Elucidating important factors and corresponding method optimization for sensitive detection of small peptide drugs in human urine by solid-phase extraction and UPLC-HRMS: The influence of MS scan modes, protein precipitants, and ammonium formate.

Small peptide hormones are widely used in sports as performance-enhancing substances, making it crucial to develop sensitive analytical methods for their detection in doping control analysis. Various factors significantly affect analytical sensitivity, such as the selection of ultra-performance liquid chromatography (UPLC) mobile phase, high-resolution mass spectrometry (HRMS) scanning modes, and extraction solvents for pretreatment. Herein, comparative study approach was utilized to investigate the sensitivity of each peptide analyte under both full scan and parallel reaction monitoring (PRM) modes of HRMS and assess the effects of some protein precipitants as a part of extraction solvents on solid-phase extraction (SPE). The results showed that full scan should be selected as the primary scan mode of HRMS, and the combination with PRM mode could effectively compensate for the limitations of full scan, and the addition of protein precipitants would adversely affect the detection of certain small peptide analytes. Meanwhile, influences of ammonium formate in reverse UPLC mobile phase on the charge state distribution of small peptides were investigated and elucidated. Based on these findings, a sensitive and reliable UPLC-HRMS analytical method combining full scan and PRM mode was validated for screening and confirmation of 63 small peptide analytes after SPE, with limits of detection (LODs) ranging between 0.010 and 0.473 ng/ml and limits of identification (LOIs) ranging between 0.015 and 1.512 ng/ml. Additionally, suggestions were provided for the detection of [Arg8]-vasopressin, dermorphin, and its analogues.

Olgotrelvir as a Single-Agent Treatment of Nonhospitalized Patients with Covid-19.

BACKGROUND: Olgotrelvir is an oral antiviral with dual mechanisms of action targeting severe acute respiratory syndrome coronavirus 2 main protease (i.e., Mpro) and human cathepsin L. It has potential to serve as a single-agent treatment of coronavirus disease 2019 (Covid-19). METHODS: We conducted a phase 3, double-blind, randomized, placebo-controlled trial to evaluate the efficacy and safety of olgotrelvir in 1212 nonhospitalized adult participants with mild to moderate Covid-19, irrespective of risk factors, who were randomly assigned to receive orally either 600 mg of olgotrelvir or placebo twice daily for 5 days. The primary and key secondary end points were time to sustained recovery of a panel of 11 Covid-19-related symptoms and the viral ribonucleic acid (RNA) load. The safety end point was incidence of treatment-emergent adverse events. RESULTS: The baseline characteristics of 1212 participants were similar in the two groups. In the modified intention-to-treat population (567 patients in the placebo group and 558 in the olgotrelvir group), the median time to symptom recovery was 205 hours in the olgotrelvir group versus 264 hours in the placebo group (hazard ratio, 1.29; 95% confidence interval [CI], 1.13 to 1.46; P<0.001). The least squares mean (95% CI) changes of viral RNA load from baseline were -2.20 (-2.59 to -1.81) log10 copies/ml in olgotrelvir-treated participants and -1.40 (-1.79 to -1.01) in participants receiving placebo at day 4. Skin rash (3.3%) and nausea (1.5%) were more frequent in the olgotrelvir group than in the placebo group; there were no treatment-related serious adverse events, and no deaths were reported. CONCLUSIONS: Olgotrelvir as a single-agent treatment significantly improved symptom recovery. Adverse effects were not dose limiting. (Funded by Sorrento Therapeutics, a parent company of ACEA Therapeutics; ClinicalTrials.gov number, NCT05716425.).

The plant is neither dumb nor deaf; it talks and hears.

Animals and insects communicate using vibrations that are frequently too low or too high for human ears to detect. Plants and trees can communicate and sense sound. Khait et al. used a dependable recording system to capture airborne sounds produced by stressed plants. In addition to allowing plants to communicate their stress, sound aids in plant defense, development, and resilience. It also serves as a warning that danger is approaching. Demey et al. and others discussed the audit examinations that were conducted to investigate sound discernment in plants at the atomic and biological levels. The biological significance of sound in plants, the morphophysiological response of plants to sound, and the airborne noises that plants make and can hear from a few meters away were all discussed.

Comparative analysis of endophytic fungal communities in bamboo species Phyllostachys edulis, Bambusa rigida, and Pleioblastus amarus.

Fungal endophytes in plant leaf mesophyll form mutually beneficial associations through carbon assimilation, synthesis of biologically active chemicals, and enhancement of aesthetic and nutritional value. Here, we compared community structure, diversity, and richness of endophytic fungi in the leaves of three bamboo species, including Phyllostachys edulis (MZ), Bambusa rigida (KZ), and Pleioblastus amarus (YT) via high-throughput Illumina sequencing. In total, 1070 operational taxonomic units (OTUs) were retrieved and classified into 7 phylum, 27 classes, 82 orders, 185 families, 310 genus, and 448 species. Dominant genera were Cladosporium, Trichomerium, Hannaella, Ascomycota, Sporobolomyces, Camptophora and Strelitziana. The highest fungal diversity was observed in Pleioblastus amarus, followed by Bambusa rigida, and Phyllostachys edulis. Comparatively, monopodial species Ph. edulis and sympodial B. rigida, mixed P. amarus revealed the highest richness of endophytic fungi. We retrieved a few biocontrol agents, Sarocladium and Paraconiothyrium, and unique Sporobolomyces, Camptophora, and Strelitziana genera. FUNGuild analysis revealed the surrounding environment (The annual average temperature is between 15 and 25 °C, and the relative humidity of the air is above 83% all year round) as a source of fungal accumulation in bamboo leaves and their pathogenic nature. Our results provide precise knowledge for better managing bamboo forests and pave the way for isolating secondary metabolites and potential bioactive compounds.

SynAI: an AI-driven cancer drugs synergism prediction platform.

Summary: The SynAI solution is a flexible AI-driven drug synergism prediction solution aiming to discover potential therapeutic value of compounds in early stage. Rather than providing a finite choice of drug combination or cell lines, SynAI is capable of predicting potential drug synergism/antagonism using in silico compound SMILE (Simplified Molecular Input Line Entry System) sequences. The AI core of SynAI platform has been trained against cell lines and compound pairs listed by NCI (National Cancer Institute)-Almanac and DurgCombDB datasets. In total, the training data consists of over 1 200 000 in vitro synergism tests on 150 cancer cell lines of different organ origins. Each cell line is tested against over 6000 pairs of FDA (Food and Drug Administration) approved compound combinations. Given one or both candidate compound in SMILE sequence, SynAI is able to predict the potential Bliss score of the combined compound test with the designated cell line without the needs of compound synthetization or structural analysis; thus can significantly reduce the candidate screening costs during the compound development. SynAI platform demonstrates a comparable performance to existing methods but offers more flexibilities for data input. Availability and implementation: The evaluation version of SynAI is freely accessible online at https://synai.crownbio.com.

Integrative application of silicon and/or proline improves Sweet corn (Zea mays L. saccharata) production and antioxidant defense system under salt stress condition.

Silicon (Si) and/or proline (Pro) are natural supplements that are considered to induce plants' stress tolerance against various abiotic stresses. Sweet corn (Zea mays L. saccharata) production is severely afflicted by salinity stress. Therefore, two field tests were conducted to evaluate the potential effects of Si and/or Pro (6mM) used as seed soaking (SS) and/or foliar spray (FS) on Sweet corn plant growth and yield, physio-biochemical attributes, and antioxidant defense systems grown in a saline (EC = 7.14dS m-1) soil. The Si and/or Pro significantly increased growth and yield, photosynthetic pigments, free proline, total soluble sugars (TSS), K+/Na+ratios, relative water content (RWC), membrane stability index (MSI), α-Tocopherol (α-TOC), Ascorbate (AsA), glutathione (GSH), enzymatic antioxidants activities and other anatomical features as compared to controls. In contrast, electrolytes, such as SS and/or FS under salt stress compared to controls (SS and FS using tap water) were significantly decreased. The best results were obtained when SS was combined with FS via Si or Pro. These alterations are brought about by the exogenous application of Si and/or Pro rendering these elements potentially useful in aiding sweet corn plants to acclimate successfully to saline soil.

Variable-Structure Proportional-Integral-Derivative Laser Solder Joint Temperature Intelligent Control Method with Adjustable Power Upper Limit.

Laser soldering is a crucial soldering technique in the realm of electronic assembly. The temperature of the solder joint is intimately connected with the quality of the solder. This paper introduces an adjustable power upper limit variable-structure Proportional-Integral-Derivative (PID) intelligent control method for regulating the temperature of the solder joint during laser soldering. Distinct laser power limits are employed for workpieces with varying heat capacities. The solder joint temperature is monitored through an infrared thermometer, which enables closed-loop temperature control via a variable-structure PID algorithm. Residual neural network (ResNet) models are utilized to predict key soldering process parameters. This method has been executed and validated on a practical testing platform. Compared to other laser soldering control techniques, the proposed method demonstrates a low overshoot, rapid dynamic response, and swift adjustment capabilities, effectively enhancing the soldering quality and production efficiency.

A comprehensive and high-throughput screening method for multiple prohibited substances by UPLC-QE Plus-HRMS and HPLC-QQQ-MS in human urine for doping control.

Since the World Anti-Doping Agency's (WADA) Prohibited List is updated on an annual basis, screening methods must be continually adapted to align with these changes. In accordance with Technical Document-MRPL 2022, a newly combined, comprehensive, rapid and high-throughput doping control screening method has been developed for the analysis of 350 substances with different polarities in human urine using ultra-high performance liquid chromatography coupled with Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer (UPLC-QE Plus-HRMS) and ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometer (UPLC-QQQ-MS). The limits of detection were in the range of 0.12-50 ng mL-1 for beta-2 agonists, hormone and metabolic modulators, narcotics, cannabinoids and glucocorticoids, 0.1-14 ng mL-1 for the manipulation of blood and blood components, beta blockers, anabolic agents and hypoxia-inducible factor (HIF) activating agents, and 2.5-100 000 ng mL-1 for substances of Appendix A, diuretics & masking agents and stimulants. The sample preparation consisted of two parts: one is the dilute & shoot part analyzed in UPLC-QQQ-MS, another is a mixture of the dilute & shoot part and a liquid-liquid extraction part of hydrolyzed human urine analyzed in UPLC-QE Plus-HRMS in full scan mode with polarity switching and parallel reaction monitoring (PRM) mode. The method has been fully validated for doping control purposes. All the substances were compliant with WADA's required 1/2 minimum requirement performance level (MRPL) or minimum reporting level (MRL), and this method was successfully used in the 2022 Beijing Winter Olympic Games and Winter Paralympic Games for anti-doping purpose.

Interplay of silymarin and clove fruit extract effectively enhances cadmium stress tolerance in wheat (Triticum aestivum).

Introduction: Osmoprotectant supplementation can be used as a useful approach to enhance plant stress tolerance. However, the effect of silymarin and clove fruit extract (CFE) on wheat plants grown under cadmium (Cd) stress has not been studied. Methods: Wheat seeds were planted in plastic pots filled with ions-free sand. A ½-strength Hoagland's nutrient solution was used for irrigation. Pots were treated with eight treatments thirteen days after sowing: 1) Control, 2) 0.5 mM silymarin foliar application [silymarin], 3) 2% CFE foliar application [CFE], 4) CFE enriched with silymarin (0.24 g silymarin L-1 of CFE) [CFE-silymarin], 5) Watering wheat seedlings with a nutritious solution of 2 mM Cd [Cd]. 6) Cadmium + silymarin, 7) Cadmium + CFE, and 8) Cadmium + CFE-silymarin. The experimental design was a completely randomized design with nine replicates. Results and discussion: The Cd stress decreased grain yield, shoot dry weight, leaf area, carotenoids, chlorophylls, stomatal conductance, net photosynthetic rate, transpiration rate, membrane stability index, nitrogen, phosphorus, and potassium content by 66.9, 60.6, 56.7, 23.8, 33.5, 48.1, 41.2, 48.7, 42.5, 24.1, 39.9, and 24.1%, respectively. On the other hand, Cd has an Application of CFE, silymarin, or CEF-silymarin for wheat plants grown under Cd stress, significantly improved all investigated biochemical, morphological, and physiological variables and enhanced the antioxidant enzyme activities. Applying CFE and/or silymarin enhanced plant tolerance to Cd stress more efficiently. Our findings suggest using CFE-silymarin as a meaningful biostimulator for wheat plants to increase wheat plants' tolerance to Cd stress via enhancing various metabolic and physiological processes.

Genome-Wide Analysis and Expression Profiling of SlHsp70 Gene Family in Solanum lycopersicum Revealed Higher Expression of SlHsp70-11 in Roots under Cd2+ Stress.

BACKGROUND: Tomato is an important part of daily food, rich source of multitude nutrients, suitable candidate for bio-pharmaceutical production due to berry size and has numerous health benefits. Transcriptional regulation of metalloregulatory heat shock protein-70 family plays pivotal role in plants tolerance against abiotic stress factors including salinity, heat, cold, drought and trace metal elements such as cadmium (Cd2+). METHODS: Here, we provide comprehensive report on in-silico identification of SlHsp70 family genes in tomato (Solanum lycopersicum) and their expression in tomato via qPCR analysis under broad range of trace metal elements. RESULTS: In-silico analysis revealed 23 SlHsp70 family genes in tomato, phylogenetically divided into four groups I-IV and displayed expression in all tissues. Gene Ontology (GO) analysis revealed that SlHSP70 proteins were membrane localized which were involved in metal ions translocation and oxidoreductase activity to counter hyper-accumlation of reactive oxygen species (ROS). CONCLUSIONS: Cd2+ is a widespread heavy metal soil contaminent which is continously polluting fertile soils, a knotty issue which has serious implications over photosynthesis, nitrogen assimilation, minerals and water absorption by plants. Plants exposure to Cd2+ and subsequent qRT-PCR analysis revealed increased expression of SlHsp70-11 in tomato roots, which can be employed in breeding low Cd2+ enriched tomato varieties.

Agri-Nanotechnology and Tree Nanobionics: Augmentation in Crop Yield, Biosafety, and Biomass Accumulation.

Nanomaterials (NMs) are the leading edge as an amazing class of materials that consists of at least one dimension in the range of 1-100 nm. NMs can be made with exceptional magnetic, electrical, and catalytic properties different from their bulk counterparts. We summarized unique features of NMs, their synthesis, and advances in agri-nanotechnology and cutting-edge nanobionics. The review describes advances in NMs including their applications, dosimetry to ensure biosafety, remote sensing of agro-forestry fields, nanofertilizers, and nanopesticides, and avoid post-harvest losses, gene delivery, and nanobionics. Tree nanobionics has enabled the synthesis and delivery of nanosensors, which enhance the rate of photosynthesis, detection of pathogens, and poisonous residues to ensure biosafety and biomass accumulation. Finally, we conclude by discussing challenges, future perspectives, and agro-ecological risks of using NMs.

Heat Stress-Mediated Constraints in Maize (Zea mays) Production: Challenges and Solutions.

An increase in temperature and extreme heat stress is responsible for the global reduction in maize yield. Heat stress affects the integrity of the plasma membrane functioning of mitochondria and chloroplast, which further results in the over-accumulation of reactive oxygen species. The activation of a signal cascade subsequently induces the transcription of heat shock proteins. The denaturation and accumulation of misfolded or unfolded proteins generate cell toxicity, leading to death. Therefore, developing maize cultivars with significant heat tolerance is urgently required. Despite the explored molecular mechanism underlying heat stress response in some plant species, the precise genetic engineering of maize is required to develop high heat-tolerant varieties. Several agronomic management practices, such as soil and nutrient management, plantation rate, timing, crop rotation, and irrigation, are beneficial along with the advanced molecular strategies to counter the elevated heat stress experienced by maize. This review summarizes heat stress sensing, induction of signaling cascade, symptoms, heat stress-related genes, the molecular feature of maize response, and approaches used in developing heat-tolerant maize varieties.

Knowledge domain and emerging trends of climate-smart agriculture: a bibliometric study.

Climate-smart agriculture (CSA) is a new agricultural development pattern to address future food crises. Since CSA was proposed in 2010, it has attracted the attention of scholars from all over the world. It is of great significance to scientifically summarize the overview and emerging trends of CSA research, providing ideas for scholars concerned about CSA to engage in research in this field. Based on bibliometrics and CSA-related literature data in the WOS database, this paper used CiteSpace software to draw knowledge maps to scientifically analyze publications in the field of CSA. Our study found that (1) CSA research is showing a rapid upward trend, focusing on the environmental sciences and agricultural economic management; (2) international organizations such as the FAO, World Bank, and the international agricultural research institute have made significant contributions to CSA research; (3) among the nine clusters in the CSA literature, CSA practice, conservation agriculture, smallholder farmers, and sub-Saharan Africa have been consistently given high attention; (4) CSA research can be divided into three phases, and the research hotspots have transferred from essential elements of CSA to household and carbon emissions. We believe that in future research, more attention should be paid to the trade-off and synergy of the three pillars of CSA, as well as the investment, finance, and evaluation criteria of CSA. Such strengthening is of great significance to the sustainable promotion of CSA.

Functional patient-derived organoid screenings identify MCLA-158 as a therapeutic EGFR × LGR5 bispecific antibody with efficacy in epithelial tumors.

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.

Genotoxicity and Trace Elements Contents Analysis in Nile Tilapia (Oreochromis niloticus) Indicated the Levels of Aquatic Contamination at Three Egyptian Areas.

The toxic waste and pollutants of heavy metals continuously pollute freshwater aquatic reservoirs, which have severe implications on aquatic life and human health. The present work aims to evaluate trace elements (Zn, Mn, Cu, Cd, and Pb) along with three sites, Mariout Lake, Abbassa, and River Nile Aswan in Egypt, using Nile tilapia (Oreochromis niloticus) as bioindicator. The quality assurance, health-risk assessment, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), quantitative polymerase chain reaction (qPCR), and micronucleus test were performed to investigate the effect of different trace elements on Hsp70 gene level and micronuclei formation. We observed the highest expression of Hsp70 protein band of 70 KD and stress-responsive Hsp70 gene in the liver followed by gills of Nile tilapia caught from Mariout and Abbassa, but the lowest expression was in Nile tilapia caught from Aswan. Obvious micronuclei were observed under the microscope in erythrocytes, and their number was gradually decreased in the following manner: Mariout > Abbassa > Aswan. Noticeably, Cu, Zn, and Mn contents were low. Still, Pb and Cd contents were higher than the toxicity level recommended by the Food and Agriculture Organization (FAO), The World Health Organization (WHO), and the European Commission (EC). These results showed that Hsp70's appearance at the two levels of mRNA and protein is an effective indicator for aquatic pollution besides the aberration at the chromosome level represented in the micronucleus test. Furthermore, these results showed that Nile tilapia of the Aswan region had comparatively low trace elements contamination and were suitable for consumption.

Quantification of Ephedrine Substances in Human Urine by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

An accurate quantitative method for four prohibited ephedrine substances in human urine has been established, based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The quantitative bias caused by pretreatment operations and matrix effects was reduced by the dilute and shoot pretreatment method. The good separation of isomers was achieved with the advantages of the UPLC instrument and Agilent Poroshell 120 EC-C8 UPLC column. Stable quantitative ions were selected during the analysis with MS/MS. The result of the method validation experiment showed an excellent linearity between 50% and 200% threshold concentration with a correlation coefficient (r2) greater than 0.999. The coefficient of variation at the limit of quantification and threshold was <20% and 10%, respectively. The uncertainty was below the maximum uncertainty specified in the technical document of the World Anti-Doping Agency (WADA). The analytical result using this method has passed the WADA-external quality assessment scheme. The anti-doping laboratory has applied the method in routine tests and reported adverse analytical finding.

Determination of Community Structure and Diversity of Seed-Vectored Endophytic Fungi in Alpinia zerumbet.

Endophytic fungi act as seed endosymbiont, thereby playing a very crucial role in the growth and development of seeds. Seed-vectored endophytic fungi establish an everlasting association with seeds and travel from generation to generation. To explore the composition and diversity of endophytic fungi in Alpinia zerumbet seeds, high-throughput Illumina MiSeq sequencing was employed for the following stages: fruit formation period (YSJ1), young fruit period (YSJ2), early mature period (YSJ3), middle mature period (YSJ4), and late mature period (YSJ5). A total of 906,694 sequence reads and 745 operational taxonomic units (OTUs) were obtained and further classified into 8 phyla, 30 classes, 73 orders, 163 families, 302 genera, and 449 species. The highest endophytic fungal diversity was observed at YSJ5. The genera with the highest abundance were Cladosporium, Kodamaea, Hannaella, Mycothermus, Gibberella, Sarocladium, and Neopestalotiopsis. Functional Guild (FUNGuild) analysis revealed that endophytic fungi were undefined saprotroph, plant pathogens, animal pathogen-endophyte-lichen parasite-plant pathogen-wood saprotroph, and soil saprotrophs. Alternaria, Fusarium, Cladosporium, and Sarocladium, which are potential probiotics and can be used as biocontrol agents, were also abundant. This study is part of the Sustainable Development Goals of United Nations Organization (UNO) to "Establish Good Health and Well-Being."

Natural resistance of tomato plants to Tomato yellow leaf curl virus.

Tomato yellow leaf curl virus (TYLCV) is one of the most harmful afflictions in the world that affects tomato growth and production. Six regular antagonistic genes (Ty-1, Ty-2, Ty-3, Ty-4, ty-5, and Ty-6) have been transferred from wild germplasms to commercial cultivars as TYLCV protections. With Ty-1 serving as an appropriate source of TYLCV resistance, only Ty-1, Ty-2, and Ty-3 displayed substantial levels of opposition in a few strains. It has been possible to clone three TYLCV opposition genes (Ty-1/Ty-3, Ty-2, and ty-5) that target three antiviral safety mechanisms. However, it significantly impacts obtaining permanent resistance to TYLCV, trying to maintain opposition whenever possible, and spreading opposition globally. Utilizing novel methods, such as using resistance genes and identifying new resistance resources, protects against TYLCV in tomato production. To facilitate the breeders make an informed decision and testing methods for TYLCV blockage, this study highlights the portrayal of typical obstruction genes, common opposition sources, and subatomic indicators. The main goal is to provide a fictitious starting point for the identification and application of resistance genes as well as the maturation of tomato varieties that are TYLCV-resistant.

Doping control analysis of small peptides in human urine using LC-HRMS with parallel reaction monitoring mode: screening and confirmation.

This study described a reliable analytical method, which combines solid-phase extraction (SPE) with liquid chromatography-high resolution mass spectrometry (LC-HRMS) employing the parallel reaction monitoring (PRM) mode, for screening 41 small peptides and 3 non-peptide growth hormone secretagogues in human urine. Additionally 36 small peptides and 3 non-peptide growth hormone secretagogues were also confirmed in the same way. For the whole screening procedure, the PRM mode was applied to the HRMS detection of small peptides, which reduces the background noise from matrix compounds to a large extent and thus improves the selectivity and reliability of the peptide analytes. Meanwhile, competent chromatographic separation was achieved within a total runtime of 14 minutes, indicating an improvement in the detection efficiency. Moreover, the PRM mode could also be applied to the confirmation procedure due to its strong identification power with a low risk of generating false positives or negatives and good selectivity. Validation was performed according to the relevant World Anti-Doping Agency (WADA) criteria, including selectivity and reliability, limit of detection (LOD), limit of identification (LOI), recovery, extraction stability and carryover. The LODs of the peptide analytes ranged between 0.20 ng mL-1 and 0.92 ng mL-1 in urine, while their LOIs ranged between 0.20 ng mL-1 and 2.00 ng mL-1, which met the corresponding Minimum Required Performance Levels (MRPLs) as defined by WADA. The developed method furnished the rapid and sensitive detection of small peptides in urine for more than 5000 samples with no false-positive or false-negative, indicating that it is an eligible method for doping control analysis.

Genome-Wide Identification and Expression Analysis of Metal Tolerance Protein Gene Family in Medicago truncatula Under a Broad Range of Heavy Metal Stress.

Metal tolerance proteins (MTPs) encompass plant membrane divalent cation transporters to specifically participate in heavy metal stress resistance and mineral acquisition. However, the molecular behaviors and biological functions of this family in Medicago truncatula are scarcely known. A total of 12 potential MTP candidate genes in the M. truncatula genome were successfully identified and analyzed for a phylogenetic relationship, chromosomal distributions, gene structures, docking analysis, gene ontology, and previous gene expression. M. truncatula MTPs (MtMTPs) were further classified into three major cation diffusion facilitator (CDFs) groups: Mn-CDFs, Zn-CDFs, and Fe/Zn-CDFs. The structural analysis of MtMTPs displayed high gene similarity within the same group where all of them have cation_efflux domain or ZT_dimer. Cis-acting element analysis suggested that various abiotic stresses and phytohormones could induce the most MtMTP gene transcripts. Among all MTPs, PF16916 is the specific domain, whereas GLY, ILE, LEU, MET, ALA, SER, THR, VAL, ASN, and PHE amino acids were predicted to be the binding residues in the ligand-binding site of all these proteins. RNA-seq and gene ontology analysis revealed the significant role of MTP genes in the growth and development of M. truncatula. MtMTP genes displayed differential responses in plant leaves, stems, and roots under five divalent heavy metals (Cd2+, Co2+, Mn2+, Zn2+, and Fe2+). Ten, seven, and nine MtMTPs responded to at least one metal ion treatment in the leaves, stems, and roots, respectively. Additionally, MtMTP1.1, MtMTP1.2, and MtMTP4 exhibited the highest expression responses in most heavy metal treatments. Our results presented a standpoint on the evolution of MTPs in M. truncatula. Overall, our study provides a novel insight into the evolution of the MTP gene family in M. truncatula and paves the way for additional functional characterization of this gene family.