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Due to the trade imbalance and poor management, a large number of empty containers are piled up after unpacking in importing regions, while the export-oriented areas are in urgent need of empty containers for replenishment. This paper aims to determine the volume of empty containers transferred from the surplus area to the shortage area and the inventory of each node from the operational level. We subdivide container freight stations into rail stations and road stations and consider using sea, road, and railway as transportation modes to transfer empty containers. A multi-period linear programing model of multimodal empty container repositioning is established aiming at minimizing the cost of empty container repositioning. A real case study of the transportation network in Northeast China is conducted to demonstrate that using multiple modes of transportation can significantly reduce the cost of empty container repositioning compared to using a single mode of transportation.

Adenosine is an endogenous molecule that plays a vital role in biological processes. Research indicates that abnormal adenosine levels are associated with a range of diseases. The development of sensors capable of detecting adenosine is pivotal for early diagnosis of disease. For example, elevated adenosine levels are closely associated with the onset and progression of cancer. In this study, we designed a novel DNA biosensor utilizing chaperone copolymer-assisted catalytic hairpin assembly for highly sensitive detection of adenosine. The functional probe comprises streptavidin magnetic beads, an aptamer, and a catalytic chain. In the presence of adenosine, it selectively binds to the aptamer, displacing the catalytic chain into the solution. The cyclic portion of H1 hybridizes with the catalytic strand, while H2 hybridizes with the exposed H1 fragment to form an H1/H2 complex containing a G-quadruplex. Thioflavin T binds specifically to the G-quadruplex, generating a fluorescent signal. As a nucleic acid chaperone, PLL-g-Dex expedites the strand exchange reaction, enhancing the efficiency of catalytic hairpin assembly, thus amplifying the signal and reducing detection time. The optimal detection conditions were determined to be a temperature of 25 °C and a reaction time of 10 min. Demonstrating remarkable sensitivity and selectivity, the sensor achieved a lowest limit of detection of 9.82 nM. Furthermore, it exhibited resilience to interference in complex environments such as serum, presenting an effective approach for rapid and sensitive adenosine detection.

The rapid increase in e-commerce and the emergence of combined passenger/freight systems in urban areas have raised the question of how best to integrate public transport services into door-to-door deliveries. This paper develops a variant of the pickup and delivery problem, called the two-echelon pickup and delivery problem using public transport (2E-PDP-PT). In the 2E-PDP-PT, the transportation network is split into two echelons. Different vehicles are utilized across the first and second echelons to ensure distribution efficiency. Parcels are delivered by public transport with free capacity or via trucks between satellites in the first echelon, and logistics vehicles are operated in the second echelon. The satellites are located at the echelon borders to transfer commodities between echelons. The 2E-PDP-PT aims to minimize total delivery costs and improve public transport capacity utilization. We formulate a new mathematical model based on a space-time network and adopt an adaptive large neighborhood search (ALNS) algorithm for the 2E-PDP-PT. The effectiveness of the ALNS algorithm is validated using newly generated small-scale instances. Furthermore, we investigate large-scale instances based on the Beijing Yizhuang transportation network. The computations show that an average total delivery cost savings of 4.5% is feasible. In addition, we analyze the impact of demand distributions and compare the ALNS algorithm and the LNS algorithm. Finally, we conclude that dynamically integrating public transport into freight transport services can benefit both logistics companies and public transport operators.

Background The occurrence of prosthesis-related complications after total shoulder arthroplasty is devastating and costly. The purpose was to determine the incidence and risk of in-hospital prosthesis-related complications after total shoulder arthroplasty utilizing a large-scale sample database. Methods A retrospective database analysis was performed based on Nationwide Inpatient Sample from 2010 to 2014. Patients who underwent total shoulder arthroplasty were included. Patient demographics, hospital characteristics, length of stay, economic indicators, in-hospital mortality, comorbidities, and peri-operative complications were evaluated. Results A total of 34,198 cases were capture from the Nationwide Inpatient Sample database. There were 343 cases of in-hospital prosthesis-related complications after total shoulder arthroplasty and the overall incidence was 1%, with a more than 2.5-fold decrease from 2010 to 2014. Dislocation was the most common category among prosthesis-related complications (0.1%). The occurrence of in-hospital prosthesis-related complications was associated with significantly more total charges and slightly longer length of stay while less usage of Medicare. Risk factors of prosthesis-related complications were identified including younger age (

P450 (cytochrome P450) is a supergene family, which is involved in various metabolic pathways in plants. Based on previous study, we found some of cucumber P450 mRNAs were systemic mobile in cucumber/pumpkin grafts. However, the reason that why P450 mRNAs were endorsed as signaling, and what specific motif (s) did they harbored is not clear yet. Here, we first identified 221 CsaP450 genes in cucumber genome-wide level. Combining with graft-transmissible mRNAs datasets in cucumber, we elucidated 15 mobile-CsaP450-coding genes, of which 5 and 10 belonged to A-type and non-A type respectively. Compared with Arabidopsis and pumpkin (Cucubit moschata) graft-induced-transmissible P450 mRNAs, a phylogenetic tree was constructed and divided into eight clans by using multiple-sequence alignment. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations indicated that the expression patterns of the mobile-mRNA-coding CsaP450 genes in different tissues of cucumber was specifically enriched in oxidoreductase activity and secondary metabolic pathways. The structures and motifs of these 15 mobile-mRNA-coding CsaP450 genes and their types of regulatory elements told that the proportion of CU-rich motifs was higher than nonmobile-mRNA-coding CsaP450 genes. The integrated analysis of mobility direction and mRNA abundance of 15 mobile-mRNA-coding CsaP450 genes allowed to conclude that there was rarely relationship between them. The study provided a new insight into the relationship between the motifs and functional characterization of mobile-mRNA-coding P450 genes of cucumber in genome-wide levels.

As an internal modification of transcripts, RNA methylation determines RNA fate by changing RNA–protein binding affinity. In plants, RNA methylation is ubiquitous and is involved in all aspects of RNA post-transcriptional regulation. For instance, long-distance mobile RNAs, strongly influenced by their methylation status, play important roles in plant growth, development and environmental adaptation. Cucumber/pumpkin heterografts are widely used to improve stress tolerance of cucumber and to study mobile RNA signals due to their strong developed vasculature system. Here, we developed the Cucume (Cucurbit RNA methylation, http://cucume.cn/) database for these two important vegetables, cucumber (Cucumis sativus L.) and pumpkin (Cucurbita moschata) with high productivity worldwide. We identified mRNAs harboring 5-methylcytosine (m5C) and N6-methyladenosine (m6A) sites in pumpkin and cucumber at the whole genome level via Methylated RNA Immunoprecipitation sequencing (MeRIP-seq) of different tissues and the vascular exudates. In addition to RNA methylation sites, the Cucume database includes graft-transmissible systemic mRNAs identified in previous studies using cucumber/pumpkin heterografts. The further integration of cucumber genome-wide association analysis (GWAS) and quantitative trait loci (QTL) allows the study of RNA methylation-related genetic and epigenetic regulation in cucurbits. Therefore, the here developed Cucume database will promote understanding the role of cucurbit RNA methylation in RNA mobility and QTL, ultimately benefitting future breeding of agronomic crop germplasms.

ObjectivesTo investigate the relationship between antibiotic exposure and asthma in adults in the United States.MethodsData was obtained from the National Health and Nutrition Examination Survey (NHANES) conducted between 1999 and 2018. A total of 51,124 participants were included, excluding those who were aged

Chrysin is a flavonoid with various biological activities. However, its low water solubility and strong metabolism render its oral bioavailability rather poor. This study aimed to develop a stable solid dispersion formulation of chrysin to improve the dissolution of chrysin, so as to increase its oral bioavailability and improve its antihyperlipidemic activities. A solid dispersion of chrysin was prepared using a solvent evaporation method, with Plasdone® S630 as the hydrophilic carrier. The formulations were characterized via X-ray diffraction, in vitro dissolution studies, and stability studies. An in-situ perfusion model was used to evaluate the absorption rates. Plasma pharmacokinetics and antihyperlipidemic performance after the oral administration of the chrysin formulations were investigated in rats. It was found that the solid dispersion of chrysin prepared using the drug–polymer mass ratio of 1:6 can form the optimized formulation. X-ray diffraction results showed that the chrysin was in an amorphous state in this optimized formulation. The cumulative release percentage of the optimized solid dispersion of chrysin at pH 1.2 and pH 6.8 was elevated to above 90% within 24 h, indicating that the formulation could enhance the dissolution rates of chrysin. Stability studies showed that the optimized formulation presented acceptable long-term storage stability, but it was susceptible to high temperature and humidity. The solid dispersion of chrysin showed higher absorption rates in the in-situ perfusion model. Pharmacokinetic studies revealed that Cmax and AUC after the intragastric administration of solid dispersion of chrysin were appreciably higher than those resulting from chrysin suspension. The oral bioavailability of the solid dispersion of chrysin was 41 times higher than that of chrysin suspension. Pharmacological studies suggested that the solid dispersion of chrysin was more powerful than chrysin raw material in improving biochemical indicators in the hyperlipidemic model in rats. This study reveals the potential use of a novel oral formulation of chrysin to reduce the currently required high dose.

The production–living–ecological functions of rural settlements are closely tied to water resources, which are the primary influencing factors of the spatial characteristics of rural settlements. However, the specific relationship between water resources and the spatial characteristics of rural settlements remains unclear. Understanding the interrelationship between the two can better safeguard the ecological pattern of the basin and optimize the living environment of settlements. This study utilized multi-source data to calculate the water yield, water demand, and ecological surplus or deficit of water resources in the Yanhe watershed. We quantified the spatial characteristics of rural settlements and employed bivariate spatial autocorrelation methods to analyze the spatial correlation between water resources and the spatial distribution, scale, and boundary form of rural settlements in the Yanhe watershed. The results show the following: ➀ Seven sub-basins in the upper reaches exhibit a severe ecological deficit in water resources, with insufficient water resources to support the demands of regional socio-economic development. The middle and lower reaches have achieved a balance between water supply and demand. ➁ Rural settlements are most densely distributed in the middle reaches, with the smallest area scale, exhibiting a transitional spatial characteristic towards the upstream and downstream ends. ➂ The Moran’s I values of spatial aggregation and morphological index of rural settlements with respect to the ecological surplus or deficit of water resources are 0.36 and 0.50, respectively, indicating a strong positive correlation. The Moran’s I value of the area scale with respect to the ecological surplus or deficit of water resources is −0.60, indicating a significant negative correlation. This research has important practical significance for guiding the spatial layout of rural settlements in the Yanhe watershed and promoting their sustainable development.

Objectives: Dual specificity phosphatase 1 (DUSP1) is regarded as an anti-inflammatory factor in cardiovascular disorders. Mitophagy removes damaged mitochondria and thus promotes mitochondrial regeneration. We investigated whether DUSP1 could attenuate inflammation-induced cardiomyopathy by improving mitophagy. Methods: Lipopolysaccharide was used to induce septic cardiomyopathy in wild-type (WT) and DUSP1 transgenic (DUSP1TG) mice. Results: Echocardiography revealed that lipopolysaccharide impaired heart function by reducing the cardiac systolic and diastolic capacities of WT mice. Freshly isolated single cardiomyocytes from lipopolysaccharide-treated WT mice also exhibited reduced contractile/relaxation parameters. However, DUSP1 overexpression not only maintained the mechanical properties of cardiomyocytes, but also improved heart performance. Lipopolysaccharide upregulated myocardial inflammatory gene transcription and adhesive factor expression, which increased myocardial neutrophil accumulation and cardiomyocyte apoptosis in WT mice. DUSP1 overexpression inhibited the inflammatory response and therefore promoted cardiomyocyte survival. Lipopolysaccharide disrupted mitochondrial respiration and metabolism in WT cardiomyocytes, but DUSP1 overexpression restored mitochondrial metabolism, maintained the mitochondrial membrane potential and inhibited mitochondrial reactive oxygen species production, possibly by increasing FUN14 domain-containing 1 (FUNDC1)-dependent mitophagy. Silencing of FUNDC1 abolished the protective effects of DUSP1 overexpression on cardiomyocytes and their mitochondria following lipopolysaccharide treatment. Conclusion: These results demonstrated that DUSP1 is a novel anti-inflammatory factor that protects against septic cardiomyopathy by improving FUNDC1-induced mitophagy.

The indefinite characteristics of gas–liquid two-phase flow limit the usage of aerated drip irrigation. Gas–liquid two-phase flow in a labyrinth channel was observed using a particle tracking velocimetry (PTV) technique in this study. The motion trajectory and velocity vector of large numbers of microbubbles were characterized and analyzed at 0.01, 0.02, 0.04 MPa inlet pressure and in three labyrinth channels with different geometries. The results indicated that bubbly flow was the typical flow pattern in a labyrinth channel, with slug flow occurring occasionally. Smooth and gliding motion trajectories of bubbles were observed in the mainstream zone, while twisted trajectories were seen in the vortex zone. Increasing the inlet pressure increased the number of bubbles and the trajectory length in the vortex zone. When the inlet pressure increased from 0.02 to 0.04 MPa, the 25th percentile of Rc-t (the Ratio of Circular path length in the vortex zone to the Total trajectory length for a single bubble) increased from 0 to 12.3%, 0 to 6.1%, and 0 to 5.2% for channels A, B, and C, respectively; the 75th percentile increased from 31.3% to 43.9%, 27.5% to 31.9%, and 18.7% to 22.3%. The velocity vectors of the bubbles showed position dependence. Bubbles with high speed were found in the mainstream zone with their directions parallel to the water flow direction. Bubbles with low speed were seen in the vortex zone, moving in all directions. With inlet pressure increased from 0.01 to 0.04 MPa, the mean instantaneous velocities of bubbles in channels A, B, and C are increased by 106.2%, 107.6%, and 116.6%, respectively. At 0.04 MPa, channel A has the longest path length and the highest instantaneous velocity of bubbles in the vortex zone among three channels, exhibiting the highest anti-clogging performance of the three channels. This study will help in the comprehensive understanding of gas–liquid two-phase flow in a labyrinth channel used for aerated drip irrigation.

Climate changes especially chilling stress affects cucurbit crops during winter seasonal production. Grafting to pumpkin rootstocks is widely used to improve the vigor of cucurbits, especially cucumber (Cucumis sativus L.) plants, in the face of chilling stress. In our study, multi-disciplinary aspect approaches were used to investigate growth changes of pumpkin under chilling stress. Firstly, the morphological and physiological characteristics of 14 pumpkin (Cucurbita moschata) varieties following different periods of chilling stress was analyzed by using physiological means. Mathematical results of principal component analysis (PCA) with chlorophyll-a, chlorophyll-b, carotenoid contents, chilling injury index and relative electrolyte permeability indicated that relative electrolyte permeability as the primary judgment index was best associated with the comparison of chilling tolerance in pumpkin rootstock varieties. Then, transcriptomic and DCMU (Diuron) application and chlorophyll fluorescence examination analysis of pumpkin leaves revealed that 390 Cucurbita moschata differentially expressed genes (CmoDEGs) that affect photosynthesis were upregulated in leaves. 127 CmoDEGs both in leaves and roots were enriched for genes involved in unsaturated fatty acid metabolism, suggesting that plasma membrane lipids are involved in chilling perception. The results of increased composition of unsaturated fatty acid in leaves and qRT-PCR analysis of relative mRNA abundance confirmed that α-linolenic acid biosynthesis was responding to pumpkin chilling tolerance. The integration of physiological, mathematical bioinformatical and biological analysis results contributes to our understanding of the molecular mechanisms underlying chilling tolerance and its improvement in cucumber grafted on pumpkin rootstocks. It provided an important theoretical basis and reference for further understanding on the impact of climate change on plant physiological changes.

A large number of RNA molecules have been found in the phloem of higher plants, and they can be transported to distant organelles through the phloem. RNA signals are important cues to be evolving in fortification strategies by long-distance transportation when suffering from various physiological challenges. So far, the mechanism of RNA selectively transportation through phloem cells is still in progress. Up to now, evidence have shown that several RNA motifs including Polypyrimidine (poly-CU) sequence, transfer RNA (tRNA)-related sequence, Single Nucleotide Mutation bound with specific RNA binding proteins to form Ribonucleotide protein (RNP) complexes could facilitate RNA mobility in plants. Furthermore, some RNA secondary structure such as tRNA-like structure (TLS), untranslation region (UTR) of mRNA, stem-loop structure of pre-miRNA also contributed to the mobility of RNAs. Latest researchs found that RNA methylation such as methylated 5′ cytosine (m5C) played an important role in RNA transport and function. These studies lay a theoretical foundation to uncover the mechanism of RNA transport. We aim to provide ideas and clues to inspire future research on the function of RNA motifs in RNA long-distance transport, furthermore to explore the underlying mechanism of RNA systematic signaling.

As the core operational issue in container terminals, yard crane scheduling problem directly affects the overall operation efficiency of port connecting highway or railway transportation and sea transportation. In practice, the scheduling of yard cranes is subject to many uncertain factors, so the scheme may be inapplicable and needs to be adjusted. From the perspective of proactive strategy, considering fluctuations in arrival time of external trucks as well as varied handling volume of yard cranes, a stochastic programming model is established in this paper to obtain a fixed scheme with the minimum expected value of yard crane makespan and total task waiting time over all the scenarios. The scheme does not require rescheduling when facing different situations. Subsequently, two algorithms based on certain rules are proposed to obtain the yard crane operation scheme in the deterministic environment, which are taken as the basic solution in the uncertain conditions, and then a tailored genetic algorithm is adopted to find the optimal solution with good adaptability to the uncertain scenarios. Finally, we use small-scale examples to compare the performance of algorithms in the deterministic and uncertain environment and then analyze the relationship between different yard crane configurations and the number of tasks. Large-scale experiments are performed to study the operation efficiency of the storage yard with different handling volumes assigned to each yard crane.

There are few studies on lead’s effect on bone mineral density (BMD) in childhood. In this study, we examined the association between lead exposure and BMD among 13,951 children and adolescents aged 8–19 years from NHANES 1999–2006 and 2011–2018. The whole blood lead levels (BLLs) were used as lead exposure biomarkers, and total BMD, subtotal BMD, lumbar spine BMD and limb BMD were used as outcome variables. The survey weighted multivariable generalized additive models (GAMs) with smoothing terms were used to explore the association between blood lead levels and BMDs, adjusted for age, sex, race/ethnicity, height, weight, family-income-to-poverty ratio and blood cadmium. Subgroup analyses stratified by sex and bony sites were further performed. We found an N-shaped curve association between BLLs and total BMD, subtotal BMD and limb BMD for males and females, whereas the association between BLLs and lumbar spine BMD was only significantly negative for females. The findings suggested that lead exposure had different effects on BMD of different bony sites (highly cortical or trabecular regions) in childhood and adolescence and had different effects on the same bone among different ages population and/or at different levels.

The purpose of this study was to develop a drug delivery system for paliperidone (PPD) in order to provide a more effective therapeutic strategy for patients with acute schizophrenia. PPD-loaded Soluplus®/TPGS mixed micelles (PPD-S/T-MM) were prepared using the thin-film hydration method. The critical micelle concentration (CMC) of blank S/T-MM was 4.77 × 10−2 mg/mL. PPD presented much higher solubility in PPD-S/T-MM formulation than that in pure water. The particle size of blank or drug loaded S/T-MM was around 60 nm. The polydispersity index (PDI) was less than 0.1. PPD-S/T-MM presented a nearly spherical shape under transmission electron microscopy. The encapsulation efficiency (EE%) of PPD-S/T-MM was higher than 94%. Based on the analysis of XRD and DSC, it was proved that PPD was incorporated in the core of the mixed micelles as amorphous dispersion or solid solution. PPD-S/T-MM were stable when they were undergoing dilution with water and the change of environmental pH. Although PPD-S/T-MM showed lower rates to release PPD than those from PPD raw material in acidic solution, they provided faster release rates in neutral conditions than those from PPD raw material who only showed modest dissolution in the same neutral condition. This proves that PPD-S/T-MM can release PPD in a more controlled manner. After oral administration of PPD-S/T-MM (dose of PPD, 6 mg/kg) in rats, the plasma concentration of PPD increased rapidly: Tmax was 0.83 ± 0.29 h, and Cmax was 844.33 ± 93.73 ng/mL. Oral administration of PPD suspension resulted in longer Tmax and lower Cmax. The relative oral bioavailability was about 158% for PPD-S/T-MM over PPD suspension. These findings confirm that PPD-S/T-MM can provide faster release in neutral conditions and better oral absorption in rats than those from PPD raw material, which should potentially benefit patients with acute schizophrenia.

Background: Trends of asthma mortality vary widely all over the world, while the trends in China over the past 15 years are unknown. The aim of this study was to assess the trends of asthma mortality in China. Methods: Asthma deaths and demographic characteristics were collected from National Death Cause Datasets of Disease Surveillance System between 2004 and 2019. The data were analyzed with joinpoint regression analysis and age-period-cohort (APC) analysis for the mortality rate due to asthma in China. Results: Asthma mortality declined from 2.4 (95% confidence interval (CI): 2.3–2.5) per 100,000 in 2004 to 1.6 (95% CI: 1.5–1.7) per 100,000 in 2019. Age-adjusted asthma mortality rates decreased for men and women in urban and rural areas from 2004 through 2019. The decreasing trend of the mortality rate has slowed down substantially during 2007 and 2009. After that, the decreasing trend has stabilized. The asthma mortality rates generally have a positive relationship with the age of the population when controlling for period and cohort. The period trend decreased and then increased when controlling for age and cohort. Conclusions: We should pay more attention to asthma management plans or treatment for aging people who are facing higher risk of asthma death.

Grafting is widely used in fruit, vegetable, and flower propagation to improve biotic and abiotic stress resistance, yield, and quality. At present, the systemic changes caused by grafting, as well as the mechanisms and effects of long-distance signal transport between rootstock and scion have mainly been investigated in model plants (Arabidopsis thaliana and Nicotiana benthamiana). However, these aspects of grafting vary when different plant materials are grafted, so the study of model plants provides only a theoretical basis and reference for the related research of grafted vegetables. The dearth of knowledge about the transport of signaling molecules in grafted vegetables is inconsistent with the rapid development of large-scale vegetable production, highlighting the need to study the mechanisms regulating the rootstock-scion interaction and long-distance transport. The rapid development of molecular biotechnology and “omics” approaches will allow researchers to unravel the physiological and molecular mechanisms involved in the rootstock–scion interaction in vegetables. We summarize recent progress in the study of the physiological aspects (e.g., hormones and nutrients) of the response in grafted vegetables and focus in particular on long-distance molecular signaling (e.g., RNA and proteins). This review provides a theoretical basis for studies of the rootstock–scion interaction in grafted vegetables, as well as provide guidance for rootstock breeding and selection to meet specific demands for efficient vegetable production.

Glutamine synthetase (GS; EC 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is the key enzyme responsible for the primary assimilation and reassimilation of nitrogen (N) in higher plants. There are two main isoforms of GS in higher plants, classified as cytosolic GS (GS1) and chloroplastic GS (GS2) by their size and subcellular localization. In order to improve the stress tolerance, quality, and yield of cucurbit crops such as cucumbers (Csa, Cucumis sativus L.), pumpkins (Cmo, Cucurbita moschata var. Rifu) are often used as rootstocks. Here, the GS family of the two species were comprehensively analyzed using bioinformatics in terms of aspects of the phylogenic tree, gene structure, chromosome location, subcellular localization, and evolutionary and expression patterns. Seven and four GS gene family members were screened in pumpkin and cucumber, respectively. GS family genes were divided into three groups (one for GS2 and two for GS1) according to their homology and phylogenetic relationships with other species. The analysis of gene ontology annotation of GS family genes, promoter regulatory elements, and tissue-specific expression patterns indicates the potential different biological roles of GS isoforms in Cucurbitaceae. In particular, we have identified a potentially available gene (GS1: CmoCh08G004920) from pumpkin that is relatively highly expressed and tissue-specifically expressed. RT-PCR analysis showed that most CmoGSs are induced by low temperature, and long-term (day 2 to day 9) cold stress has a more obvious effect on the RNA abundance of CmoGS. Our work presents the structure and expression patterns of all candidate members of the pumpkin and cucumber GS gene family, and to the best of our knowledge, this is the first time such work has been presented. It is worth focusing on the candidate genes with strong capacity for improving pumpkin rootstock breeding in order to increase nitrogen-use efficiency in cold conditions, as well as rootstock-scion communication.

Cucumber (Cucumis sativus L.) is one of the main vegetable crops in China. The physiological cultivation mechanism and gene function characteristics of cucumber are of great significance to the construction of modern agriculture. Due to the low genetic transformation rate of cucumber, only in situ hybridization, which does not involve the progress of gene modified transformation, is convenient to study mRNA localization, so it is more suitable for determination on mRNA localization in the mature tissue of cucumber. At present, the existing in situ hybridization technology system is more suitable for cucumber meristem than for the mature tissue of cucumber seedlings. Therefore, we optimized the traditional plant in situ hybridization protocol. Taking a known gene CsNPF7.2 (Nitrate Transporter Families protein) as an example, we then optimized the steps of plant tissue culture, gene probe preparation, plant material sampling and fixation, preparation of cross section, hybridization pretreatment, hybridization incubation, chromogenic reaction, microscopy examination, and treatment after reaction termination in order to obtain a new RNA in situ hybridization technique suitable for identification on mRNA localization in mature tissues of cucumber seedlings. This optimized technique will ensure the yield of probes, the integrity of RNA molecules, and the clarity and integrity of plant tissue structure, which is conducive to the study of gene function and screening of key genes in cucumber.

Grafting has been widely used to improve plant growth and tolerance in crop production, as well as for clarifying systemic mRNA signaling from donor to recipient tissues in organ-to-organ communication. In this study, we investigated graft partner interaction mechanisms of Cucumis sativus (Csa) and Cucurbita moschata (Cmo) using a large-scale endogenous mRNA transport. The results indicated that most mobile transcripts followed an allocation pathway from source to sink. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that mRNA mobility functions are universally common and individually specific. Identification of mRNA mobility between distant tissues in heterografts with RT-PCR (reverse transcription PCR), RT-qPCR (reverse transcriptional quantitative real time PCR), and clone sequencing were used to estimate 78.75% of selected mobile transcripts. Integration of bioinformatic analysis and RT-qPCR identification allowed us to hypothesize a scion-to-rootstock-to-scion feedback signal loop of Csa move-down and Cmo move-up mRNAs, where Csa scion move-down mRNAs were involved in carbon fixation and biosynthesis of amino acid pathways, and Cmo root received Csa move-down mRNA and then delivered the corresponding Cmo upward mRNA to scion to improve photosynthesis of cucumber scion. This formed a feedback signal loop of scion-to-rootstock-to scion to explain why pumpkin rootstock enhanced cucumber production in the industry, which was utilized for organ communication and mediates photosynthesis processes in heterograft cucurbit crops.