Proteasomal degradation of MaMYB60 mediated by the E3 ligase MaBAH1 causes high temperature-induced repression of chlorophyll catabolism and green ripening in banana.

Banana (Musa acuminata) fruits ripening at 30 °C or above fail to develop yellow peels; this phenomenon, called green ripening, greatly reduces their marketability. The regulatory mechanism underpinning high temperature-induced green ripening remains unknown. Here we decoded a transcriptional and post-translational regulatory module that causes green ripening in banana. Banana fruits ripening at 30 °C showed greatly reduced expression of 5 chlorophyll catabolic genes (CCGs), MaNYC1 (NONYELLOW COLORING 1), MaPPH (PHEOPHYTINASE), MaTIC55 (TRANSLOCON AT THE INNER ENVELOPE MEMBRANE OF CHLOROPLASTS 55), MaSGR1 (STAY-GREEN 1), and MaSGR2 (STAY-GREEN 2), compared to those ripening at 20 °C. We identified a MYB transcription factor, MaMYB60, that activated the expression of all 5 CCGs by directly binding to their promoters during banana ripening at 20 °C, while showing a weaker activation at 30 °C. At high temperatures, MaMYB60 was degraded. We discovered a RING-type E3 ligase MaBAH1 (benzoic acid hypersensitive 1) that ubiquitinated MaMYB60 during green ripening and targeted it for proteasomal degradation. MaBAH1 thus facilitated MaMYB60 degradation and attenuated MaMYB60-induced transactivation of CCGs and chlorophyll degradation. By contrast, MaMYB60 upregulation increased CCG expression, accelerated chlorophyll degradation, and mitigated green ripening. Collectively, our findings unravel a dynamic, temperature-responsive MaBAH1-MaMYB60-CCG module that regulates chlorophyll catabolism, and the molecular mechanism underpinning green ripening in banana. This study also advances our understanding of plant responses to high-temperature stress.

Banana MaNAC1 activates secondary cell wall cellulose biosynthesis to enhance chilling resistance in fruit.

Chilling injury has a negative impact on the quantity and quality of crops, especially subtropical and tropical plants. The plant cell wall is not only the main source of biomass production, but also the first barrier to various stresses. Therefore, improving the understanding of the alterations in cell wall architecture is of great significance for both biomass production and stress adaptation. Herein, we demonstrated that the cell wall principal component cellulose accumulated during chilling stress, which was caused by the activation of MaCESA proteins. The sequence-multiple comparisons show that a cold-inducible NAC transcriptional factor MaNAC1, a homologue of Secondary Wall NAC transcription factors, has high sequence similarity with Arabidopsis SND3. An increase in cell wall thickness and cellulosic glucan content was observed in MaNAC1-overexpressing Arabidopsis lines, indicating that MaNAC1 participates in cellulose biosynthesis. Over-expression of MaNAC1 in Arabidopsis mutant snd3 restored the defective secondary growth of thinner cell walls and increased cellulosic glucan content. Furthermore, the activation of MaCESA7 and MaCESA6B cellulose biosynthesis genes can be directly induced by MaNAC1 through binding to SNBE motifs within their promoters, leading to enhanced cellulose content during low-temperature stress. Ultimately, tomato fruit showed greater cold resistance in MaNAC1 overexpression lines with thickened cell walls and increased cellulosic glucan content. Our findings revealed that MaNAC1 performs a vital role as a positive modulator in modulating cell wall cellulose metabolism within banana fruit under chilling stress.

MaHsf24, a novel negative modulator, regulates cold tolerance in banana fruits by repressing the expression of HSPs and antioxidant enzyme genes.

Transcriptional regulation mechanisms underlying chilling injury (CI) development have been widely investigated in model plants and cold-sensitive fruits, such as banana (Musa acuminata). However, unlike the well-known NAC and WRKY transcription factors (TFs), the function and deciphering mechanism of heat shock factors (HSFs) involving in cold response are still fragmented. Here, we showed that hot water treatment (HWT) alleviated CI in harvested banana fruits accomplishing with reduced reactive oxygen species (ROS) accumulation and increased antioxidant enzyme activities. A cold-inducible but HWT-inhibited HSF, MaHsf24, was identified. Using DNA affinity purification sequencing (DAP-seq) combined with RNA-seq analyses, we found three heat shock protein (HSP) genes (MaHSP23.6, MaHSP70-1.1 and MaHSP70-1.2) and three antioxidant enzyme genes (MaAPX1, MaMDAR4 and MaGSTZ1) were the potential targets of MaHsf24. Subsequent electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and dual-luciferase reporter (DLR) analyses demonstrated that MaHsf24 repressed the transcription of these six targets via directly binding to their promoters. Moreover, stably overexpressing MaHsf24 in tomatoes increased cold sensitivity by suppressing the expressions of HSPs and antioxidant enzyme genes, while HWT could recover cold tolerance, maintaining higher levels of HSPs and antioxidant enzyme genes, and activities of antioxidant enzymes. In contrast, transiently silencing MaHsf24 by virus-induced gene silencing (VIGS) in banana peels conferred cold resistance with the upregulation of MaHSPs and antioxidant enzyme genes. Collectively, our findings support the negative role of MaHsf24 in cold tolerance, and unravel a novel regulatory network controlling bananas CI occurrence, concerning MaHsf24-exerted inhibition of MaHSPs and antioxidant enzyme genes.

E3 ligase MaNIP1 degradation of NON-YELLOW COLORING1 at high temperature inhibits banana degreening.

Banana (Musa acuminata) fruit ripening under high temperatures (>24 °C) undergoes green ripening due to failure of chlorophyll degradation, which greatly reduces marketability. However, the mechanism underlying high temperature-repressed chlorophyll catabolism in banana fruit is not yet well understood. Here, using quantitative proteomic analysis, 375 differentially expressed proteins were identified in normal yellow and green ripening in banana. Among these, one of the key enzymes involved in chlorophyll degradation, NON-YELLOW COLORING 1 (MaNYC1), exhibited reduced protein levels when banana fruit ripened under high temperature. Transient overexpression of MaNYC1 in banana peels resulted in chlorophyll degradation under high temperature, which weakens the green ripening phenotype. Importantly, high temperature induced MaNYC1 protein degradation via the proteasome pathway. A banana RING E3 ligase, NYC1-interacting protein 1 (MaNIP1), was found to interact with and ubiquitinate MaNYC1, leading to its proteasomal degradation. Furthermore, transient overexpression of MaNIP1 attenuated MaNYC1-induced chlorophyll degradation in banana fruits, indicating that MaNIP1 negatively regulates chlorophyll catabolism by affecting MaNYC1 degradation. Taken together, the findings establish a post-translational regulatory module of MaNIP1-MaNYC1 that mediates high temperature-induced green ripening in bananas.

E3 ubiquitin ligase MaRZF1 modulates high temperature-induced green ripening of banana by degrading MaSGR1.

High temperatures (>24°C) prevent the development of a yellow peel on bananas called green ripening, owing to the inhibition of chlorophyll degradation. This phenomenon greatly reduces the marketability of banana fruit, but the mechanisms underlining high temperature-repressed chlorophyll catabolism need to be elucidated. Herein, we found that the protein accumulation of chlorophyll catabolic enzyme MaSGR1 (STAY-GREEN 1) was reduced when bananas ripened at high temperature. Transiently expressing MaSGR1 in banana peel showed its positive involvement in promoting chlorophyll degradation under high temperature, thereby weakening green ripening phenotype. Using yeast two-hybrid screening, we identified a RING-type E3 ubiquitin ligase, MaRZF1 (RING Zinc Finger 1), as a putative MaSGR1-interacting protein. MaRZF1 interacts with and targets MaSGR1 for ubiquitination and degradation via the proteasome pathway. Moreover, upregulating MaRZF1 inhibited chlorophyll degradation, and attenuated MaSGR1-promoted chlorophyll degradation in bananas during green ripening, indicating that MaRZF1 negatively regulates chlorophyll catabolism via the degradation of MaSGR1. Taken together, MaRZF1 and MaSGR1 form a regulatory module to mediate chlorophyll degradation associated with high temperature-induced green ripening in bananas. Therefore, our findings expand the understanding of posttranslational regulatory mechanisms of temperature stress-caused fruit quality deterioration.

The changes of intestinal microbiota and metabolomics during the inhibition of bladder cancer by liquiritigenin.

Liquiritigenin is a natural medicine. However, its inhibitory effect and its potential mechanism on bladder cancer (BCa) remain to be explored. It was found that it could be visualized that the transplanted tumours in the low-dose liquiritigenin -treated group and the high-dose liquiritigenin -treated group were smaller than those in the model group. Liquiritigenin treatment led to alterations in Lachnoclostridium, Escherichia-Shigella, Alistipes and Akkermansia. Non-targeted metabolomics analysis showed that a total of multiple differential metabolites were identified between the model group and the high-dose liquiritigenin-treated group. This provides a new direction and rationale for the antitumour effects of liquiritigenin.

[Buzhong Yiqi Decoction ameliorates spleen deficiency syndrome by regulating gut microbiota].

This study aims to decipher the mechanism of Buzhong Yiqi Decoction(BZYQD) in the treatment of spleen deficiency syndrome via gut microbiota. The mouse models of spleen deficiency syndrome were established by fecal microbiota transplantation(FMT, from patients with spleen deficiency syndrome) and administration of Sennae Folium(SF, 10 g·kg~(-1)), respectively, and treated with BZYQD for 5 d. The pseudosterile mice(administrated with large doses of antibiotics) and the mice transplanted with fecal bacteria from healthy human were taken as the controls. The levels of IgA, interleukin(IL)-2, IL-1ß, interferon(IFN)-γ, tumor necrosis factor-alpha(TNF-α), and 5-hydroxytryptamine(5-HT) in the intestinal tissue of two models were measured by enzyme-linked immunosorbent assay, and the CD8~+/CD3~+ ratio was determined by flow cytometry. The composition and changes of the gut microbiota were determined by 16S rRNA high-throughput sequencing and qPCR. Furthermore, the correlation analysis was performed to study the mediating role of gut microbiota in the treatment. The results showed that BZYQD elevated the IgA level, lowered the IL-1ß, TNF-α, and 5-HT levels, and decreased the CD8~+/CD3~+ ratio in the intestinal tissue of the two models. Moreover, BZYQD had two-way regulatory effects on the levels of IL-2 and IFN-γ. BZYQD inhibited the overgrowth and reduced the richness of gut microbiota in the SF model, and improved the gut microbiota structure in the two models. Algoriphagus, Mycobacterium, and CL500＿29＿marine＿group were the common differential genera in the two models compared with the control. Acinetobacter, Parabacteroides, and Ruminococcus were the differential genera unique to the FMT model, and Sphingorhabdus, Lactobacillus, and Anaeroplasma were the unique differential genera in the SF model. BZYQD was capable of regulating all these genera. The qPCR results showed that BZYQD increased the relative abundance of Akkermansia muciniphila and decreased that of Bacteroides uniformis in the two models. The correlation analysis revealed that the levels of above intestinal cytokines were significantly correlated with characteristic gut microorganisms in different mo-dels. The IL-1ß level had a significantly positive correlation with Acinetobacter and CL500＿29＿marine＿group in the two models, while the different levels of IL-2 and IFN-γ in the two models may be related to its different gut microbiota structures. In conclusion, BZYQD could regulate the disordered gut microbiota structure in different animal models of spleen deficiency syndrome to improve the intestinal immune status, which might be one of the mechanisms of BZYQD in treating spleen deficiency syndrome.

Construction of Si-Stereogenic Silanols by Palladium-Catalyzed Enantioselective C-H Alkenylation.

The construction of silicon-stereogenic silanols via Pd-catalyzed intermolecular C-H alkenylation with the assistance of a commercially available L-pyroglutamic acid has been realized for the first time. Employing oxime ether as the directing group, silicon-stereogenic silanol derivatives could be readily prepared with excellent enantioselectivities, featuring a broad substrate scope and good functional group tolerance. Moreover, parallel kinetic resolution with unsymmetric substrates further highlighted the generality of this protocol. Mechanistic studies indicate that L-pyroglutamic acid could stabilize the Pd catalyst and provide excellent chiral induction. Preliminary computational studies unveil the origin of the enantioselectivity in the C-H bond activation step.

MaMYB4 is a negative regulator and a substrate of RING-type E3 ligases MaBRG2/3 in controlling banana fruit ripening.

Fruit ripening is a complex developmental process, which is modulated by both transcriptional and post-translational events. Control of fruit ripening is important in maintaining moderate quality traits and minimizing postharvest deterioration. In this study, we discovered that the transcription factor MaMYB4 acts as a negative regulator of fruit ripening in banana. The protein levels of MaMYB4 decreased gradually with banana fruit ripening, paralleling ethylene production, and decline in firmness. DNA affinity purification sequencing combined with RNA-sequencing analyses showed that MaMYB4 preferentially binds to the promoters of various ripening-associated genes including ethylene biosynthetic and cell wall modifying genes. Furthermore, ectopic expression of MaMYB4 in tomato delayed tomato fruit ripening, which was accompanied by downregulation of ethylene biosynthetic and cell wall modifying genes. Importantly, two RING finger E3 ligases MaBRG2/3, whose protein accumulation increased progressively with fruit ripening, were found to interact with and ubiquitinate MaMYB4, contributing to decreased accumulation of MaMYB4 during fruit ripening. Transient overexpression of MaMYB4 and MaBRG2/3 in banana fruit ripening delayed or promoted fruit ripening by inhibiting or stimulating ethylene biosynthesis, respectively. Taken together, we demonstrate that MaMYB4 negatively modulates banana fruit ripening, and that MaMYB4 abundance could be regulated by protein ubiquitination, thus providing insights into the role of MaMYB4 in controlling fruit ripening at both transcriptional and post-translational levels.

Phosphorylation of transcription factor bZIP21 by MAP kinase MPK6-3 enhances banana fruit ripening.

Ripening of fleshy fruits involves both diverse post-translational modifications (PTMs) and dynamic transcriptional reprogramming, but the interconnection between PTMs, such as protein phosphorylation and transcriptional regulation, in fruit ripening remains to be deciphered. Here, we conducted a phosphoproteomic analysis during banana (Musa acuminata) ripening and identified 63 unique phosphopeptides corresponding to 49 proteins. Among them, a Musa acuminata basic leucine zipper transcription factor21 (MabZIP21) displayed elevated phosphorylation level in the ripening stage. MabZIP21 transcript and phosphorylation abundance increased during banana ripening. Genome-wide MabZIP21 DNA binding assays revealed MabZIP21-regulated functional genes contributing to banana ripening, and electrophoretic mobility shift assay, chromatin immunoprecipitation coupled with quantitative polymerase chain reaction, and dual-luciferase reporter analyses demonstrated that MabZIP21 stimulates the transcription of a subset of ripening-related genes via directly binding to their promoters. Moreover, MabZIP21 can be phosphorylated by MaMPK6-3, which plays a role in banana ripening, and T318 and S436 are important phosphorylation sites. Protein phosphorylation enhanced MabZIP21-mediated transcriptional activation ability, and transient overexpression of the phosphomimetic form of MabZIP21 accelerated banana fruit ripening. Additionally, MabZIP21 enlarges its role in transcriptional regulation by activating the transcription of both MaMPK6-3 and itself. Taken together, this study reveals an important machinery of protein phosphorylation in banana fruit ripening in which MabZIP21 is a component of the complex phosphorylation pathway linking the upstream signal mediated by MaMPK6-3 with transcriptional controlling of a subset of ripening-associated genes.

Promising Cytotoxic butenolides from the Soybean endophytic fungus Aspergillus terreus: a combined molecular docking and in-vitro studies.

AIM: This study aimed to use one strain many compounds approach (OSMAC) to investigate the cytotoxic potential of Aspergillus terreus associated with soybean versus several cancer cell lines, by means of in-silico and in vitro approaches. METHODS AND RESULTS: Fermentation of the isolated strain was done on five media. The derived extracts were investigated for their inhibitory activities against three human cancer cell lines; mammary gland breast cancer (MCF-7), colorectal adenocarcinoma (Caco-2), and hepatocellular carcinoma (HepG2) using MTT Assay. The fungal mycelia fermented in Modified Potato Dextrose Broth (MPDB) was the most cytotoxic extract against HepG2, MCF-7, and Caco-2 cell lines with IC50 4.2 ± 0.13, 5.9 ± 0.013 and 7.3 ± 0.004 µg mL-1, respectively. MPDB extract was scaled up resulting in the isolation of six metabolites; three fatty acids (1, 2, and 4), one sterol (3) and two butenolides (5 and 6) by column chromatography. The isolated compounds (1-6) were screened through a molecular docking approach for their binding aptitude to various active sites. butyrolactone-I (5) revealed a significant interaction within the CDK2 active site, while aspulvinone E (6) showed promising binding affinity to FLT3 and EGFR active sites that was confirmed by in vitro CDK2, FLT3 and EGFR inhibitory activity. Finally, the in vitro cytotoxic activities of butyrolactone-I (5) and aspulvinone E (6) revealed the antiproliferative activity of butyrolactone-I (5), against HepG2 cell line (IC50 = 17.85 ± 0.32 µM). CONCLUSION: Molecular docking analysis and in vitro assays suggested the CDK2/A2 inhibitory potential of butyrolactone-I (5) in addition to the promising interaction abilities of aspulvinone E (6) with EGFR and FLT3 active sites as a possible mechanism of their biological activities.

[Rapid identification of components in Wuzhuyu Decoction using UHPLC Q-Exactive Orbitrap MS~n and molecular network technology].

Wuzhuyu Decoction, the classical formula recorded in the Treatise on Febrile Diseases(Shang Han Lun), has been included in the Catalogue of Ancient Classic Prescriptions(the First Batch). Consisting of Euodiae Fructus, Ginseng Radix et Rhizoma, Zingiberis Rhizoma Recens, and Jujubae Fructus, it is effective in warming the middle, tonifying deficiency, dispelling cold, and descending adverse Qi, and is widely applied clinically with remarkable efficacies. For a classical formula, the chemical composition is the material basis and an important premise for quantity value transfer. This study aimed to establish a rapid identification method of chemical components in Wuzhuyu Decoction by high-resolution mass spectrometry(HR-MS) and molecular network. AQUITY UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 µm) was used for sample separation, and acetonitrile-0.1% formic acid in water was used as mobile phases for gradient elution. Q-Exactive Orbitrap MS data were collected in positive and negative ion modes, and GNPS molecular network was plotted according to the similarity of MS/MS fragmentation modes. Cytoscape 3.6.1 was used to screen molecular clusters with similar structures. Finally, the chemical components of Wuzhuyu Decoction were rapidly identified according to the controls, as well as the information of retention time, accurate relative molecular weight of HR-MS, and MS/MS multistage fragments. A total of 105 chemical components were identified in Wuzhuyu Decoction. This study can provide data for the follow-up quality control, standard substance research, and pharmacodynamic material research on Wuzhuyu Decoction, as well as references for the rapid qualitative analysis of the chemical components of Chinese medicine.

[Plasma components of Danzhi Xiaoyao Formula and its mechanism of action in treating perimenopausal depression based on UPLC-Q-TOF-MS~E integrated with network pharmacology].

In this study, ultra-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS~E) was used to analyze the plasma components of Danzhi Xiaoyao Formula after oral administration. Forty-nine plasma components were found in the serum of rats by comparing the compound extract, drug-containing serum, and blank serum. Components, such as 6-hydroxycoumarin, poricoic acid F, deoxoglabrolide, 30-norhederagenin, kanzonol R, 3',6'-di-O-galloylpaeoniflorin, 16α-hydroxytrametenolic acid, 16-deoxyporicoic acid B, 3-O-acetyl-16α-hydroxytrametenolic acid, and 16α,25-dihydroxydehydroeburiconic acid, were first found in rat serum. Behavioral tests, including the tail suspension test, novel object recognition test, and novelty-suppressed feeding test, were conducted for behavioral analysis. It was confirmed that this formula had therapeutic effects on perimenopausal depression. Furthermore, in combination with the network pharmacology method, 53 core targets including MAPK1, HRAS, AKT1, EGFR, and ESR1 were screened, and these targets participated in 165 signaling pathways, including PI3K-AKT, AMPK, VEGFA, MAPK, and HIF-1. In summary, the potential effects of Danzhi Xiaoyao Formula in treating perimenopausal depression are associated with mechanisms in accelerating inflammation repair, improving neuroplasticity, affecting neurotransmitters, regulating estrogen levels, and promoting new blood vessel formation.

A tomato NAC transcription factor, SlNAM1, positively regulates ethylene biosynthesis and the onset of tomato fruit ripening.

Fruit ripening in tomato (Solanum lycopersicum) is the result of selective expression of ripening-related genes, which are regulated by transcription factors (TFs). The NAC (NAM, ATAF1/2, and CUC2) TF family is one of the largest families of plant-specific TFs and members are involved in a variety of plant physiological activities, including fruit ripening. Fruit ripening-associated NAC TFs studied in tomato to date include NAC-NOR (non-ripening), SlNOR-like1 (non-ripening like1), SlNAC1, and SlNAC4. Considering the large number of NAC genes in the tomato genome, there is little information about the possible roles of other NAC members in fruit ripening, and research on their target genes is lacking. In this study, we characterize SlNAM1, a NAC TF, which positively regulates the initiation of tomato fruit ripening via its regulation of ethylene biosynthesis. The onset of fruit ripening in slnam1-deficient mutants created by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) technology was delayed, whereas fruit ripening in OE-SlNAM1 lines was accelerated compared with the wild type. The results of RNA-sequencing (RNA-seq) and promoter analysis suggested that SlNAM1 directly binds to the promoters of two key ethylene biosynthesis genes (1-aminocyclopropane-1-carboxylate synthase: SlACS2 and SlACS4) and activates their expression. This hypothesis was confirmed by electrophoretic mobility shift assays and dual-luciferase reporter assay. Our findings provide insights into the mechanisms of ethylene production and enrich understanding of the tomato fruit ripening regulatory network.

Cholesterol promotes EGFR-TKIs resistance in NSCLC by inducing EGFR/Src/Erk/SP1 signaling-mediated ERRα re-expression.

BACKGROUND: The use of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) brings remarkable benefits for the survival of patients with advanced NSCLC harboring EGFR mutations. Unfortunately, acquired resistance seems to be inevitable and limits the application of EGFR-TKIs in clinical practice. This study reported a common molecular mechanism sustaining resistance and potential treatment options to overcome EGFR-TKIs resistance. METHODS: EGFR-TKIs resistant NSCLC cells were established and confirmed by MTT assay. Cholesterol content was detected and the promotional function of cholesterol on NSCLC growth was determined in vivo. Then, we identified ERRα expression as the downstream factor of cholesterol-mediated drug resistance. To dissect the regulatory mechanism, we conducted experiments, including immunofluorescence, co-immunoprecipitation, luciferase reporter assay and chromatin immunoprecipitation assay. RESULTS: Long-term exposure to EGFR-TKIs generate drug resistance with the characteristic of cholesterol accumulation in lipid rafts, which promotes EGFR and Src to interact and lead EGFR/Src/Erk signaling reactivation-mediated SP1 nuclear translocation and ERRα re-expression. Further investigation identifies ERRα as a target gene of SP1. Functionally, re-expression of ERRα sustains cell proliferation by regulating ROS detoxification process. Lovastatin, a drug used to decrease cholesterol level, and XCT790, an inverse agonist of ERRα, overcome gefitinib and osimertinib resistance both in vitro and in vivo. CONCLUSIONS: Our study indicates that cholesterol/EGFR/Src/Erk/SP1 axis-induced ERRα re-expression promotes survival of gefitinib and osimertinib-resistant cancer cells. Besides, we demonstrate the potential of lowing cholesterol and downregulation of ERRα as effective adjuvant treatment of NSCLC.

CDK6-PI3K signaling axis is an efficient target for attenuating ABCB1/P-gp mediated multi-drug resistance (MDR) in cancer cells.

BACKGROUND: Multidrug resistance (MDR) mediated by ATP binding cassette subfamily B member 1 (ABCB1/P-gp) is a major cause of cancer chemotherapy failure, but the regulation mechanisms are largely unknown. METHODS: Based on single gene knockout, we studied the regulation of CDK6-PI3K axis on ABCB1-mediated MDR in human cancer cells. CRISPR/Cas9 technique was performed in KB-C2 cells to knockout cdk6 or cdk4 gene. Western blot, RT-PCR and transcriptome analysis were performed to investigate target gene deletion and expression of critical signaling factors. The effect of cdk4 or cdk6 deficiency on cell apoptosis and the cell cycle was analyzed using flow cytometry. In vivo studies were performed to study the sensitivity of KB-C2 tumors to doxorubicin, tumor growth and metastasis. RESULTS: Deficiency of cdk6 led to remarkable downregulation of ABCB1 expression and reversal of ABCB1-mediated MDR. Transcriptomic analysis revealed that CDK6 knockout regulated a series of signaling factors, among them, PI3K 110α and 110ß, KRAS and MAPK10 were downregulated, and FOS-promoting cell autophagy and CXCL1-regulating multiple factors were upregulated. Notably, PI3K 110α/110ß deficiency in-return downregulated CDK6 and the CDK6-PI3K axis synergizes in regulating ABCB1 expression, which strengthened the regulation of ABCB1 over single regulation by either CDK6 or PI3K 110α/110ß. High frequency of alternative splicing (AS) of premature ABCB1 mRNA induced by CDK6, CDK4 or PI3K 110α/110ß level change was confirmed to alter the ABCB1 level, among them 10 common skipped exon (SE) events were found. In vivo experiments demonstrated that loss of cdk6 remarkably increased the sensitivity of KB-C2 tumors to doxorubicin by increasing drug accumulation of the tumors, resulting in remarkable inhibition of tumor growth and metastasis, as well as KB-C2 survival in the nude mice. CONCLUSIONS: CDK6-PI3K as a new target signaling axis to reverse ABCB1-mediated MDR is reported for the first time in cancers. Pathways leading to inhibition of cancer cell proliferation were revealed to be accompanied by CDK6 deficiency.

The prognostic nutritional index predicts the biochemical recurrence of patients treated with robot-assisted laparoscopic radical prostatectomy.

OBJECTIVE: To evaluate the prognostic nutritional index (PNI) in predicting the biochemical recurrence (BCR) of patients treated with robot-assisted laparoscopic radical prostatectomy (RALP). METHODS: The clinical data of 136 patients treated with RALP in the Department of Urology, The Third Xiangya Hospital of Central South University were retrospectively analyzed. The endpoint of observation was BCR. The area under the receiver operating characteristic (ROC) curve was evaluated to determine the optimal cutoff value of PNI. The correlation of the PNI with BCR was estimated using Kaplan-Meier analysis and Cox proportional hazards model. RESULTS: The optimal cutoff value of the PNI was 46.03 according to the ROC curve. (95% confidence interval: 0.604-0.805, Youden index = 0.401, sensitivity = 82.5%, specificity = 57.6%, p < 0.01). Multivariate Cox analysis showed that clinical staging, prostate-specific antigen, and PNI were independent prognostic factors for predicting BCR in patients treated with RALP. CONCLUSION: PNI is an independent prognostic factor for predicting BCR in patients treated with RALP. The incorporation of the PNI into risk assessments may provide additional prognostic information.

GAF domain is essential for nitrate-dependent AtNLP7 function.

Nitrate is an essential nutrient and an important signaling molecule in plants. However, the molecular mechanisms by which plants perceive nitrate deficiency signaling are still not well understood. Here we report that AtNLP7 protein transport from the nucleus to the cytoplasm in response to nitrate deficiency is dependent on the N-terminal GAF domain. With the deletion of the GAF domain, AtNLP7ΔGAF always remains in the nucleus regardless of nitrate availability. AtNLP7 ΔGAF also shows reduced activation of nitrate-induced genes due to its impaired binding to the nitrate-responsive cis-element (NRE) as well as decreased growth like nlp7-1 mutant. In addition, AtNLP7ΔGAF is unable to mediate the reduction of reactive oxygen species (ROS) accumulation upon nitrate treatment. Our investigation shows that the GAF domain of AtNLP7 plays a critical role in the sensing of nitrate deficiency signal and in the nitrate-triggered ROS signaling process.

Sex-related differences in safety profiles, pharmacokinetics and tissue distribution of sinomenine hydrochloride in rats.

Sinomenine is a bioactive alkaloid isolated from the Chinese medicinal plant Sinomenium acutum (Thunb.) Rehd. et Wils which exhibits significant analgesic, anti-inflammatory, and immunosuppressive effects. Sinomenine hydrochloride (SH) preparations, classified as natural disease-modifying antirheumatic drugs, are currently available for the treatment of rheumatoid arthritis and other rheumatic diseases. Our toxicity evaluation demonstrated that the median lethal dose of SH in female Sprague-Dawley (SD) rats was over 11 times greater than that in male SD rats, revealing striking sex-linked differences in the safety profile of SH. The present study was designed to investigate differences in the pharmacokinetics (PKs) and tissue distribution of SH between male and female SD rats after a single oral dose of 25 mg/kg. PK and tissue distribution studies were performed using a validated UPLC-MS/MS method. The results showed that SH-treated SD female rats displayed markedly greater drug exposure, and SH exhibited a longer half-life and slower clearance rate than comparable studies in male rats. Moreover, the tissue distribution study confirmed that the sinomenine concentration in female rats was considerably greater in the internal organs than in male rats. Our study demonstrates, for the first time, significant sex-related differences in the safety profile and PKs of SH, which may be associated with a distinct sex-dependent metabolic mechanism of sinomenine.

Presence of secondary bladder cancer following radical nephroureterectomy for upper tract urothelial carcinoma: characteristics, risk factors, and predictive value.

BACKGROUND: To assess the characteristics, predictive risk factors, and prognostic effect of secondary bladder cancer (SBCa) following radical nephroureterectomy (RNU) in upper tract urothelial carcinoma (UTUC). METHODS: Using the Surveillance, Epidemiology, and End Results database, the authors analyzed clinicopathologic characteristics and survival data from 472 UTUC patients with SBCa after RNU, between 2004 and 2017. Cox's proportional hazard regression model was implemented to identify independent predictors associated with post-recurrence outcomes. The threshold for statistical significance was p < 0.05. RESULTS: In total, 200 Ta-3N0M0 localized UTUC patients with complete data were finally included. With a median follow-up of 71.0 months (interquartile ranges [IQR] 36.0 -103.8 months), 52.5% (n = 105) had died, with 30.5% (n = 61) dying of UTUC. The median time interval from UTUC to SBCa was 13.5 months (IQR 6.0-40.8 months). According to multivariable Cox regression analysis, patients with SBCa located at multiple sites, advanced SBCa stage, higher SBCa grade, elderly age and a shorter recurrence time, encountered worse cancer-specific survival (CSS), all p < 0.05. CONCLUSION: For primary UTUC patients with SBCa after radical surgery, advanced age, multiple SBCa sites, shorter recurrence time, higher SBCa stage, and grade proved to be significant independent prognostic factors of CSS. We ought to pay more attention to SBCa prevention as well as to earlier signs which may increase the likelihood of early detection. Having the ability to manage what may be seen as the superficial SBCa signs may enable us to improve survival but further research is required.