Combination of facial and nose features of Amur tigers to determine age.

We found that the area of black round or irregular-shaped spots on the tiger's nose increased with age, indicating a positive relationship between age and nose features. We used the deep learning model to train the facial and nose image features to identify the age of Amur tigers, using a combination of classification and prediction methods to achieve age determination with an accuracy of 87.81%.

FLOWERING LOCUS T1 and TERMINAL FLOWER1 regulatory networks mediate flowering initiation in apple.

Flower bud formation is a critical process that directly determines yield and fruit quality in fruit crops. Floral induction is modulated by the balance between 2 flowering-related proteins, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1); however, the mechanisms underlying the establishment and maintenance of this dynamic balance remain largely elusive. Here, we showed that in apple (Malus × domestica Borkh.), MdFT1 is predominantly expressed in spur buds and exhibits an increase in expression coinciding with flower induction; in contrast, MdTFL1 exhibited downregulation in apices during flower induction, suggesting that MdTFL1 has a role in floral repression. Interestingly, both the MdFT1 and MdTFL1 transcripts are directly regulated by transcription factor basic HELIX-LOOP-HELIX48 (MdbHLH48), and overexpression of MdbHLH48 in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) results in accelerated flowering. Binding and activation analyses revealed that MdbHLH48 functions as a positive regulator of MdFT1 and a negative regulator of MdTFL1. Further studies established that both MdFT1 and MdTFL1 interact competitively with MdWRKY6 protein to facilitate and inhibit, respectively, MdWRKY6-mediated transcriptional activation of target gene APPLE FLORICAULA/LFY (AFL1, an apple LEAFY-like gene), ultimately regulating apple flower bud formation. These findings illustrate the fine-tuned regulation of flowering by the MdbHLH48-MdFT1/MdTFL1-MdWRKY6 module and provide insights into flower bud formation in apples.

Chitinase-Like Protein PpCTL1 Contributes to Maintaining Fruit Firmness by Affecting Cellulose Biosynthesis during Peach Development.

The firmness of the flesh fruit is a very important feature in the eating process. Peach fruit is very hard during development, but its firmness slightly decreases in the later stages of development. While there has been extensive research on changes in cell wall polysaccharides during fruit ripening, little is known about the changes that occur during growth and development. In this study, we investigated the modifications in cell wall components throughout the development and ripening of peach fruit, as well as its impact on firmness. Our findings revealed a significant positive correlation between fruit firmness and cellulose content at development stage. However, the correlation was lost during the softening process, suggesting that cellulose might be responsible for the fruit firmness during development. Members of the chitinase-like protein (CTL) group are of interest because of their possible role in plant cell wall biosynthesis. Here, two CTL homologous genes, PpCTL1 and PpCTL2, were identified in peach. Spatial and temporal expression patterns of PpCTLs revealed that PpCTL1 exhibited high expression abundance in the fruit and followed a similar trend to cellulose during fruit growth. Furthermore, silencing PpCTL1 expression resulted in reduced cellulose content at 5 DAI (days after injection), this change that would have a negative effect on fruit firmness. Our results indicate that PpCTL1 plays an important role in cellulose biosynthesis and the maintenance of peach firmness during development.

The longitudinal and regional analysis of bleomycin-induced pulmonary fibrosis in mice by microcomputed tomography.

Introduction: Microcomputed tomography (micro-CT) is powerful for assessment of the progression of lung fibrosis in animal model, but current whole lung analysis (WLA) methods are time-consuming. Here, a longitudinal and regional analysis (LRA) method was developed to assess fibrosis easily and quickly by micro-CT. Method: Firstly, we investigated the distribution pattern of lesions in BLM-induced pulmonary fibrosis mice. Then, the VOIs for LRA were selected based on the anatomical locations and we compared the robustness, accuracy, repeatability, analysis time of LRA to WLA. Additionally, LRA was applied to assess different stages of pulmonary fibrosis, and was validated with conventional endpoint measurements (such as lung hydroxyproline and histopathology). Results: The lesions of fibrosis in 66 bleomycin (BLM)-induced pulmonary fibrosis mice were mostly in the middle and upper parts of lungs. By applying LRA, the percentages of high-density voxels in selected volumes of interest (VOIs) were well correlated with that in WLA both at Day 7 and Day 21 after bleomycin induction (R2 = 0.8784 and 0.8464, respectively). The relative standard deviation (RSD) of the percentage of high-density voxels in the VOIs was lower than that of WLA (P < 0.05). The cost time of LRA was shorter than that of WLA (P < 0.05) and the accuracy of LRA was further confirmed by the histological analysis and biochemical quantification of hydroxyproline. Conclusion: LRA is probably an easier and more time-saving method to assess fibrosis formation and evaluate treatment efficacy.

Myricetin possesses the potency against SARS-CoV-2 infection through blocking viral-entry facilitators and suppressing inflammation in rats and mice.

BACKGROUND: Myricetin (3,5,7-trihydroxy-2-(3,4,5-tri hydroxyphenyl)-4-benzopyrone) is a common flavonol extracted from many natural plants and Chinese herb medicines and has been demonstrated to have multiple pharmacological activities, such as anti-microbial, anti-thrombotic, neuroprotective, and anti-inflammatory effects. Previously, myricetin was reported to target Mpro and 3CL-Pro-enzymatic activity to SARS-CoV-2. However, the protective value of myricetin on SARS-Cov-2 infection through viral-entry facilitators has not yet been comprehensively understood. PURPOSE: The aim of the current study was to evaluate the pharmacological efficacy and the mechanisms of action of myricetin against SARS-CoV-2 infection both in vitro and in vivo. METHODS: The inhibitory effects of myricetin on SARS-CoV-2 infection and replication were assessed on Vero E6 cells. Molecular docking analysis and bilayer interferometry (BLI) assays, immunocytochemistry (ICC), and pseudoviruses assays were performed to evaluate the roles of myricetin in the intermolecular interaction between the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and angiotensin-converting enzyme 2 (ACE2). The anti-inflammatory potency and mechanisms of myricetin were examined in THP1 macrophages in vitro, as well as in carrageenan-induced paw edema, delayed-type hypersensitivity (DTH) induced auricle edema, and LPS-induced acute lung injury (ALI) animal models. RESULTS: The results showed that myricetin was able to inhibit binding between the RBD of the SARS-CoV-2 S protein and ACE2 through molecular docking analysis and BLI assay, demonstrating its potential as a viral-entry facilitator blocker. Myricetin could also significantly inhibit SASR-CoV-2 infection and replication in Vero E6 cells (EC50 55.18 µM), which was further validated with pseudoviruses containing the RBD (wild-type, N501Y, N439K, Y453F) and an S1 glycoprotein mutant (S-D614G). Moreover, myricetin exhibited a marked suppressive action on the receptor-interacting serine/threonine protein kinase 1 (RIPK1)-driven inflammation and NF-kappa B signaling in THP1 macrophages. In animal model studies, myricetin notably ameliorated carrageenan-induced paw edema in rats, DTH induced auricle edema in mice, and LPS-induced ALI in mice. CONCLUSION: Our findings showed that myricetin inhibited HCoV-229E and SARS-CoV-2 replication in vitro, blocked SARS-CoV-2 virus entry facilitators and relieved inflammation through the RIPK1/NF-κB pathway, suggesting that this flavonol has the potential to be developed as a therapeutic agent against COVID-19.

Deciphering the molecular mechanisms of Maxing Huoqiao Decoction in treating pulmonary fibrosis via transcriptional profiling and circRNA-miRNA-mRNA network analysis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung condition with unknown etiology and high mortality. Chinese herbal medicine has been used for more than a thousand years to treat various lung diseases. PURPOSE: The current study aimed to examine whether Chinese herbal Maxing Huoqiao Decoction (MXHQD) exerts therapeutic effects on IPF and to further uncover its underlying molecular mechanisms. METHODS: Mouse model of acute lung injury (ALI) or IPF was induced by intratracheal instillation of LPS or bleomycin, respectively. ALI mice were treated with MXHQD for 7 days, and lung tissues were taken for test after modeling 24 h. IPF mice were gavaged for 21 days after modeling. Lung tissues were subjected to whole transcriptome detection, and the differential RNAs were experimentally verified. RESULTS: The results showed that MXHQD alleviated the computed tomography (CT) and the pathological degree changes in mice with IPF, improved changes in the expression of fibrosis related genes and reduced the hydroxyproline expression in IPF mice. MXHQD also decreased the cell numbers in bronchoalveolar lavage fluids, and the expression levels of the inflammatory factors in the ALI mice lung tissues were significantly inhibited. By applying whole transcriptome analysis, results showed that MXHQD acted on 40 mRNAs, 15 miRNAs, 25 novel lncRNAs and 17 circRNAs to resist pulmonary fibrosis. The competing endogenous RNA (ceRNA) network diagram showed that the multiple components of MXHQD against fibrosis through a network of multiple targets. The differential mRNAs were mainly related to the innate immune response and the defense response to virus. Then the expression of mRNAs in the differential mRNA-miRNA-differential circRNA network in the lung tissue of IPF was verified. The expression of ZBP1 and ISG15 related to immune system and anti virus was verified at both gene and protein expressions. MXHQD could significantly inhibit the elevation of ZBP1 and ISG15 factors induced by the fibrosis model. CONCLUSION: Overall, our findings provide compelling evidence that MXHQD can alleviate IPF by modulating innate immunity. This is the first study to reveal the molecular mechanism underlying the multi-components, multi-channels and multi-targets anti-IPF immune injury of MXHQD, and supports its potential clinical application for IPF.

PpHY5 is involved in anthocyanin coloration in the peach flesh surrounding the stone.

Red coloration around the stone (Cs) is an important trait of canned peaches (Prunus persica). In this study, an elongated hypocotyl 5 gene in peach termed PpHY5 was identified to participate in the regulation of the Cs trait. The E3 ubiquitin ligase PpCOP1 was expressed in the flesh around the stone and could interact with PpHY5. Although HY5 is known to be degraded by COP1 in darkness, the PpHY5 gene was activated in the flesh tissue surrounding the stone at the ripening stages and its expression was consistent with anthocyanin accumulation. PpHY5 was able to promote the transcription of PpMYB10.1 through interacting with its partner PpBBX10. Silencing of PpHY5 in the flesh around the stone caused a reduction in anthocyanin pigmentation, while transient overexpression of PpHY5 and PpBBX10 resulted in anthocyanin accumulation in peach fruits. Moreover, transgenic Arabidopsis seedlings overexpressing PpHY5 showed increased anthocyanin accumulation in leaves. Our results improve our understanding of the mechanisms of anthocyanin coloration in plants.

Cytokinin-responsive MdTCP17 interacts with MdWOX11 to repress adventitious root primordium formation in apple rootstocks.

Adventitious root (AR) formation plays an important role in vegetatively propagated plants. Cytokinin (CK) inhibits AR formation, but the molecular mechanisms driving this process remain unknown. In this study, we confirmed that CK content is related to AR formation and further revealed that a high auxin/CK ratio was beneficial to AR formation in apple (Malus domestica). A correlation between expression of CK-responsive TEOSINTE BRANCHED1, CYCLOIDEA, and PCF17 (MdTCP17) and AR formation in response to CK was identified, and overexpression of MdTCP17 in transgenic apple inhibited AR formation. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays revealed an interaction between MdTCP17 and WUSCHEL-RELATED HOMEOBOX11 (MdWOX11), and a significant correlation between the expression of MdWOX11 and AR ability. Overexpression of MdWOX11 promoted AR primordium formation in apple, while interference of MdWOX11 inhibited AR primordium production. Moreover, a positive correlation was found between MdWOX11 and LATERAL ORGAN BOUNDARIES DOMAIN29 (MdLBD29) expression, and yeast one-hybrid, dual luciferase reporter, and ChIP-qPCR assays verified the binding of MdWOX11 to the MdLBD29 promoter with a WOX-box element in the binding sequence. Furthermore, MdTCP17 reduced the binding of MdWOX11 and MdLBD29 promoters, and coexpression of MdTCP17 and MdWOX11 reduced MdLBD29 expression. Together, these results explain the function and molecular mechanism of MdTCP17-mediated CK inhibition of AR primordium formation, which could be used to improve apple rootstocks genetically.

The East Asian wild apples, Malus baccata (L.) Borkh and Malus hupehensis (Pamp.) Rehder., are additional contributors to the genomes of cultivated European and Chinese varieties.

The domestication process in long-lived plant perennials differs dramatically from that of annuals, with a huge amount of genetic exchange between crop and wild populations. Though apple is a major fruit crop grown worldwide, the contribution of wild apple species to the genetic makeup of the cultivated apple genome remains a topic of intense study. We used population genomics approaches to investigate the contributions of several wild apple species to European and Chinese rootstock and dessert genomes, with a focus on the extent of wild-crop gene flow. Population genetic structure inferences revealed that the East Asian wild apples, Malus baccata (L.) Borkh and M. hupehensis (Pamp.), form a single panmictic group, and that the European dessert and rootstock apples form a specific gene pool whereas the Chinese dessert and rootstock apples were a mixture of three wild gene pools, suggesting different evolutionary histories of European and Chinese apple varieties. Coalescent-based inferences and gene flow estimates indicated that M. baccata - M. hupehensis contributed to the genome of both European and Chinese cultivated apples through wild-to-crop introgressions, and not as an initial contributor as previously supposed. We also confirmed the contribution through wild-to-crop introgressions of Malus sylvestris Mill. to the cultivated apple genome. Apple tree domestication is therefore one example in woody perennials that involved gene flow from several wild species from multiple geographical areas. This study provides an example of a complex protracted process of domestication in long-lived plant perennials, and is a starting point for apple breeding programmes.

Multi-omics analyses reveal MdMYB10 hypermethylation being responsible for a bud sport of apple fruit color.

Apple bud sports offer a rich resource for clonal selection of numerous elite cultivars. The accumulation of somatic mutations as plants develop may potentially impact the emergence of bud sports. Previous studies focused on somatic mutation in the essential genes associated with bud sports. However, the rate and function of genome-wide somatic mutations that accumulate when a bud sport arises remain unclear. In this study, we identified a branch from a 10-year-old tree of the apple cultivar 'Oregon Spur II' as a bud sport. The mutant branch showed reduced red coloration on fruit skin. Using this plant material, we assembled a high-quality haplotype reference genome consisting of 649.61 Mb sequences with a contig N50 value of 2.04 Mb. We then estimated the somatic mutation rate of the apple tree to be 4.56 × 10 -8 per base per year, and further identified 253 somatic single-nucleotide polymorphisms (SNPs), including five non-synonymous SNPs, between the original type and mutant samples. Transcriptome analyses showed that 69 differentially expressed genes between the original type and mutant fruit skin were highly correlated with anthocyanin content. DNA methylation in the promoter of five anthocyanin-associated genes was increased in the mutant compared with the original type as determined using DNA methylation profiling. Among the genetic and epigenetic factors that directly and indirectly influence anthocyanin content in the mutant apple fruit skin, the hypermethylated promoter of MdMYB10 is important. This study indicated that numerous somatic mutations accumulated at the emergence of a bud sport from a genome-wide perspective, some of which contribute to the low coloration of the bud sport.

MdNup62 interactions with MdHSFs involved in flowering and heat-stress tolerance in apple.

Because of global warming, the apple flowering period is occurring significantly earlier, increasing the probability and degree of freezing injury. Moreover, extreme hot weather has also seriously affected the development of apple industry. Nuclear pore complexes (NPCs) are main channels controlling nucleocytoplasmic transport, but their roles in regulating plant development and stress responses are still unknown. Here, we analysed the components of the apple NPC and found that MdNup62 interacts with MdNup54, forming the central NPC channel. MdNup62 was localized to the nuclear pore, and its expression was significantly up-regulated in 'Nagafu No. 2' tissue-cultured seedlings subjected to heat treatments. To determine MdNup62's function, we obtained MdNup62-overexpressed (OE) Arabidopsis and tomato lines that showed significantly reduced high-temperature resistance. Additionally, OE-MdNup62 Arabidopsis lines showed significantly earlier flowering compared with wild-type. Furthermore, we identified 62 putative MdNup62-interacting proteins and confirmed MdNup62 interactions with multiple MdHSFs. The OE-MdHSFA1d and OE-MdHSFA9b Arabidopsis lines also showed significantly earlier flowering phenotypes than wild-type, but had enhanced high-temperature resistance levels. Thus, MdNUP62 interacts with multiple MdHSFs during nucleocytoplasmic transport to regulate flowering and heat resistance in apple. The data provide a new theoretical reference for managing the impact of global warming on the apple industry.

Integrated RNA-sequencing and network pharmacology approach reveals the protection of Yiqi Huoxue formula against idiopathic pulmonary fibrosis by interfering with core transcription factors.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a refractory disease. Therefore, developing effective therapies for IPF is the need of the hour. PURPOSE: Yiqi Huoxue Formula (YQHX) is an herbal formula comprising three herbal medicines: Ligusticum chuanxiong Hort. (Chuanxiong Rhizoma, CR), Panax notoginseng (Burk.) F. H. Chen (Notoginseng Radix Et Rhizoma, NR) and Panax ginseng C. A. Mey. (Ginseng Radix Et Rhizoma, GR). This study aims to determine the anti-pulmonary fibrosis effect of YQHX and explore its mechanism of action. STUDY: Design and Methods: The chemical components in the GR, CR and NR extracts were identified by High Performance Liquid Chromatography. A TGF-ß1-induced myofibroblast cell model was used to test the anti-fibrosis effect of GR, CR, NR and YQHX. RNA-sequencing was used to identify the differentially expressed genes (DEGs) after YQHX treatment. Subsequently, gene enrichment analysis and key transcription factors (TFs) prediction for YQHX-regulated DEGs was performed. The active constituents of GR, CR and NR were obtained from the Traditional Chinese Medicine Database and Analysis Platform. Targets of the active constituents were predicted using the similarity ensemble approach search server and Swiss Target Prediction tool. YQHX-targeted key TFs that transcribed the DEGs were screened out. Then, the effect of YQHX on the bleomycin-induced pulmonary fibrosis mouse model was studied. Finally, one of the predicted TFs, STAT3, was selected to validate the prediction accuracy. RESULTS: Seven, two, and five compounds were identified in the GR, CR, and NR extracts, respectively. YQHX and its constituents-GR, CR and NR-inhibited the expression of fibrotic markers, including α -SMA and fibronectin, indicating that YQHX inhibited TGF-ß1-induced myofibroblast activation. RNA-sequencing identified 291 genes that were up-regulated in the TGF-ß1 group but down-regulated after YQHX treatment. In total, 55 key TFs that transcribed YQHX-regulated targets were predicted. A regulatory network of 24 active ingredients and 232 corresponding targets for YQHX was established. Among YQHX's predicted targets, 20 were TFs. On overlapping YQHX-targeted TFs and DEGs' key TFs, six key TFs, including HIF1A, STAT6, STAT3, PPARA, DDIT3 and AR, were identified as the targets of YQHX. Additionally, YQHX alleviated bleomycin-induced pulmonary fibrosis in a mouse model by inhibiting the phosphorylation of STAT3 in the lungs of pulmonary fibrosis mice. CONCLUSIONS: This study provides pharmacological support for the use of YQHX in the treatment of IPF. The potential mechanism of action of YQHX is speculated to involve the modulation of core TFs and inhibition of pathogenetic gene expressions in IPF.

MdNup54 Interactions With MdHSP70 Involved in Flowering in Apple.

Flowering-related problems in "Fuji" apple have severely restricted the development of China's apple industry. Nuclear pore complexes (NPCs) control nucleoplasmic transport and play an important role in the regulation of plant growth and development. However, the effects of NPCs on apple flowering have not been reported. Here, we analysed the expression and function of MdNup54, a component of apple NPC. MdNup54 expression was the highest in flower buds and maintained during 30-70 days after flowering. MdNup54-overexpressing (OE) Arabidopsis lines displayed significantly earlier flowering than that of the wild type. We further confirmed that MdNup54 interacts with MdHSP70, MdMYB11, and MdKNAT4/6. Consistent with these observations, flowering time of MdHSP70-OE Arabidopsis lines was also significantly earlier. Therefore, our findings suggest a possible interaction of MdNup54 with MdHSP70 to mediate its nuclear and cytoplasmic transport and to regulate apple flowering. The results enhance the understanding of the flowering mechanism in apple and propose a novel strategy to study nucleoporins.

A novel and robust analytical method for the quantification of perospirone in human plasma using an LC-MS/MS system with self-internal standard calibration.

OBJECTIVES: This study aims to establish a novel method for measuring perospirone in human plasma for therapeutic drug monitoring (TDM) by liquid chromatography-mass spectrometry (LC-MS) coupled with an automatic liquid chromatograph mass spectrometer coupler 9500 (LC-MS/MS-Mate 9500), which has been equipped with self-internal standard (SIS) calibration technology. DESIGN & METHODS: A novel and attractive analytical calibration method designed for perospirone, calibration with SIS, was reported. After protein precipitation with acetonitrile-cyclopentanol (9:1, v/v) containing 1% NH3·H2O, LC-MS quantification of perospirone was performed by multiple reaction monitoring in the positive mode with quantitative and qualitative analysis of the ion pairs m/z 427.30 â†’ 177.15 and 427.30 â†’ 166.15 for perospirone and SIS. Chromatographic separation was accomplished in < 2.0 min on an Hypersil GOLDTM C18 column (2.1 mm × 50 mm, 3.0 µm) using a mobile methanol phase and 0.1% formic acid in water. RESULTS: This method showed good selectivity because no interfering peaks were observed in the plasma samples during the 2.0-min run time. The calibration curve range was 0.05-20 ng/mL, with a correlation coefficient of ≥ 0.9995. Intraday and interday accuracies were 98.3%-107.9%, respectively, with precision relative standard deviation values of < 10%. The matrix effects ranged from 92.7% to 96.1%, and extraction recoveries were between 97.3% and 108.8%. Finally, this method was successfully applied to routine clinical TDM for 142 patients. The perospirone plasma concentrations of the patients ranged between 0.07 and 10.96 ng/mL. CONCLUSIONS: This bioanalytical method can be used for the quantification of perospirone in human plasma by LC-MS/MS-Mate 9500 using perospirone itself as the SIS.

PpePL1 and PpePL15 Are the Core Members of the Pectate Lyase Gene Family Involved in Peach Fruit Ripening and Softening.

Pectin is the major component in the primary cell wall and middle lamella, maintaining the physical stability and mechanical strength of the cell wall. Pectate lyase (PL), a cell wall modification enzyme, has a major influence on the structure of pectin. However, little information and no comprehensive analysis is available on the PL gene family in peach (Prunus persica L. Batsch). In this study, 20 PpePL genes were identified in peach. We characterized their physicochemical characteristics, sequence alignments, chromosomal locations, and gene structures. The PpePL family members were classified into five groups based on their phylogenetic relationships. Among those, PpePL1, 9, 10, 15, and 18 had the higher expression abundance in ripe fruit, and PpePL1, 15, and 18 were upregulated during storage. Detailed RT-qPCR analysis revealed that PpePL1 and PpePL15 were responsive to ETH treatment (1 g L-1 ethephon) with an abundant transcript accumulation, which suggested these genes were involved in peach ripening and softening. In addition, virus-induced gene silencing (VIGS) technology was used to identify the roles of PpePL1 and PpePL15. Compared to controls, the RNAi fruit maintained greater firmness in the early storage stage, increased acid-soluble pectin (ASP), and reduced water-soluble pectin (WSP). Moreover, transmission electron microscopy (TEM) showed that cell wall degradation was reduced in the fruit of RNAi-1 and RNAi-15, which indicated that softening of the RNAi fruit has been delayed. Our results indicated that PpePL1 and PpePL15 play an important role in peach softening by depolymerizing pectin and degrading cell wall.

Selection and Validation of Reliable Reference Genes for Gene Expression Studies in Different Genotypes and TRV-Infected Fruits of Peach (Prunus persica L. Batsch) during Ripening.

Real-time quantitative PCR (RT-qPCR) is a powerful tool to detect and quantify transcription abundance, and the stability of the reference gene determines its success. However, the most suitable reference gene for different genotypes and tobacco rattle virus (TRV) infected fruits was unclear in peach (Prunus persica L. Batsch). In this study, 10 reference genes were selected and gene expression was characterized by RT-qPCR across all samples, including different genotypes and TRV-infected fruits during ripening. Four statistical algorithms (geNorm, NormFinder, BestKeeper, and RefFinder) were used to calculate the stability of 10 reference genes. The geNorm analysis indicated that two suitable reference genes should be used for gene expression normalization. In general, the best combination of reference genes was CYP2 and Tua5 for TRV-infected fruits and CYP2 and Tub1 for different genotypes. In 18S, GADPH, and TEF2, there is an unacceptable variability of gene expression in all experimental conditions. Furthermore, to confirm the validity of the reference genes, the expression levels of PpACO1, PpEIN2, and PpPL were normalized at different fruit storage periods. In summary, our results provide guidelines for selecting reliable reference genes in different genotypes and TRV-infected fruits and lay the foundation for accurate evaluation of gene expression for RT-qPCR analysis in peach.

Elucidation of the anti-inflammatory mechanism of Er Miao San by integrative approach of network pharmacology and experimental verification.

Traditional Chinese medicine (TCM) has been long time used in China and gains ever-increasing worldwide acceptance. Er Miao San (EMS), a TCM formula, has been extensively used to treat inflammatory diseases, while its bioactive components and therapeutic mechanisms remain unclear. In this study, we conducted an integrative approach of network pharmacology and experimental study to elucidate the underlying mechanisms of EMS in treating human rheumatoid arthritis (RA) and other inflammatory conditions. Quercetin, wogonin and rutaecarpine were probably the main active compounds of EMS in RA treatment as they affected the most RA-related targets, and TNF-α, IL-6 and IL-1ß were considered to be the core target proteins. The main compounds in EMS bound to these core proteins, which was further confirmed by molecular docking and bio-layer interferometry (BLI) analysis. Moreover, the potential molecular mechanisms of EMS predicted from network pharmacology analysis, were validated in vivo and in vitro experiments. EMS was found to inhibit the production of NO, TNF-α and IL-6 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells; reduce xylene-induced mouse ear edema; and decrease the incidence of carrageenan-induced rat paw edema. The carrageenan-induced up-regulation of TNF-α, IL-6 and IL-1ß mRNA expression in rat paws was down-regulated by EMS, consistent with the network pharmacology results. This study provides evidence that EMS plays a critical role in anti-inflammation via suppressing inflammatory cytokines, indicating that EMS is a candidate herbal drug for further investigation in treating inflammatory and arthritic conditions.

Development of a Novel Analytical Method for Determining Trazodone in Human Plasma by Liquid Chromatography Coupled With Mass Spectrometry Coupled With Automatic 2-Dimensional Liquid Chromatograph-Mass Spectrometer Coupler 9500 and Its Application to Therapeutic Drug Monitoring.

BACKGROUND: Trazodone (TZD) is a tetracyclic serotonin antagonist and reuptake inhibitor that is used as a second-generation phenylpiperazine antidepressant. However, the plasma concentrations of TZD have shown individual variations in clinical practice. Quantification of TZD plasma concentrations may be an effective and valuable method to balance the clinical efficacy and adverse reactions. This study aimed to establish a novel liquid chromatography coupled with mass spectrometry (LC-MS) assay for measuring TZD concentrations in human plasma for therapeutic drug monitoring (TDM). METHODS: After protein precipitation with acetonitrile, LC-MS quantification of TZD was performed in the multiple reaction monitoring mode with chromatographic separation using a mobile phase of MeOH and 0.1% formic acid in water. This method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect, and stability according to United states food and drug administration guidelines. RESULTS: This method showed good selectivity because no interfering peaks were observed in the plasma samples during the 2-minute run time. The range of the calibration curve was 1-3000 ng/mL. The detection and quantification limits were 0.3 and 1 ng/mL, respectively. The intraday and interday accuracies were 96.5%-103.4%, with precision relative SD% values of <5%, except for the limit of quality. The mean TZD recovery from human plasma was 95.4%-104.5%. Finally, this method was successfully applied to TDM in 20 patients. The TZD plasma concentrations of the patients ranged between 21.5 and 2267.3 ng/mL. CONCLUSIONS: A novel analytical method was established to measure TZD by LC-MS coupled with an automatic 2-dimensional liquid chromatograph mass spectrometer coupler 9500 (LC-MS/MS-Mate 9500), which is superior to the ordinary LC-MS system in separation, transport, anti-interference, sensitivity, and quantitative analysis stability.

Identification of apple TFL1-interacting proteins uncovers an expanded flowering network.

KEY MESSAGE: MdTFL1, a floral repressor, forms protein complexes with several proteins and could compete with MdFT1 to regulate reproductive development in apple. Floral transition is a key developmental stage in the annual growth cycle of perennial fruit trees that directly determines the fruit development in the subsequent stage. FLOWERING LOCUS T (FT)/TERMINAL FLOWER1 (TFL1) family is known to play a vital regulatory role in plant growth and flowering. In apple, the two TFL1-like genes (MdTFL1-1 and MdTFL1-2) function as floral inhibitors; however, their mechanism of action is still largely unclear. This study aimed to functionally validate MdTFL1 and probe into its mechanism of action in apple. MdTFL1-1 and MdTFL1-2 were expressed mainly in stem and apical buds of vegetative shoots, with little expression in flower buds and young fruit. Expression of MdTFL1-1 and MdTFL1-2 rapidly decreased during floral induction. On the other hand, transgenic Arabidopsis, which ectopically expressed MdTFL1-1 or MdTFL1-2, flowered later than wild-type plants; demonstrating their in planta capability to function redundantly as flower repressors. Furthermore, we identified hundreds of novel interaction proteins of the two apple MdTFL1 proteins using yeast two-hybrid screens. Independent experiments for several proteins confirmed the yeast two-hybrid interactions. Among them, the transcription factor Nuclear Factor-Y subunit C (MdNF-YC2) functions as a promoter of flowering in Arabidopsis by activating LEAFY (LFY) and APETALA1 (AP1) expression. MdFT1 showed a similar interaction pattern as MdTFL1, implying a possible antagonistic action in the regulation of flowering. These newly identified TFL1-interacting proteins (TIPs) not only expand the floral regulatory network, but may also introduce new roles for TFL1 in plant development.

The downregulation of PpPG21 and PpPG22 influences peach fruit texture and softening.

MAIN CONCLUSION: The downregulation of PpPG21 and PpPG22 expression in melting-flesh peach delays fruit softening and hinders texture changes by influencing pectin solubilization and depolymerization. The polygalacturonase (PG)-catalyzed solubilization and depolymerization of pectin plays a central role in the softening and texture formation processes in peach fruit. In this study, the expression characteristics of 15 PpPG members in peach fruits belonging to the melting flesh (MF) and non-melting flesh (NMF) types were analyzed, and virus-induced gene silencing (VIGS) technology was used to identify the roles of PpPG21 (ppa006839m) and PpPG22 (ppa006857m) in peach fruit softening and texture changes. In both MF and NMF peaches, the expression of PpPG1, 10, 12, 23, and 25 was upregulated, whereas that of PpPG14, 24, 35, 38, and 39 was relatively stable or downregulated during shelf life. PpPG1 was highly expressed in NMF fruit, whereas PpPG21 and 22 were highly expressed in MF peaches. Suppressing the expression of PpPG21 and 22 by VIGS in MF peaches significantly reduced PG enzyme activity, maintained the firmness of the fruit during the late shelf life stage, and suppressed the occurrence of the "melting" stage compared with the control fruits. Moreover, the downregulation of PpPG21 and 22 expression also reduced the water-soluble pectin (WSP) content, increased the contents of ionic-soluble pectin (ISP) and covalent-soluble pectin (CSP) and affected the expression levels of ethylene synthesis- and pectin depolymerization-related genes in the late shelf life stage. These results indicate that PpPG21 and 22 play a major role in the development of the melting texture trait of peaches by depolymerizing cell wall pectin. Our results provide direct evidence showing that PG regulates peach fruit softening and texture changes.