SlPP2C2 interacts with FZY/SAUR and regulates tomato development via signaling crosstalk of ABA and auxin.

Abscisic acid (ABA) signaling interacts frequently with auxin signaling when it regulates plant development, affecting multiple physiological processes; however, to the best of our knowledge, their interaction during tomato development has not yet been reported. Here, we found that type 2C protein phosphatase (SlPP2C2) interacts with both flavin monooxygenase FZY, an indole-3-acetic acid (IAA) biosynthetic enzyme, and small auxin upregulated RNA (SAUR) of an IAA signaling protein and regulates their activity, thereby affecting the expression of IAA-responsive genes. The expression level of SlPP2C2 was increased by exogenous ABA, IAA, NaCl, or dehydration treatment of fruits, leaves, and seeds, and it decreased in imbibed seeds. Manipulating SlPP2C2 with overexpression, RNA interference, and CRISPR/Cas9-mediated genome editing resulted in pleiotropic changes, such as morphological changes in leaves, stem trichomes, floral organs and fruits, accompanied by alterations in IAA and ABA levels. Furthermore, the RNA-seq analysis indicated that SlPP2C2 regulates the expression of auxin-/IAA-responsive genes in different tissues of tomato. The results demonstrate that SlPP2C2-mediated ABA signaling regulates the development of both vegetative and reproductive organs via interaction with FZY/SAUR, which integrates the cross-talk of ABA and auxin signals during development and affects the expressions of development-related genes in tomato.

Metabolomics Analysis and Diagnosis of Lung Cancer: Insights from Diverse Sample Types.

Lung cancer is a highly fatal disease that poses a significant global health burden. The absence of characteristic clinical symptoms frequently results in the diagnosis of most patients at advanced stages of lung cancer. Although low-dose computed tomography (LDCT) screening has become increasingly prevalent in clinical practice, its high rate of false positives continues to present a significant challenge. In addition to LDCT screening, tumor biomarker detection represents a critical approach for early diagnosis of lung cancer; unfortunately, no tumor marker with optimal sensitivity and specificity is currently available. Metabolomics has recently emerged as a promising field for developing novel tumor biomarkers. In this paper, we introduce metabolic pathways, instrument platforms, and a wide variety of sample types for lung cancer metabolomics. Specifically, we explore the strengths, limitations, and distinguishing features of various sample types employed in lung cancer metabolomics research. Additionally, we present the latest advances in lung cancer metabolomics research that utilize diverse sample types. We summarize and enumerate research studies that have investigated lung cancer metabolomics using different metabolomic sample types. Finally, we provide a perspective on the future of metabolomics research in lung cancer. Our discussion of the potential of metabolomics in developing new tumor biomarkers may inspire further study and innovation in this dynamic field.

Aspartate Transaminase-to-Albumin Ratio (ATAR), a Novel Prognostic Index, Predicts Outcomes in Patients with Small-Cell Lung Cancer.

BACKGROUND: Small cell lung cancer (SCLC) is characterized by high invasion rates, rapid progression, and poor prognoses. Thus, identifying SCLC patients at high risk of progression and death is critical to improve long-term survival. In this study, the aspartate transaminase-to-albumin ratio (ATAR) was examined as a prognostic factor for SCLC patients. METHODS: We screened 196 SCLC patients from December 2013 to September 2022 at the Sichuan Cancer Hospital. The data was collected from patients' medical information as well as from their blood results during diagnosis. Using the Youden index as a cutoff value, patients were divided into high-risk（> 0.54) and low-risk (≤ 0.54) ATAR groups. We analyzed the prognostic factors for overall survival (OS) using the Kaplan-Meier method, univariate and multivariate analyses, Cox regression, and the C-index. RESULTS: There were 109 (55.6%) smokers among the patients, and the median OS was 17.55 months. The Kaplan-Meier analysis indicated that patients with high-risk ATAR had significantly lower OS (p < 0.0001). A multivariate analysis demonstrated that elevated ATAR is an independent adverse predictor of OS (p < 0.001, HR = 1.907). Our study found that ATAR is an independent predictor of survival outcomes in SCLC, which was superior to ALB, PNI, and SII in predicting outcomes in low-risk and high-risk groups (all p < 0.05). Models combining ATAR with ALB, PNI, and SII showed more powerful prognostic value than their corresponding original models. Moreover, the prognostic indicator ATAR can significantly stratify stage I - II and III - IV SCLC patients (p ＜ 0.05). CONCLUSIONS: Peripheral blood ATAR prognostic index can be used as an independent predictor of SCLC patients before treatment.

Chromosome-scale genome assembly and insights into the metabolome and gene regulation of leaf color transition in an important oak species, Quercus dentata.

Quercus dentata Thunb., a dominant forest tree species in northern China, has significant ecological and ornamental value due to its adaptability and beautiful autumn coloration, with color changes from green to yellow into red resulting from the autumnal shifts in leaf pigmentation. However, the key genes and molecular regulatory mechanisms for leaf color transition remain to be investigated. First, we presented a high-quality chromosome-scale assembly for Q. dentata. This 893.54 Mb sized genome (contig N50 = 4.21 Mb, scaffold N50 = 75.55 Mb; 2n = 24) harbors 31 584 protein-coding genes. Second, our metabolome analyses uncovered pelargonidin-3-O-glucoside, cyanidin-3-O-arabinoside, and cyanidin-3-O-glucoside as the main pigments involved in leaf color transition. Third, gene co-expression further identified the MYB-bHLH-WD40 (MBW) transcription activation complex as central to anthocyanin biosynthesis regulation. Notably, transcription factor (TF) QdNAC (QD08G038820) was highly co-expressed with this MBW complex and may regulate anthocyanin accumulation and chlorophyll degradation during leaf senescence through direct interaction with another TF, QdMYB (QD01G020890), as revealed by our further protein-protein and DNA-protein interaction assays. Our high-quality genome assembly, metabolome, and transcriptome resources further enrich Quercus genomics and will facilitate upcoming exploration of ornamental values and environmental adaptability in this important genus.

Exploration of the link between gut microbiota and purinergic signalling.

Growing evidence reveals that microorganisms in the gut are linked to metabolic health and disease risk in human beings to a considerable extent. The focus of research at this stage must tend to focus on cause-and-effect studies. In addition to being a component of DNA and RNA, purine metabolites can be involved in purine signalling in the body as chemical messengers. Abnormalities in purinergic signalling may lead to neuropathy, rheumatic immune diseases, inflammation, tumors, and a wide range of other diseases. It has proved that gut microbes are involved in purinergic signalling. The relationship between these gut-derived purinergic signalling molecules and host metabolism may be one of the important clues to our understanding of the mechanisms by which the microbiota affects host metabolism.

Factors influencing platelet isolation: a prospective multicenter study from Western China.

Tumor-educated platelets (TEPs) have been widely reported to have promising application potential; nonetheless, platelet isolation from peripheral blood is an important but neglected step in TEPs research for platelet-based liquid biopsy. In this article, we discussed some common influence factors for platelet isolation. To investigate the factors involved in platelet isolation, a prospective multicenter study was conducted on healthy Han Chinese adults (18 to 79 years of age). A total of 208 individuals were included in the final statistical analysis out of the 226 healthy volunteers who were prospectively enrolled from four hospitals. The primary study metric was the platelet recovery rate (PRR). The similar pattern was observed in the four hospitals, The PRR at room temperature (23°C±2°C) was slightly higher than the PRR at cold temperature (4°C±2°C). Moreover, the PRR gradually decreased as the storage time increased. The PRR for samples within 2 hours of storage is significantly higher than for samples beyond 2 hours (p < .05). Additionally, PRR was also affected by the equipment used in different centers. This study confirmed several factors that influence platelet isolation. In our study, we indicated that platelet isolation should be performed within two hours of peripheral blood draw and held at room temperature until isolation, and that centrifuge models should be fixed during the extraction process, which will further improve the research progress of platelet-based liquid biopsy in cancer.

What is the context? Globally, cancer is one of the leading cause of premature death. Early screening is important for cancer diagnosis and treatment and can even significantly lower cancer mortalityGlobally, cancer is one of the leading cause of premature death. Early screening is important for cancer diagnosis and treatment and can even significantly lower cancer mortalityFor the liquid biopsy, isolation is an important step. Early studies have explored the influencing factors of exosome, circulating tumor cells (CTCs), and other components extraction in liquid biopsy.Despite platelet also being an excellent source of liquid biopsy, few studies have explored the factors that influence platelet isolation.Considering the importance of platelet isolation in tumor-based platelet liquid biopsy, our aim is to optimize platelet isolation conditions as much as possible to obtain a high platelet recovery rate.What is new? In this study, we conducted a prospective multicenter study ofhealthy adults from four centers, combining whole blood with platelet-richplasma to investigate factors influencing platelet recovery rate (PRR) during platelet isolation.In our study, we indicated that platelet isolation should be performed within two hours at room temperature, and that centrifuge models should be fixed during the extraction process, which will further improve the research progress of platelet-based liquid biopsy in cancer.What is the impact? In future platelet-related studies, we should fix the sample storage temperature, storage time and centrifuge model in the process of platelet extraction, so as to reduce the variables affecting platelet extraction as much as possible and ensure the stable recovery rate of platelet extraction.

Clinlabomics: leveraging clinical laboratory data by data mining strategies.

The recent global focus on big data in medicine has been associated with the rise of artificial intelligence (AI) in diagnosis and decision-making following recent advances in computer technology. Up to now, AI has been applied to various aspects of medicine, including disease diagnosis, surveillance, treatment, predicting future risk, targeted interventions and understanding of the disease. There have been plenty of successful examples in medicine of using big data, such as radiology and pathology, ophthalmology cardiology and surgery. Combining medicine and AI has become a powerful tool to change health care, and even to change the nature of disease screening in clinical diagnosis. As all we know, clinical laboratories produce large amounts of testing data every day and the clinical laboratory data combined with AI may establish a new diagnosis and treatment has attracted wide attention. At present, a new concept of radiomics has been created for imaging data combined with AI, but a new definition of clinical laboratory data combined with AI has lacked so that many studies in this field cannot be accurately classified. Therefore, we propose a new concept of clinical laboratory omics (Clinlabomics) by combining clinical laboratory medicine and AI. Clinlabomics can use high-throughput methods to extract large amounts of feature data from blood, body fluids, secretions, excreta, and cast clinical laboratory test data. Then using the data statistics, machine learning, and other methods to read more undiscovered information. In this review, we have summarized the application of clinical laboratory data combined with AI in medical fields. Undeniable, the application of Clinlabomics is a method that can assist many fields of medicine but still requires further validation in a multi-center environment and laboratory.

Inhibitory effects of temozolomide on glioma cells is sensitized by RSL3-induced ferroptosis but negatively correlated with expression of ferritin heavy chain 1 and ferritin light chain.

Invasive growth of glioblastoma makes residual tumor unremovable by surgery and leads to disease relapse. Temozolomide is widely used first-line chemotherapy drug to treat glioma patients, but development of temozolomide resistance is almost inevitable. Ferroptosis, an iron-dependent form of non-apoptotic cell death, is found to be related to temozolomide response of gliomas. However, whether inducing ferroptosis could affect invasive growth of glioblastoma cells and which ferroptosis-related regulators were involved in temozolomide resistance are still unclear. In this study, we treated glioblastoma cells with RSL3, a ferroptosis inducer, in vitro (cell lines) and in vivo (subcutaneous and orthotopic animal models). The treated glioblastoma cells with wild-type or mutant IDH1 were subjected to RNA sequencing for transcriptomic profiling. We then analyze data from our RNA sequencing and public TCGA glioma database to identify ferroptosis-related biomarkers for prediction of prognosis and temozolomide resistance in gliomas. Analysis of transcriptome data from RSL3-treated glioblastoma cells suggested that RSL3 could inhibit glioblastoma cell growth and suppress expression of genes involved in cell cycle. RSL3 effectively reduced mobility of glioblastoma cells through downregulation of critical genes involved in epithelial-mesenchymal transition. Moreover, RSL3 in combination with temozolomide showed suppressive efficacy on glioblastoma cell growth, providing a promising therapeutic strategy for glioblastoma treatment. Although temozolomide attenuated invasion of glioblastoma cells with mutant IDH1 more than those with wild-type IDH1, the combination of RSL3 and temozolomide similarly impaired invasive ability of glioblastoma cells in spite of IDH1 status. Finally, we noticed that both ferritin heavy chain 1 and ferritin light chain predicted unfavorable prognosis of glioma patients and were significantly correlated with mRNA levels of methylguanine methyltransferase as well as temozolomide resistance. Altogether, our study provided rationale for combination of RSL3 with temozolomide to suppress glioblastoma cells and revealed ferritin heavy chain 1 and ferritin light chain as biomarkers to predict prognosis and temozolomide resistance of glioma patients.

Crosstalk between Methylation and ncRNAs in Breast Cancer: Therapeutic and Diagnostic Implications.

Breast cancer, as a highly heterogeneous malignant tumor, is one of the primary causes of death among females worldwide. The etiology of breast cancer involves aberrant epigenetic mechanisms and abnormal expression of certain non-coding RNA (ncRNAs). DNA methylation, N6-methyladenosine(m6A), and histone methylation are widely explored epigenetic regulation types in breast cancer. ncRNAs are a group of unique RNA transcripts, mainly including microRNA (miRNAs), long non-coding RNA (lncRNAs), circular RNA (circRNAs), small interfering RNA (siRNAs), piwi-interacting RNA (piRNAs), etc. Different types of methylation and ncRNAs mutually regulate and interact to form intricate networks to mediate precisely breast cancer genesis. In this review, we elaborate on the crosstalk between major methylation modifications and ncRNAs and discuss the role of their interaction in promoting breast cancer oncogenesis. This review can provide novel insights into establishing a new diagnostic marker system on methylation patterns of ncRNAs and therapeutic perspectives of combining ncRNA oligonucleotides and phytochemical drugs for breast cancer therapy.

Overexpression of the persimmon abscisic acid ß-glucosidase gene (DkBG1) alters fruit ripening in transgenic tomato.

ß-Glucosidases (BG) are present in many plant tissues. Among these, abscisic acid (ABA) ß-glucosidases are thought to take part in the adjustment of cellular ABA levels, however the role of ABA-BG in fruits is still unclear. In this study, through RNA-seq analysis of persimmon fruit, 10 full-length DkBG genes were isolated and were all found to be expressed. In particular, DkBG1 was highly expressed in persimmon fruits with a maximum expression 95 days after full bloom (DAFD). We verified that, in vitro, DkBG1 protein can hydrolyze ABA-glucose ester (ABA-GE) to release free ABA. Compared with wild-type, tomato plants that overexpressed DkBG1 significantly upregulated the expression of ABA receptor PYL3/7 genes and showed typical symptoms of ABA hypersensitivity in fruits. DkBG1 overexpression (DkBG1-OE) accelerated fruit ripening onset by 3-4 days by increasing ABA levels at the pre-breaker stage and induced early ethylene release compared with wild-type fruits. DkBG1-OE altered the expression of ripening regulator NON-RIPENING (NOR) and its target genes; this in turn altered fruit quality traits such as coloration. Our results demonstrated that DkBG1 plays an important role in fruit ripening and quality by adjusting ABA levels via hydrolysis of ABA-GE.

Tomato SlPP2C5 Is Involved in the Regulation of Fruit Development and Ripening.

Abscisic acid (ABA) regulates plant development mainly through its signaling, in which ABA binds to receptors to inhibit type 2C protein phosphatases (PP2Cs). The exact roles of PP2Cs in fruit development are still unclear. In this work, we verify that tomato SlPP2C5 works as a negative regulator in ABA signaling during fruit development. SlPP2C5 was inhibited by both monomeric and dimeric ABA receptors SlPYLs through ABA dose-dependent way, and it interacted physically with SlPYLs and SlSnRK2s. SlPP2C5 was highly expressed in fruits induced by exogenous ABA. Plants with overexpressed SlPP2C5 had lower sensitivity to ABA, which showed faster seed germination and primary root growth compared to Wild type (WT), while SlPP2C5-suppressed plants were more sensitive to ABA. SlPP2C5-over-expression (OE) delayed fruit ripening onset, while SlPP2C5-RNAi advanced fruit ripening. Alteration of SlPP2C5 expression impacts fruit quality parameters as well, including pericarp thickness, fruit shape index, seed number and weight and the soluble solid content. RNA-seq analysis revealed that there were significant expression differences of genes related to ethylene release and lycopene synthesis between WT and both SlPP2C5-OE and SlPP2C5-RNAi lines with an inversed variation. Taken together, our findings demonstrate that SlPP2C5 plays an important role in the regulation of fruit development, ripening and quality.

G protein coupled estrogen receptor attenuates mechanical stress-mediated apoptosis of chondrocyte in osteoarthritis via suppression of Piezo1.

BACKGROUND: Apoptosis of chondrocyte is involved in osteoarthritis (OA) pathogenesis, and mechanical stress plays a key role in this process by activation of Piezo1. However, the negative regulation of signal conduction mediated by mechanical stress is still unclear. Here, we elucidate that the critical role of G protein coupled estrogen receptor (GPER) in the regulation of mechanical stress-mediated signal transduction and chondrocyte apoptosis. METHODS: The gene expression profile was detected by gene chip upon silencing Piezo1. The expression of GPER in cartilage tissue taken from the clinical patients was detected by RT-PCR and Western blot as well as immunohistochemistry, and the correlation between GPER expression and OA was also investigated. The chondrocytes exposed to mechanical stress were treated with estrogen, G-1, G15, GPER-siRNA and YAP (Yes-associated protein)-siRNA. The cell viability of chondrocytes was measured. The expression of polymerized actin and Piezo1 as well as the subcellular localization of YAP was observed under laser confocal microscope. Western blot confirmed the changes of YAP/ Rho GTPase activating protein 29 (ARHGAP29) /RhoA/LIMK /Cofilin pathway. The knee specimens of osteoarthritis model were stained with safranin and green. OARSI score was used to evaluate the joint lesions. The expressions of GPER and YAP were detected by immunochemistry. RESULTS: Expression profiles of Piezo1- silenced chondrocytes showed that GPER expression was significantly upregulated. Moreover, GPER was negatively correlated with cartilage degeneration during OA pathogenesis. In addition, we uncovered that GPER directly targeted YAP and broadly restrained mechanical stress-triggered actin polymerization. Mechanism studies revealed that GPER inhibited mechanical stress-mediated RhoA/LIMK/cofilin pathway, as well as the actin polymerization, by promoting expression of YAP and ARHGAP29, and the YAP nuclear localization, eventually causing the inhibition of Piezo1. YAP was obviously decreased in degenerated cartilage. Silencing YAP caused significantly increased actin polymerization and activation of Piezo1, and an increase of chondrocyte apoptosis. In addition, intra-articular injection of G-1 to OA rat effectively attenuated cartilage degeneration. CONCLUSION: We propose a novel regulatory mechanism underlying mechanical stress-mediated apoptosis of chondrocyte and elucidate the potential application value of GPER as therapy targets for OA.

Tomato protein phosphatase 2C influences the onset of fruit ripening and fruit glossiness.

Abscisic acid (ABA) plays a vital role in coordinating physiological processes during fresh fruit ripening. Binding of ABA to receptors facilitates the interaction and inhibition of type 2C phosphatase (PP2C) co-receptors. However, the exact mechanism of PP2C during fruit ripening is unclear. In this study, we determined the role of the tomato ABA co-receptor type 2C phosphatase SlPP2C3, a negative regulator of ABA signaling and fruit ripening. SlPP2C3 selectively interacted with monomeric ABA receptors and SlSnRK2.8 kinase in both yeast and tobacco epidermal cells. Expression of SlPP2C3 was ABA-inducible, which was negatively correlated with fruit ripening. Tomato plants with suppressed SlPP2C3 expression exhibited enhanced sensitivity to ABA, while plants overexpressing SlPP2C3 were less sensitive to ABA. Importantly, lack of SlPP2C3 expression accelerated the onset of fruit ripening and affected fruit glossiness by altering the outer epidermis structure. There was a significant difference in the expression of cuticle-related genes in the pericarp between wild-type and SlPP2C3-suppressed lines based on RNA sequencing (RNA-seq) analysis. Taken together, our findings demonstrate that SlPP2C3 plays an important role in the regulation of fruit ripening and fruit glossiness in tomato.

Establishment of Serum TSH Reference Intervals by an Indirect Method for Healthy Population in Southwest China.

BACKGROUND: Serum thyroid stimulating hormone (TSH) detection is of great clinical significance in monitoring thyroid function. The aim of the present study was to establish reference intervals (RIs) for serum TSH in healthy Han population in Southwest China using data from routine health check-up individuals. METHODS: Healthy subjects (n = 7,116) were enrolled from January 2019 to September 2020. Serum TSH values were determined in the Beckman Coulter UniCel™ DxI 800 Access® immunoassay system. Outliers were identified and removed using Dixon's interactive principle. The 95th percentile range was calculated as RIs for serum TSH. All the statistical analyses were run on R statistical software version 4.0.3. RESULTS: A total of 6,668 (1,324 female and 5,344 male) suitable individuals were included in this study. Serum TSH results showed a non-Gaussian distribution by Kolmogorov-Smirnov test. According to Mann-Whitney U analysis, the serum TSH values for the female group differ from the male group's (p < 0.05). Besides, Spearman's rank correlation test disclosed that no obvious correlation was observed between serum TSH levels and age (r = -0.0039, p > 0.05). Accordingly, the RIs for serum TSH in Southwest China Han population were 0.64 (95% CI: 0.62 to 0.65) to 4.05 (95% CI: 4.02 to 4.09) mIU/L in male and 0.72 (95% CI: 0.67 to 0.77) to 5.66 (95% CI: 5.58 to 5.75) mIU/L in female, respectively. CONCLUSIONS: In this study, the laboratory specific RIs for serum TSH were successfully established by indirect method using the data from health check-up population. It implies that the indirect method is an easy and lowcost pathway for each laboratory to establish its own RIs.

PYL9 is involved in the regulation of ABA signaling during tomato fruit ripening.

Abscisic acid (ABA) regulates fruit ripening, yet little is known about the exact roles of ABA receptors in fruit. In this study, we reveal the role of SlPYL9, a tomato pyrabactin resistance (PYR)/pyrobactin resistance-like (PYL)/regulatory component of ABA receptors (RCAR) protein, as a positive regulator of ABA signaling and fruit ripening. SlPYL9 inhibits protein phosphatase-type 2C (PP2C2/6) in an ABA dose-dependent way, and it interacts physically with SlPP2C2/3/4/5 in an ABA-dependent manner. Expression of SlPYL9 was observed in the seeds, flowers, and fruits. Overexpression and suppression of SlPYL9 induced a variety of phenotypes via altered expression of ABA signaling genes (SlPP2C1/2/9, SlSnRK2.8, SlABF2), thereby affecting expression of ripening-related genes involved in ethylene release and cell wall modification. SlPYL9-OE/RNAi plants showed a typical ABA hyper-/hypo-sensitive phenotype in terms of seed germination, primary root growth, and response to drought. Fruit ripening was significantly accelerated in SlPYL9-OE by 5-7 d as a result of increased endogenous ABA accumulation and advanced release of ethylene compared with the wild-type. In the SlPYL9-RNAi lines, fruit ripening was delayed, mesocarp thickness was enhanced, and petal abscission was delayed compared with the wild-type, resulting in conical/oblong and gourd-shaped fruits. These results suggest that SlPYL9 is involved in ABA signaling, thereby playing a role in the regulation of flower abscission and fruit ripening in tomato.

The functional analysis of SlNCED1 in tomato pollen development.

Abscisic acid (ABA) regulates plant growth and development, but the role of ABA in the development of reproductive organs in tomato has rarely been addressed. In the present study, the role of ABA in the regulation of male and female gametogenesis as well as pollen development and germination is tested in tomato. qRT-PCR and in situ hybridization analysis of 9-cis-epoxycarotenoid dioxygenase (SlNCED1), a key enzyme in the ABA biosynthetic pathway, showed high expression of SlNCED1 primarily in the meristem during gametogenesis and mainly in ovule, stigma, anther/pollen and vascular tissues during floral organ development. SlNCED1 expression and ABA accumulation in anther peak at stages 13-14, suggesting that ABA plays a role in the primary formation of pollen grains. Over expression and suppression of SlNCED1 led to the abnormal development of anther/pollen, especially in SlNCED1-OE lines, which have serious pollen deterioration. The percentage of pollen germination in wild type is 91.47%, whereas it is 6.85% in OE transgenic lines and 38.4% at anthesis in RNAi lines. RNA-Seq of anthers shows that SlNCED1-OE can significantly enhance the expression of SlPP2Cs and down-regulate the expression of SlMYB108 and SlMYB21, which are anther/flower-specific transcriptional factors in tomato. Finally, anther transcriptome data indicate that SlNCED1 is involved in ABA-mediated regulation in pollen/anther metabolism, cell wall modification, and transcription levels. These results support an important role for ABA in the development of reproductive organs in tomato and contribute to the elucidation of the underlying regulatory mechanisms.

Quantification of 1D, a novel derivative of curcumin with potential antitumor activity, in rat plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study in rats.

CONTEXT: 1 D is a novel derivative of curcumin and shows very promising antitumor activities in various cancer cell lines. OBJECTIVE: To characterize its preclinical pharmacokinetic profiles, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of 1 D in rat plasma. MATERIALS AND METHODS: An aliquot of 50 µL plasma sample was processed by protein precipitation with methanol. Chromatographic separation was accomplished on a Zorbax Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 µm) with a gradient elution system (water/0.1% formic acid and methanol). Detection was performed by multiple reaction monitoring (MRM) mode using electrospray ionization in the positive ion mode. The optimized fragmentation transition for 1 D was m/z 491.2 â†’ 361.2. RESULTS: The method was linear over the concentration range of 5-1000 ng/mL. The intra- and inter-day precisions were less than 9.8% and the accuracy was within ± 14.5%. The mean recovery of 1 D ranged from 102.5 to 105.9%. No matrix effects and significant sample loss during sample processing were observed. The validated method has been successfully applied to a pharmacokinetic study in rats after intravenous administration of 1 D. Non-compartmental pharmacokinetic parameters, including half-life (t1/2), apparent volume of distribution (Vz), clearance (CLz), and area under the concentration-time curve (AUC(0-t)) were 4.92 h, 46.56 L/kg, 6.33 L/h/kg, and 806.70 µg/L/h, respectively. DISCUSSION AND CONCLUSIONS: Results demonstrated that 1 D displayed favourable pharmacokinetic properties for further in vivo pharmacologic evaluation, which could be facilitated by the validated LC-MS/MS method.

[Effect of Autophagy Inhibitor Hydroxychloroquine on Chemosensitivity of Castration-resistant Prostate Cancer].

OBJECTIVE: To determine the effects of autophagy inhibitor hydroxychloroquine (HCQ) on chemosensitivity of castration-resistant prostate cancer 22RV1 cell line in vitro and in vivo, and changes in its mRNA expressions of autophagy gene Bcelin-1, autophagy specific substrate P62 gene, pro-apoptotic gene Bax. METHODS: 22RV1 cells were cultured in vitro and divided into blank control (no drug), DOC, and HCQ (20 µmol/L)+DOC groups. The concentration of DOC was set at 10 -6 mol/L, 10 -7 mol/L, and 10 -8 mol/L in the tests. Cell proliferation activities were detected by CCK-8 method 72 h after drug treatments. The 22RV1 cell suspension was injected subcutaneously into nude mice to establish transplanted tumor. The successfully modeled mice were randomly divided into three groups (five each) treated by physiological saline, DOC and HCQ+DOC (injected intraperitoneally for 4 weeks), respectively. Changes in growth of the transplanted tumor were observed. The mRNA expressions of Beclin-1, P62, and Bax were detected by qPCR. The protein expressions of Beclin-1, LC3B, and Bax were detected by Western blot. RESULTS: In vitro: compared with the blank control, the DOC and HCQ+DOC groups showed decrease proliferation of cells( P<0.05); HCQ further lowered cell proliferation in the presence of DOC ( P<0.05), resulting in reduced half maximal inhibitory concentration (IC 50) of DOC. In vivo: compared with the model mice, the DOC and HCQ+DOC groups had decreased volume of transplanted tumor. HCQ slowed the weekly growth of tumor in the presence of DOC ( P<0.05), most obvious at the 4th week. In vitro and in vivo, HCQ+DOC upregulated the mRNA and protein expressions of Beclin-1, P62 and Bax ( P<0.05). CONCLUSION: HCQ can interfere with the autophagy of castration-resistant prostate cancer cells, inhibiting its proliferation and enhancing its sensitivity to chemotherapeutic drugs.

Suppressing ABA uridine diphosphate glucosyltransferase (SlUGT75C1) alters fruit ripening and the stress response in tomato.

Abscisic acid (ABA) glucose conjugation mediated by uridine diphosphate glucosyltransferases (UGTs) is an important pathway in regulating ABA homeostasis. In the present study, we investigated three tomato SlUGTs that are highly expressed in fruit during ripening, and these SlUGTs were localized to the cytoplasm and cell nucleus. Among these three UGTs, SlUGT75C1 catalyzes the glucosylation of both ABA and IAA in vitro; SlUGT76E1 can only catalyze the conjugation of ABA; and SlUGT73C4 cannot glycosylate either ABA or IAA. Therefore, SlUGT75C1 was selected for further investigation. SlUGT75C1 RNA interference significantly up-regulated the expression level of SlCYP707A2, which encodes an ABA 8'-hydroxylase but did not affect the expression of SlNCED1, which encodes a key enzyme in ABA biosynthesis. Suppression of SlUGT75C1 significantly accelerated fruit ripening by enhancing ABA levels and promoting the early release of ethylene. SlUGT75C1-RNAi altered the expression of fruit ripening genes (genes involved in ethylene release and cell wall catabolism). SlUGT75C1-RNAi seeds showed delayed germination and root growth compared with wild-type as well as increased sensitivity to exogenous ABA. SlUGT75C1-RNAi plants were also more resistant to drought stress. These results demonstrated that SlUGT75C1 plays a crucial role in ABA-mediated fruit ripening, seed germination, and drought responses in tomato.

SlPti4 Affects Regulation of Fruit Ripening, Seed Germination and Stress Responses by Modulating ABA Signaling in Tomato.

Although the role of the ethylene response factor (ERF) Pti4 in disease resistance has been demonstrated in higher plants, it is presently unknown whether the tomato SlPti4 protein plays a role in the regulation of fruit development and the stress response. Here, we show that SlPti4 is involved in the regulation of fruit ripening, seed germination, and responses to drought and Botrytis cinerea infection through adjustments to ABA metabolism and signaling. SlPti4 gene expression is very low early in fruit development, but increases rapidly during ripening and can be induced by exogenous ABA and 1-aminocyclopropane 1-carboxylate (ACC). RNA interference (RNAi)-induced silencing of SlPti4 leads to an increase of ABA accumulation together with a decrease of ethylene release, which causes the high expression level of SlBcyc, and thus the transgenic fruit is orange instead of red as in wild-type fruit during ripening. SlPti4-RNAi seeds accumulate less ABA and mRNA for ABA receptor SlPYL genes, which causes insensitivity to ABA treatment. SlPti4-RNAi transgenic plants with low ABA levels and high ethylene release were more sensitive to drought stress. SlPti4-RNAi plants also showed weaker resistance to B. cinerea infection than the wild type. Thus, SlPti4 is an important regulator of tomato fruit ripening, seed germination and abiotic/biotic stress responses. This study expands our knowledge on diverse plant physiologies which are regulated by ABA signaling and the function of SlPti4.