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High altitude and strong ultraviolet radiation in Qinghai Tibet Plateau induces the brittleness and hardening of asphalt, which leads to temperature cracks, pits and other diseases of asphalt pavement. In this paper, the accelerated UV aging test of rubber asphalt was carried out by simulating the UV radiation intensity in Xining, Qinghai Province, and the rheological properties and microstructure changes of rubber asphalt were analyzed. The results show that the complex modulus of rubber asphalt increases exponentially with the extension of UV aging time. The phase angle of rubber asphalt decreases linearly with UV aging, and the deformation recovery performance of rubber asphalt increases; UV radiation has the most significant effect on the micro morphology of rubber asphalt. With the further extension of UV aging time, micro cracks are formed in rubber asphalt, and the low-temperature crack resistance and fatigue failure resistance of rubber asphalt are gradually reduced.

Purpose: Histologic tumor grade is a well-established prognostic factor for breast cancer, and tumor grade–associated genes are the common denominator of many prognostic gene signatures. The objectives of this study are as follows: (a) to develop a simple gene expression index for tumor grade (molecular grade index or MGI), and (b) to determine whether MGI and our previously described HOXB13:IL17BR index together provide improved prognostic information.Experimental Design: From our previously published list of genes whose expression correlates with both tumor grade and tumor stage progression, we selected five cell cycle–related genes to build MGI and evaluated MGI in two publicly available microarray data sets totaling 410 patients. Using two additional cohorts (n = 323), we developed a real-time reverse transcription PCR assay for MGI, validated its prognostic utility, and examined its interaction with HOXB13:IL17BR.Results: MGI performed consistently as a strong prognostic factor and was comparable with a more complex 97-gene genomic grade index in multiple data sets. In patients treated with endocrine therapy, MGI and HOXB13:IL17BR modified each other's prognostic performance. High MGI was associated with significantly worse outcome only in combination with high HOXB13:IL17BR, and likewise, high HOXB13:IL17BR was significantly associated with poor outcome only in combination with high MGI.Conclusions: We developed and validated a five-gene reverse transcription PCR assay for MGI suitable for analyzing routine formalin-fixed paraffin-embedded clinical samples. The combination of MGI and HOXB13:IL17BR outperforms either alone and identifies a subgroup (∼30%) of early stage estrogen receptor–positive breast cancer patients with very poor outcome despite endocrine therapy.

Supplementary Data from A Five-Gene Molecular Grade Index and HOXB13:IL17BR Are Complementary Prognostic Factors in Early Stage Breast Cancer

Supplementary Tables 1-2 from Ligand-Dependent Platelet-Derived Growth Factor Receptor (PDGFR)-α Activation Sensitizes Rare Lung Cancer and Sarcoma Cells to PDGFR Kinase Inhibitors

Supplementary Figure 6 from Ligand-Dependent Platelet-Derived Growth Factor Receptor (PDGFR)-α Activation Sensitizes Rare Lung Cancer and Sarcoma Cells to PDGFR Kinase Inhibitors

Platelet-derived growth factor (PDGF) receptors (PDGFR) and their ligands play critical roles in several human malignancies. Sunitinib is a clinically approved multitargeted tyrosine kinase inhibitor that inhibits vascular endothelial growth factor receptor, c-KIT, and PDGFR, and has shown clinical activity in various solid tumors. Activation of PDGFR signaling has been described in gastrointestinal stromal tumors (PDGFRA mutations) as well as in chronic myeloid leukemia (BCR-PDGFRA translocation), and sunitinib can yield clinical benefit in both settings. However, the discovery of PDGFR activating mutations or gene rearrangements in other tumor types could reveal additional patient populations who might benefit from treatment with anti-PDGFR therapies, such as sunitinib. Using a high-throughput cancer cell line screening platform, we found that only 2 of 637 tested human tumor-derived cell lines show significant sensitivity to single-agent sunitinib exposure. These two cell lines [a non–small-cell lung cancer (NSCLC) and a rhabdomyosarcoma] showed expression of highly phosphorylated PDGFRA. In the sunitinib-sensitive adenosquamous NSCLC cell line, PDGFRA expression was associated with focal PFGRA gene amplification, which was similarly detected in a small fraction of squamous cell NSCLC primary tumor specimens. Moreover, in this NSCLC cell line, focal amplification of the gene encoding the PDGFR ligand PDGFC was also detected, and silencing PDGFRA or PDGFC expression by RNA interference inhibited proliferation. A similar codependency on PDGFRA and PDGFC was observed in the sunitinib-sensitive rhabdomyosarcoma cell line. These findings suggest that, in addition to gastrointestinal stromal tumors, rare tumors that show PDGFC-mediated PDGFRA activation may also be clinically responsive to pharmacologic PDGFRA or PDGFC inhibition. [Cancer Res 2009;69(9):3937–46]

Supplementary Methods, Figures 1-8, Tables 1-3 from A Genome-Wide Screen for Microdeletions Reveals Disruption of Polarity Complex Genes in Diverse Human Cancers

Supplementary Figure 3 from Ligand-Dependent Platelet-Derived Growth Factor Receptor (PDGFR)-α Activation Sensitizes Rare Lung Cancer and Sarcoma Cells to PDGFR Kinase Inhibitors

Supplementary Figures 1-2 from Ligand-Dependent Platelet-Derived Growth Factor Receptor (PDGFR)-α Activation Sensitizes Rare Lung Cancer and Sarcoma Cells to PDGFR Kinase Inhibitors

Supplementary Figure 4 from Ligand-Dependent Platelet-Derived Growth Factor Receptor (PDGFR)-α Activation Sensitizes Rare Lung Cancer and Sarcoma Cells to PDGFR Kinase Inhibitors

Supplementary Figure 7 from Ligand-Dependent Platelet-Derived Growth Factor Receptor (PDGFR)-α Activation Sensitizes Rare Lung Cancer and Sarcoma Cells to PDGFR Kinase Inhibitors

Supplementary Figure 5 from Ligand-Dependent Platelet-Derived Growth Factor Receptor (PDGFR)-α Activation Sensitizes Rare Lung Cancer and Sarcoma Cells to PDGFR Kinase Inhibitors

One of the main challenges for multi-wheel hub motor driven vehicles is the coordination of individual drivetrains to improve mobility and stability in the steering process. This paper proposes a dual-steering mode based on direct yaw moment control for enhancing vehicle steering ability in complex environments. The control system is designed as a hierarchical structure, with a yaw moment decision layer and a driving force distribution layer. In the higher-level layer, the objective optimization function is constructed to obtain the slip steering ratio, which represents the degree of vehicle slip steering in the dual-steering mode. A yaw moment controller using active disturbance rejection control theory is designed for continuous yaw rate control. When the actual yaw rate of the vehicle deviates from the reference yaw rate obtained by the vehicle reference model and the slip steering ratio, the yaw moment controller is actuated to determine the yaw moment demand for vehicle steering. In the lower-level layer, there is a torque distribution controller based on distribution rules, which meets the requirement of yaw moment demand without affecting the total longitudinal driving force of the vehicle. For verifying the validity and feasibility of the dual-steering mode, simulations were conducted on the hardware-in-loop real-time simulation platform. Additionally, corresponding real vehicle tests were carried out on an eight-wheel prototype vehicle. Test results were generally consistent with the simulation results, thereby demonstrating that the proposed dual-steering mode reduces steering radius and enhances the steering performance of the vehicle.

Because of the complexities of tire-road interaction, the wheels of a multi-wheel distributed electric drive vehicle can easily slip under certain working conditions. As wheel slip affects the dynamic performance and stability of the vehicle, it is crucial to control it and coordinate the driving force. With this aim, this paper presents a driving force coordination control strategy with road identification for eight-wheeled electric vehicles equipped with an in-wheel motor for each wheel. In the proposed control strategy, the road identification module estimates tire-road forces using an unscented Kalman filter algorithm and recognizes the road adhesion coefficient by employing the recursive least-square method. According to road identification, the optimal slip ratio under the current driving condition is obtained, and a controller based on sliding mode control with a conditional integrator uses this value for acceleration slip regulation. The anti-slip controller obtains the adjusting torque, which is integrated with the driver-command-based feedforward control torque to implement driving force coordination control. The results of hardware-in-loop simulation show that this control strategy can accurately estimate tire-road forces as well as the friction coefficient, and thus, can effectively fulfill the purpose of driving force coordinated control under different driving conditions.

Imported medicinal materials are an important part of Chinese medicinal resources. To be specific, about 10% of the around 600 commonly used Chinese medicinal materials are from abroad, and the introduction of foreign medicinal materials has promoted the development of Chinese medicine. Amid the advancement of reform and opening up and the "Belt and Road" Initiative, major headway has been made in the cross-border trade in China, bringing opportunities for the import of medicinal materials from border ports. However, for a long time, there is a lack of systematic investigation on the types of exotic medicinal materials at border ports. In the fourth national census of traditional Chinese medicine resources, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, together with several organizations, investigated the nearly 40 border ports, Chinese medicinal material markets, and border trade markets in 6 provinces/autonomous regions in China for the first time and recorded the types, sources, circulation, and the transaction characteristics of imported medicinal materials. Moreover, they invited experts to identify the origins of the collected 237 medicinal materials. In addition, the status quo and the problems of the medicinal materials were summarized. This study is expected to lay a basis for clarifying the market and origins of imported medicinal materials as well as the scientific research on and supervision of them.

Elevated thioredoxin-interacting protein (TXNIP)-induced pyroptosis contributes to the pathology of diabetic kidney disease (DKD). However, the molecular mechanisms in dysregulated TXNIP in DKD remain largely unclear.Transcriptomic analysis identified a novel long noncoding RNA-Prader Willi/Angelman region RNA, SNRPN neighbour (PWARSN)-which was highly expressed in a proximal tubular epithelial cell (PTEC) under high glucose conditions. We focused on revealing the functions of PWARSN in regulating TXNIP-mediated pyroptosis in PTECs by targeting PWARSN expression via lentivirus-mediated overexpression and CRISPR-Cas9-based knockout in vitro and overexpressing PWARSN in the renal cortex by AAV-9 targeted injection in vivo. A number of molecular techniques disclosed the mechanisms of PWARSN in regulating TXNIP induced-pyroptosis in DKD.TXNIP-NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome and PTEC pyroptosis were activated in the renal tubules of patients with DKD and in diabetic mice. Then we explored that PWARSN enhanced TXNIP-driven PTECs pyroptosis in vitro and in vivo. Mechanistically, cytoplasmic PWARSN sponged miR-372-3p to promote TXNIP expression. Moreover, nuclear PWARSN interacted and facilitated RNA binding motif protein X-linked (RBMX) degradation through ubiquitination, resulting in the initiation of TXNIP transcription by reducing H3K9me3-enrichment at the TXNIP promoter. Further analysis indicated that PWARSN might be a potential biomarker for DKD.These findings illustrate distinct dual molecular mechanisms for PWARSN-modulated TXNIP and PTECs pyroptosis in DKD, presenting PWARSN as a promising therapeutic target for DKD.

The lack of a load power prediction function in conventional multi-wheel electric drive vehicles leads to lags in control action. To address this issue, we developed a real-time energy management strategy with improved load power prediction accuracy. Based on an assessment of the overall vehicle structure, a mathematical model for each power source was established using theoretical analysis and data fitting. A method for the joint prediction of non-stationary load power combining Kalman filter and Markov chain forecasting methods was established, and a multi-objective optimization function was constructed under the nonlinear model predictive control framework. To enable real-time optimal control command, a sequential quadratic programming method in the finite time domain was applied. Finally, the multi-power source was optimized and coordinated. Multi-road driving experiments were carried out using a hardware-in-the-loop simulation platform. Comparisons of energy management control strategies with and without power prediction revealed that applying the former enhances the predictability of future load power, significantly optimizes the coordinated control of multiple power sources, improves vehicle fuel economy, and stabilizes bus voltage and battery state of charge. Moreover, it has specific reference significance in engineering application scenarios under conventional model predictive control.

This work aimed to assess the role of TLR8 in cerebral I/R injury and its in-depth pathogenesis. Bioinformatics analysis indicated that TLR8 was up-regulated in patients with ischemic stroke than that in healthy control, and miR-18a-5p was the upstream regulatory of TLR8. Then, the rat pheochromocytoma PC12 cells were exposed in oxygen-glucose-deprivation/reoxygenation (OGD/R) conditions to construct a model in vitro. The functional experiments indicated that OGD/R can decline the viability and elevate the apoptosis of PC12 cells, while up-regulation of miR-18a-5p can alleviate OGD/R-induced cell injury. Notably, overexpression of TLR8 reverses the miR-18a-5p-mediated protection on OGD/R-induced cells injury. Finally, we found that up-regulation of miR-18a-5p obviously declined the protein levels of TLR4 and TLR7 as well as the phosphorylation of NF-κB, while overexpression of TLR8 canceled the decrease caused by miR-18a-5p up-regulation. In summing, our results illustrated that miR-18a-5p/TLR8 axis can mitigate OGD/R-induced cells injury through TLRs and NF-κB pathway. Overexpression of TLR8 reverses the miR-18a-5p-mediated protection on OGD/R-induced cells injury.

The southwestern margin of the North China Craton (NCC) is located between the Alxa Terrane to the northwest, the North Qilian Orogen to the west and the North Qinling Orogen to the south. However, the paleogeographic and tectonic evolution for the southwestern part of the NCC in the Late Paleozoic is still poorly constrained. In order to constrain the Late Paleozoic tectonic evolution of the southwestern NCC, we carried out detailed field work and detrital zircon U-Pb geochronological research on Middle–Late Permian sedimentary rocks at the southwestern margin of the NCC. The U-Pb age spectra of detrital zircons from six samples are similar, showing four populations of 2.6–2.4 Ga, 2.0–1.7 Ga, 500–360 Ma and 350–250 Ma. Moreover, on the basis of the weighted-mean age of the youngest detrital zircons (257 ± 4 Ma), combined with the published results and volcanic interlayers, we propose that the Shangshihezi Formation formed during the Middle–Late Permian. Our results and published data indicate that the detrital zircons with age groups of 2.6–2.4 Ga and 2.0–1.7 Ga were likely derived from the Khondalite Belt and Yinshan Block in the northwestern NCC. The junction part between the North Qinling and North Qilian Orogen may provide the 500–360 Ma detrital zircons for the study area. The 350–250 Ma detrital zircons were probably derived from the northwestern part of the NCC. The majority of materials from Shangshihezi Formation within the study area were derived from the northwestern part of the NCC, indicating that the northwestern part of the NCC was strongly uplifted possibly resulting from the progressive subduction and closure of the Paleo-Asian Ocean. A small amount of materials were sourced from southwestern part of the NCC, indicating that the North Qinling Orogen experienced a minor uplift resulting from the northward subduction of the South Qinling terrane.

Purpose Patients with type 1 diabetes (T1D) are associated with a high risk of multiple complications, so the development of T1D treatment is urgently needed. This study was set out to explore the molecular mechanism of metformin in the treatment of T1D insulin resistance. Patients and methods Subcutaneous adipose tissues were collected from 68 T1D patients and 51 healthy controls. Insulin resistance model rats and cells were constructed and treated with metformin respectively. Western blot was used to detect p53 and RAP2A protein levels, and qPCR was utilized to measure p53 and RAP2A mRNA levels. SiRNA and RAP2A siRNA vectors were constructed to observe their effects on insulin resistance model cells. Results In T1D, p53 was up-regulated, while RAP2A was down-regulated. Metformin could effectively improve insulin resistance and inflammatory response while down-regulating p53 and up-regulating RAP2A. P53 induced insulin resistance and inflammatory response by inhibiting RAP2A and promoted apoptosis. Conclusion Metformin improves T1D insulin resistance and inflammatory response through p53/RAP2A pathway, and the regulation of p53/RAP2A pathway is conducive to improving the efficacy of metformin in the treatment of insulin resistance.

Background Spatially resolved gene expression has emerged as a crucial technique to understand complex multicellular interactions within the tumor and its microenvironment. Interrogation of complex cellular interactions within the tumor microenvironment (TME) requires a multi-omics approach where multiple RNA and protein targets can be visualized within the same tumor sample and be feasible in FFPE sample types. Simultaneous detection of RNA and protein can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. Examination of RNA by in situ hybridization (ISH) and protein by immunohistochemistry (IHC) or immunofluorescence (IF) are widely used and accepted techniques for the detection of biomarkers in tumor samples. Given the similarities in workflow, co-detection of RNA and protein by combining ISH and IHC/IF in a single assay can be a powerful multi-omics solution for interrogating the complex tumor and its microenvironment. Methods In this report we combined the single cell, single molecule RNA ISH technology known as RNAscope with IHC/IF to simultaneously detect RNA and protein in the same FFPE tumor section using both chromogenic and fluorescence detection methods. Results We demonstrate co-localization of target mRNA and the corresponding protein in human cancer samples, visualize infiltration of immune cells into the TME, characterize the activation state of immune cells in the TME, identify single cell gene expression within cellular boundaries demarcated by IHC/IF, examine cell type-specific expression of multiple immune checkpoint markers, and distinguish endogenous T cells from activated CAR+ T cells. Overall, we show that co-detection of RNA by the RNAscope ISH assay and protein by the IHC/IF assay in the same FFPE section is a feasible methodology. The combined RNAscope ISH-IHC/IF workflow is a powerful technique that can be used to study gene expression signatures at the RNA and protein level with spatial and single cell resolution. Conclusions By leveraging the strength of the similar workflows of RNAscope ISH and IHC/IF assays, this methodology combines transcriptomics and proteomics in the same tissue section, providing a multi-omics approach for characterizing complex tissues and revealing cell type specific gene expression with spatial and single cell resolution.

Background The canonical WNT-β-catenin signaling pathway is vital for development and tissue homeostasis but becomes strongly tumorigenic when dysregulated. and alter the transcriptional signature of a cell to promote malignant transformation. However, thorough characterization of these transcriptomic signatures has been challenging because traditional methods lack either spatial information, multiplexing, or sensitivity/specificity. To overcome these challenges, we developed a novel workflow combining the single molecule and single cell visualization capabilities of the RNAscope in situ hybridization (ISH) assay with the highly multiplexed spatial profiling capabilities of the GeoMx™ Digital Spatial Profiler (DSP) RNA assays. Using these methods, we sought to spatially profile and compare gene expression signatures of tumor niches with high and low CTNNB1 expression. Methods After screening 120 tumor cores from multiple tumors for CTNNB1 expression by the RNAscope assay, we identified melanoma as the tumor type with the highest CTNNB1 expression while prostate tumors had the lowest expression. Using the RNAscope Multiplex Fluorescence assay we selected regions of high CTNNB1 expression within 3 melanoma tumors as well as regions with low CTNNB1 expression within 3 prostate tumors. These selected regions of interest (ROIs) were then transcriptionally profiled using the GeoMx DSP RNA assay for a set of 78 genes relevant in immuno-oncology. Target genes that were differentially expressed were further visualized and spatially assessed using the RNAscope Multiplex Fluorescence assay to confirm GeoMx DSP data with single cell resolution. Results The GeoMx DSP analysis comparing the melanoma and prostate tumors revealed that they had significantly different gene expression profiles and many of these genes showed concordance with CTNNB1 expression. Furthermore, immunoregulatory targets such as ICOSLG, CTLA4, PDCD1 and ARG1, also demonstrated significant correlation with CTNNB1 expression. On validating selected targets using the RNAscope assay, we could distinctly visualize that they were not only highly expressed in melanoma compared to the prostate tumor, but their expression levels changed proportionally to that of CTNNB1 within the same tumors suggesting that these differentially expressed genes may be regulated by the WNT-β-catenin pathway. Conclusions In summary, by combining the RNAscope ISH assay and the GeoMx DSP RNA assay into one joint workflow we transcriptionally profiled regions of high and low CTNNB1 expression within melanoma and prostate tumors and identified genes potentially regulated by the WNT- β-catenin pathway. This novel workflow can be fully automated and is well suited for interrogating the tumor and stroma and their interactions.GeoMx Assays are for RESEARCH ONLY, not for diagnostics.

Background The ability of T cells to mediate anti-tumor immunity has been harnessed to develop successful immunotherapies in recent years. Although direct presentation of tumor antigens by tumor cells plays an important role in the effector function of cytotoxic T lymphocytes (CTLs), cross-presentation by professional antigen presenting cells such as dendritic cells (DCs) is vital for priming naive CD8+ T cells and developing a sustainable cytotoxic response. Natural killer (NK) cells within the tumor microenvironment (TME) recruit a specific population of DCs called conventional type 1 DCs (cDC1s) into the TME by secreting chemokines such as CCL5 and XCL1. However, these cells are very low in abundance and are characterized by the expression of numerous markers, making their detection in the tissue context challenging. Methods Therefore, to interrogate the presence of cDC1 and NK cells in the TME and reveal their spatial relationship we utilized the highly sensitive and specific RNAscope Multiplex Fluorescence in situ hybridization (ISH) assay. Probes for XCR1, THBD, CLEC9A, and CCR5 were used to identify cDC1 cells within 4 cervical cancer tumors. These tumors were then assessed for the presence of NK cells by using specific marker probes such as CD56 and NCR1 and chemokines XCL1 and CCL5. Finally, CTLs were visualized to determine if there is a correlation between the presence of cDC1 and NK cells and CTL infiltration within the cervical cancer tumors. Results Our results revealed a strong correlation between the presence of NK cells, cDC1 cells, and CTLs within 3 out of 4 cervical cancer samples. The NK cells showed expression of the chemokines XCL1 and CCL5, which are the ligands for XCR1 and CCR5 respectively, suggesting that the XCR1+/CCR5+ cDC1 cells may have been potentially recruited by these NK cells. Regions high in cDC1 and NK cells also showed significantly higher levels of CTL recruitment, as indicated by the presence of CD8+/IFNG+ T cells. Conversely, 1 of the 4 cervical cancer samples demonstrated relatively lower levels of NK cells which correlated with lower cDC1 cells and in turn lower CTL infiltration. Conclusions In conclusion, by utilizing the RNAscope Multiplex ISH assay we were able to identify and visualize the spatial relationship between NK cells, CTLs, and cDC1 cells, a rare subset of DC cells that excel at presenting tumor antigens to T cells. Using this technology, it is possible to spatially interrogate the TME and detect specialized immune cells when assessing response to immunotherapies.

NF-YA transcription factors function in modulating tolerance to abiotic stresses that are serious threats to crop yields. In this study, GmNFYA13, an NF-YA gene in soybean, was strongly induced by salt, drought, ABA, and H2O2, and suppressed by tungstate, an ABA synthesis inhibitor. The GmNFYA13 transcripts were detected in different tissues in seedling and flowering stages, and the expression levels in roots were highest. GmNFYA13 is a nuclear localization protein with self-activating activity. Transgenic Arabidopsis plants overexpressing GmNFYA13 with higher transcript levels of stress-related genes showed ABA hypersensitivity and enhanced tolerance to salt and drought stresses compared with WT plants. Moreover, overexpression of GmNFYA13 resulted in higher salt and drought tolerance in OE soybean plants, while suppressing it produced the opposite results. In addition, GmNFYA13 could bind to the promoters of GmSALT3, GmMYB84, GmNCED3, and GmRbohB to regulate their expression abundance in vivo. The data in this study suggested that GmNFYA13 enhanced salt and drought tolerance in soybean plants.

Walking on complex surface conditions in outdoor environments is important for active aging. This study aimed at examining whether fall prevention exercise integrated with an outdoor multisurface terrain compared with indoor solid ground was more beneficial for older adults. Twenty-two older nursing home residents were randomly assigned to outdoor multisurface terrain (n = 11, 79.5 &plusmn, 2.1 years) or indoor solid ground (n = 11, 78.8 &plusmn, 5.2 years) groups. Training occurred five times per week (30 min) for 3 weeks. The following performance test outcomes were measured: 10 m walk test (10 mWT), multisurface terrain walk test (MTWT), 2 min walk test (2 MWT), timed up and go test (TUGT), single-leg standing test with eyes open (SLSTEO), single-leg standing test with eyes closed (SLSTEC), and closed cycles test (CCT). Compared with baseline, the outdoor multisurface terrain training significantly improved performance in all tests (p &lt, 0.01). The improvements of the outdoor multisurface terrain group after intervention were significantly higher than those of the indoor solid group in the 10 mWT (p = 0.049), MTWT (p = 0.02), and 2 MWT (p = 0.000). Exercise combined with outdoor multisurface terrain training may be an efficacious approach and a feasible environmental intervention for fall prevention in older adults.

Plant growth regulator is a kind of synthetic pesticide with similar physiological activity to plant hormones. It has been widely used in grain, vegetables, fruits, flowers and other crops, and become an important technical guarantee for high yield, stable yield, high quality and efficient production of crops. In recent years, plant growth regulator is widely used in Chinese herbal medicine production for regulating the growth and development and increasing production of traditional. However the crop is different from general Chinese medicinal materials, the use of plant growth regulator should not only consider the effect of Chinese herbal medicine production, and also pay special attention to the influence of Chinese traditional medicine efficacy and safety. This paper reviewed the application of plant growth regulator in the traditional Chinese medicine, the impact on the quality and safety of Chinese medicinal materials, as well as plant growth regulator of residue limits standards and testing technology, so as to the scientific use of plant growth regulator, to promote Chinese standardization planting, provide the scientific basis to protect the safety of herbal medicine. At present, the indiscriminate use and abuse of plant growth regulators such as Zhuanggenling and bulking element are common in the production of Chinese crude drugs, which has led to a significant decline in the quality of some Chinese crude drugs, and resulted in the dual residual harm to the Chinese crude drugs and the cultivation environment, causing serious safety risks to human health. In the future, it is necessary to strengthen the registration management, use norms and limit standards of plant growth regulators in traditional Chinese medicinal materials, and strengthen the supervision and regulations on the use of fertilizer instead of medicine to avoid pesticide registration and other disorders, so as to provide a basis for the quality and safety monitoring of traditional Chinese medicinal materials. Simultaneously, it is encouraged to reduction or non-application of plant growth regulators in the production of Chinese medicinal materials, especially for traditional Chinese medicine which contains a variety of active ingredients. Therefore, it is actively advocated to cultivate Chinese medicinal materials through organic or ecological method.

Gene expression analysis is critical to precisely characterize complex tissues and provide insight into a disease condition. Techniques like PCR, sequencing, and northern blotting are highly sensitive and specific but are unable to provide information about spatial positioning of target genes. Visualization of gene expression with a spatial context can be critical in identifying complex milieus in heterogenous tissues like tumors. The RNAscope in situ hybridization (ISH) technology detects target RNA expression with high sensitivity and specificity at single-cell resolution. To understand the cellular cross talk between different cell populations, it is essential to simultaneously study gene and protein expression within a complex tissue. This chapter details combining the RNAscope ISH assay with immunofluorescence (IF) in one protocol to simultaneously visualize gene expression and protein expression in human tumor tissue and mouse brain tissue.