Comparative transcriptome analysis and identification of candidate R2R3-MYB genes involved in anthraquinone biosynthesis in Rheum palmatum L.

BACKGROUND: Rheum palmatum L. has important medicinal value because it contains biologically active anthraquinones. However, the key genes and TFs involved in anthraquinone biosynthesis and regulation in R. palmatum remain unclear. METHODS: Based on full length transcriptome data, in this study, we screened the differentially expressed genes in the anthraquinone biosynthesis pathway. The R2R3-MYB family genes of R. palmatum were systematically identified based on full-length transcriptome sequencing followed by bioinformatics analyses. The correlation analysis was carried out by using co-expression analysis, protein interaction analysis, and real-time fluorescence quantitative analysis after MeJA treatment. The RpMYB81 and RpMYB98 genes were amplified by RT-PCR, and their subcellular localization and self-activation characteristics were analyzed. RESULTS: Comparative transcriptome analysis results revealed a total of 3525 upregulated and 6043 downregulated DEGs in the CK versus MeJA group; 28 DEGs were involved in the anthraquinone pathway. Eleven CHS genes that belonged to the PKS family were differentially expressed and involved in anthraquinone biosynthesis. Twelve differentially expressed MYBs genes were found to be co-expressed and interact with CHS genes. Furthermore, 52 MYB genes were identified as positive regulators of anthraquinone biosynthesis and were further characterized. Three MYB genes including RpMYB81, RpMYB98, and RpMYB100 responded to MeJA treatment in R. palmatum, and the levels of these genes were verified by qRT-PCR. RpMYB81 was related to anthraquinone biosynthesis. RpMYB98 had an interaction with genes in the anthraquinone biosynthesis pathway. RpMYB81 and RpMYB98 were mainly localized in the nucleus. RpMYB81 had self-activation activity, while RpMYB98 had no self-activation activity. CONCLUSION: RpMYB81, RpMYB98, and RpMYB100 were significantly induced by MeJA treatment. RpMYB81 and RpMYB98 are located in the nucleus, and RpMYB81 has transcriptional activity, suggesting that it might be involved in the transcriptional regulation of anthraquinone biosynthesis in R. palmatum.

Analysis of clinical characteristics of 66 pediatric patients with B.1.617.2 (Delta) variant of COVID-19.

BACKGROUND: The aim of this study was to analyze the clinical characteristics of 66 pediatric patients with B.1.617.2 (Delta) variant of coronavirus disease 2019 (COVID-19). METHODS: Sixty-six pediatric patients with B.1.617.2 (Delta) variant of COVID-19 admitted to the hospital from July to August 2021 were classified into mild (n = 41) and moderate groups (n = 25). Clinical characteristics, laboratory data and dynamic trends in different time periods were analyzed retrospectively. RESULTS: There were no statistically significant differences in age, gender ratios and clinical symptoms between the mild group and the moderate group. All the patients in the moderate group had clusters of onsets, and the incubation period was shorter than that of the mild group. Within 24 hours of admission, the levels of erythrocyte sedimentation rate, cardiac troponin I, D-dimer in the moderate group were higher than that in the mild group (P < 0.05). The titers of immunoglobulin (Ig) G and IgM antibodies gradually increased after disease onset. Thirty-five (53.03%) children were tested positive for antibodies in 4-12 days. IgG increased gradually, while IgM decreased obviously in about 15 days after disease onset. The cycle threshold values of open reading frame 1ab and nucleocapsid protein gene in the severe acute respiratory syndrome coronavirus 2 genomes increased gradually on the 3rd, 6th, 9th, and 12th days after disease onset, compared with those in day 0. CONCLUSIONS: The symptoms of children with B.1.617.2 (Delta) variant of COVID-19 were mild. The description and analysis of the clinical characteristics and laboratory data can help medical staff to evaluate the condition of children with COVID-19 and to accumulate more clinical experience.

Chest CT features of children infected by B.1.617.2 (Delta) variant of COVID-19.

BACKGROUND: This study aimed to explore the imaging characteristics, diversity and changing trend in CT scans of pediatric patients infected with Delta-variant strain by studying imaging features of children infected with Delta and comparing the results to those of children with original COVID-19. METHODS: A retrospective, comparative analysis of initial chest CT manifestations between 63 pediatric patients infected with Delta variant in 2021 and 23 pediatric patients with COVID-19 in 2020 was conducted. Corresponding imaging features were analyzed. In addition, the changing trend in imaging features of COVID-19 Delta-variant cases were explored by evaluating the initial and follow-up CT scans. RESULTS: Among 63 children with Delta-variant COVID-19 in 2021, 34 (53.9%) showed positive chest CT presentation; and their CT score (1.10 ± 1.41) was significantly lower than that in 2020 (2.56 ± 3.5) (P = 0.0073). Lesion distribution: lung lesions of Delta cases appear mainly in the lower lungs on both sides. Most children had single lobe involvement (18 cases, 52.9%), 14 (41.2%) in the right lung alone, and 14 (41.2%) in both lungs. A majority of Delta cases displayed initially ground glass (23 cases, 67.6%) and nodular shadows (13 cases, 38.2%) in the first CT scan, with few extrapulmonary manifestations. The 34 children with abnormal chest CT for the first time have a total of 92 chest CT examinations. These children showed a statistically significant difference between the 0-3 day group and the 4-7 day group (P = 0.0392) and a significant difference between the 4-7 day group and the more than 8 days group (P = 0.0003). CONCLUSIONS: The early manifestations of COVID-19 in children with abnormal imaging are mostly small subpleural nodular ground glass opacity. The changes on the Delta-variant COVID-19 chest CT were milder than the original strain. The lesions reached a peak on CT in 4-7 days and quickly improved and absorbed after a week. Dynamic CT re-examination can achieve a good prognosis.

Cell-free DNA in blood and urine as a diagnostic tool for bladder cancer: a meta-analysis.

OBJECTIVE: To assess the diagnostic performance of cell-free DNA assays in the detection of bladder cancer. PATIENTS AND METHODS: The quality of the studies included in this meta-analysis was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Statistical analyses were performed using the software RevMan 5.3 and Stata 14.0. We assessed the pooled sensitivity and specificity, positive/negative likelihood ratios (PLRs/NLRs), diagnostic odds ratios (DORs), and corresponding 95% confidence intervals (95% CIs). Summary receiver operating characteristic curve (ROC curve) and area under the curve (AUC) were used to summarize the overall test performance. Heterogeneity and publication bias were also examined. RESULTS: Eleven studies included 802 bladder cancer patients and 668 controls met the eligibility criteria. The overall diagnostic accuracy was measured as follows: sensitivity 0.71 (95% CI = 0.64-0.77), specificity 0.78 (95% CI = 0.70-0.85), PLR 3.3 (95% CI = 2.4-54.5), NLR 0.37 (95% CI = 0.30-0.46), DOR 9 (95% CI = 6-14), and AUC 0.80 (95% CI = 0.77-0.83). Subgroup analysis suggested that ethnicity significantly accounted for the heterogeneity of specificity. The Deeks' funnel plot asymmetry test (P = 0.97) suggested no potential publication bias. CONCLUSIONS: Cell-free DNA has a high diagnostic value in bladder cancer.

A highly integrated precision nanomedicine strategy to target esophageal squamous cell cancer molecularly and physically.

The prognosis of esophageal squamous cell carcinoma is poor. We hereby presented a highly integrated and clinically relevant precision nanomedicine strategy to target ESCC molecularly and physically for significant improvement of the treatment efficacy. We firstly identified PI3K overexpression in patient samples and its relation to poor patient survival. With our highly versatile tumor-targeted drug delivery platform (DCM), we were able to load a potent but toxic docetaxel (DTX) and a PI3K inhibitor (AZD8186) with favorable physical properties. The combination of the DTX-DCM and AZD8186-DCM showed a highly efficacious and synergistic anti-tumor effect and decreased hematotoxicity. A pro-apoptotic protein, Bax was significantly upregulated in ESCC cells treated with combination therapy compared to that with monotherapy. This study utilized a highly integrated precision nano-medicine strategy that combines the identification of cancer molecular target from human patients, precision drug delivery and effective combination therapy for the development of better ESCC treatment.

Nanoformulated paclitaxel and AZD9291 synergistically eradicate non-small-cell lung cancers in vivo.

AIM: This study aims to develop new nanoformulations of EGFR T790M targeted inhibitor AZD9291 and paclitaxel (PTX) for combination therapy of lung cancer. MATERIALS & METHODS: We prepared and characterized PTX- and AZD9291-loaded disulfide cross-linking micelles (DCMs), and evaluate their combination effect and toxicity in vitro and in lung cancer-bearing mice. RESULTS: Drug-loaded DCMs were relatively small in size, and possessed glutathione-responsive drug release. The combination of PTX-DCMs and AZD92921-DCMs exhibited strong synergistic effects in both cell line and in vivo without additional toxicity. Molecular studies demonstrated the synergistic modification in both IKB-α/NF-κB/Bcl-2 and EGFR/Akt pathways. CONCLUSION: The combination of DCM-loaded AZD9291 and PTX could potentially offer more effective and less toxicity treatment options for lung cancer patients.

A versatile nanoplatform for synergistic combination therapy to treat human esophageal cancer.

One of the major goals of precision oncology is to promote combination therapy to improve efficacy and reduce side effects of anti-cancer drugs based on their molecular mechanisms. In this study, we aimed to develop and validate new nanoformulations of docetaxel (DTX) and bortezomib (BTZ) for targeted combination therapy to treat human esophageal cancer. By leveraging our versatile disulfide cross-linked micelles (DCMs) platform, we developed nanoformulations of DTX and BTZ (named DTX-DCMs and BTZ-DCMs). Their physical properties were characterized; their anti-cancer efficacies and mechanisms of action were investigated in a human esophageal cancer cell line in vitro. Furthermore, the in vitro anti-tumor activities of combination therapies (concurrent drug treatment, sequential drug treatment, and treatment using different ratios of the drugs) were examined in comparison with the single drug treatment and free drug strategies. These drug-loaded nanoparticles were spherical in shape and relatively small in size of approximately 20-22 nm. The entrapment efficiencies of DTX and BTZ into nanoparticles were 82.4% and 84.1%, respectively. The drug release rates of DTX-DCMs and BTZ-DCMs were sustained, and greatly increased in the presence of GSH. These nanodrugs were effectively internalized by KYSE30 esophageal cancer cells, and dose-dependently induced cell apoptosis. We further revealed a strong synergistic effect between DTX-DCMs and BTZ-DCMs against KYSE30 esophageal cancer cells. Sequential combination therapy with DTX-DCMs followed by BTZ-DCMs exhibited the best anti-tumor efficacy in vitro. This study demonstrates that DTX and BTZ could be successfully nanoformulated into disulfide cross-linked micelles. The nanoformulations of DTX and BTZ demonstrate an immense potential for synergistic combination therapy to treat human esophageal cancer.

Prognostic significance of PD-L1 expression in patients with gastric cancer in East Asia: a meta-analysis.

The overexpression of programmed cell death-ligand 1(PD-L1) has been observed in gastric cancer (GC). However, whether the expression of PD-L1 in tumor cells or blood serum is associated with the prognosis of patients with GC remains unclear. Therefore, we performed a meta-analysis to evaluate the prognostic significance of PD-L1 expression in GC. Electronic databases were searched systematically. Studies that met the inclusion criteria were included in the meta-analysis. Data concerning the hazard ratio (HR) for overall survival and disease-free survival with a 95% confidence interval (CI) according to the expression status of PD-L1 evaluated by immunohistochemistry or enzyme-linked immunosorbent assay were extracted. The data were analyzed using a random effects model. Subgroup analyses were proposed. Our results showed that eight studies with 950 patients met the inclusion criteria and were included in the meta-analysis. The pooled HR for overall survival indicated that patients with PD-L1-positive expression had significantly shorter survival time compared with the PD-L1-negative group (HR 1.60, 95% CI 1.09-2.36, P=0.012). The pooled HR for disease-free survival demonstrated that the difference between the two groups was not statistically significant (HR 1.02, 95% CI 0.32-3.20, P=0.98). In conclusion, our results indicate that the evaluation of PD-L1 overexpression in GC tissue or blood serum may be useful in the future as a novel prognostic factor.

Effect of alpha-lipoic acid on boar spermatozoa quality during freezing-thawing.

Alpha-lipoic acid (ALA) is known to be a natural antioxidant. The aim of the present study was to evaluate the cryoprotective effect of ALA on the motility of boar spermatozoa and its antioxidant effect on boar spermatozoa during freezing-thawing. Different concentrations (2.0, 4.0, 6.0, 8.0 or 10.0 mg/ml) of ALA were added to the extender used to freeze boar semen, and the effects on the quality and endogenous antioxidant enzyme activities of frozen-thawed spermatozoa were assessed. The results indicated that the addition of ALA to the extender resulted in a higher percentage of motile spermatozoa post-thaw (P < 0.05). The activities of superoxide dismutase, lactate dehydrogenase, glutamic-oxaloacetic transaminase and catalase improved after adding ALA to the extender (P < 0.05). Artificial insemination results showed that pregnancy rate and litter size were significantly higher at 6.0 mg/ml in the ALA group than in the control group (P < 0.05). In conclusion, ALA conferred a cryoprotective capacity to the extender used for boar semen during the process of freezing-thawing, and the optimal concentration of ALA for the frozen extender was 6.0 mg/ml.

Effects of vitexin on the pharmacokinetics and mRNA expression of CYP isozymes in rats.

In traditional therapy with Chinese medicine, vitexin has several pharmacological properties, including antinociceptive, antispasmodic, antioxidant, antimyeloperoxidase, and α-glucosidase inhibitory activities. Recently, vitexin was shown to protect the heart against ischemia/reperfusion injury in an in vitro model by inhibiting apoptosis. The purpose of this study was to find out whether vitexin influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, and CYP3A1) by using cocktail probe drugs in vivo; the influence on the levels of CYP mRNA was also studied. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (10 mg/kg), tolbutamide (1 mg/kg), and midazolam (5 mg/kg), was given as oral administration to rats treated with short or long period of intravenous vitexin via the caudal vein. Blood samples were collected at a series of time points, and the concentrations of probe drugs in plasma were determined by HPLC-mass spectrometry (MS)/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. In addition, real-time reverse transcription-polymerase chain reaction was performed to determine the effects of vitexin on the mRNA expression of CYP1A2, CYP2C11, and CYP3A1 in rat liver. Treatment with short or long period of vitexin had no effects on rat CYP1A2. However, CYP3A1 enzyme activity was inhibited by vitexin in a concentration-dependent and time-dependent manner. Furthermore, CYP2C11 enzyme activity was induced after short period treatment but inhibited after long period of vitexin treatment. The mRNA expression results were in accordance with the pharmacokinetic results. In conclusion, vitexin can either inhibit or induce activities of CYP2C11 and CYP3A1. Therefore, caution is needed when vitexin is co-administered with some CYP2C11 or CYP3A1 substrates in clinic, which may result in treatment failure and herb-drug interactions.

Activation of AKT/ERK confers non-small cell lung cancer cells resistance to vinorelbine.

Vinorelbine is a semi-synthetic vinca-alkaloid approved for the treatment of non-small cell lung cancer (NSCLC). However, the lower objective response rate and higher adverse effects of vinorelbine hinder its wide use in treatment of advanced NSCLC. Therefore, it is of great interest to uncover the biomarkers for sensitivity of NSCLC cells to vinorelbine to allow the identification of patients most likely to benefit from vinorelbine-based chemotherapy and to improve the therapy. In present work, four NSCLC cell lines were divided into vinorelbine-sensitive (VS) group and vinorelbine-resistant (VR) group according to their sensitivities to vinorelbine. And then the gene expression profiles of these two groups was compared, the differentially expressed genes (expression difference higher than 100% and p<0.05, totally 496 genes) were applied to Ingenuity Pathway Analysis (IPA). IPA results showed that NF-κB and PTEN signaling were predicted to be inactivated in VR cell lines, which was partially validated by quantitative PCR or western blotting experiments. The higher expression of RAF1 mRNA and the activation of AKT/ERK proteins in VR NSCLC cell lines may confer resistance to vinorelbine. Our work may provide potential pathway signature for vinorelbine sensitivity and some therapeutic targets for combined therapy.

Cryoprotective effects of low-density lipoproteins, trehalose and soybean lecithin on murine spermatogonial stem cells.

Spermatogonial stem cells (SSCs) have the ability to self-renew and offer a pathway for genetic engineering of the male germ line. Cryopreservation of SSCs has potential value for the treatment of male infertility, spermatogonial transplantation, and so on. In order to investigate the cryopreservation effects of different cryoprotectants on murine SSCs, 0.2 M of low-density lipoproteins (LDL), trehalose and soybean lecithin were added to the cryoprotective medium, respectively, and the murine SSCs were frozen at -80°C or -196°C. The results indicated that the optimal recovery rates of murine SSCs in the cryoprotective medium supplemented with LDL, trehalose and soybean lecithin were 92.53, 76.35 and 75.48% at -80°C, respectively. Compared with freezing at -196°C, the optimum temperature for improvement of recovery rates of frozen murine SSCs, cryopreservation in three different cryoprotectants at -80°C, were 17.11, 6.68 and 10.44% respectively. The recovery rates of murine SSCs in the cryoprotective medium supplemented with 0.2 M LDL were significantly higher than that of other cryoprotectants (P < 0.05). Moreover, the recovery rates were demonstrated to be greater at -80°C compared with at -196°C (P < 0.05). In conclusion, 0.2 M of LDL could significantly protect murine SSCs at -80°C. In the freezing-thawing process, LDL is responsible for the cryopreservation of murine SSCs because it can form a protective film at the surface of membranes. However, more research is needed to evaluate and understand the precise role of LDL during the freezing-thawing of SSCs.

The effect of Laminaria japonic polysaccharide on sperm characteristics and biochemical parameters in cryopreserved boar sperm.

The aim of the present study was to evaluate the cryoprotective effect of Laminaria japonic polysaccharide (LJP) on boar sperm. Semen samples were collected from seven mature Yorkshire boars once a week by the gloved hand technique and frozen-thawed in the extender with LJP added. Extender with LJP added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0mg/mL to the extender and its effects on the quality of frozen-thawed boar sperm were assessed. Results showed: (i) sperm motility and plasma membrane integrity were greater in the extender containing 0.5 and 1.0mg/mL LJP, as compared to other groups (P<0.05); (ii) extender added 1.0mg/mL LJP showed the greatest plasma membrane and acrosomal integrity percentages in comparison with other groups (P<0.05); (iii) mitochondrial activity was significantly higher at the concentration of 0.5 and 1.0mg/mL LJP than those of other groups (P<0.05); (iv) in terms of biochemical assessments, 0.5 and 1.0mg/mL LJP improved SOD (superoxide dismutase) and CAT (catalase) concentrations, compared to other groups (P<0.05). However, no significant difference was found in GSH-Px (glutathione peroxidase) concentration when supplemented with LJP. Interestingly, LJP exhibited a dose-related response and the lesser concentration represented greater protective effects. It is also important to note that 1.0mg/mL LJP provides for an enhanced cryoprotective effect in boar semen.

Involvement of calpain/p35-p25/Cdk5/NMDAR signaling pathway in glutamate-induced neurotoxicity in cultured rat retinal neurons.

We investigated possible involvement of a calpain/p35-p25/cyclin-dependent kinase 5 (Cdk5) signaling pathway in modifying NMDA receptors (NMDARs) in glutamate-induced injury of cultured rat retinal neurons. Glutamate treatment decreased cell viability and induced cell apoptosis, which was accompanied by an increase in Cdk5 and p-Cdk5(T15) protein levels. The Cdk5 inhibitor roscovitine rescued the cell viability and inhibited the cell apoptosis. In addition, the protein levels of both calpain 2 and calpain-specific alpha-spectrin breakdown products (SBDPs), which are both Ca(2+)-dependent, were elevated in glutamate-induced cell injury. The protein levels of Cdk5, p-Cdk5(T15), calpain 2 and SBDPs tended to decline with glutamate treatments of more than 9 h. Furthermore, the elevation of SBDPs was attenuated by either D-APV, a NMDAR antagonist, or CNQX, a non-NMDAR antagonist, but was hardly changed by the inhibitors of intracellular calcium stores dantrolene and xestospongin. Moreover, the Cdk5 co-activator p35 was significantly up-regulated, whereas its cleaved product p25 expression showed a transient increase. Glutamate treatment for less than 9 h also considerably enhanced the ratio of the Cdk5-phosphorylated NMDAR subunit NR2A at Ser1232 site (p-NR2A(S1232)) and NR2A (p-NR2A(S1232)/NR2A), and caused a translocation of p-NR2A(S1232) from the cytosol to the plasma membrane. The enhanced p-NR2A(S1232) was inhibited by roscovitine, but augmented by over-expression of Cdk5. Calcium imaging experiments further showed that intracellular Ca(2+) concentrations ([Ca(2+)](i)) of retinal cells were steadily increased following glutamate treatments of 2 h, 6 h and 9 h. All these results suggest that the activation of the calpain/p35-p25/Cdk5 signaling pathway may contribute to glutamate neurotoxicity in the retina by up-regulating p-NR2A(S1232) expression.