use of direct ddPCR for screening and nested ddPCR for confirmation may complement each other and prevent the occurrence of false positives, allowing the reliable detection of gene-doped substances. One advantage of these methods is the small amount of sample required, approximately 2.2-5.0 μl, owing to the lack of a DNA extraction step. Therefore, these tests could be applied to small volume samples as an alternative to conventional gene doping tests., (© 2024 John Wiley & Sons Ltd.)