Synergistic lethality in chronic myeloid leukemia - targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment.

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.

Circular RNA CDR1as disrupts the p53/MDM2 complex to inhibit Gliomagenesis.

BACKGROUND: Inactivation of the tumor suppressor p53 is critical for pathogenesis of glioma, in particular glioblastoma multiforme (GBM). MDM2, the main negative regulator of p53, binds to and forms a stable complex with p53 to regulate its activity. Hitherto, it is unclear whether the stability of the p53/MDM2 complex is affected by lncRNAs, in particular circular RNAs that are usually abundant and conserved, and frequently implicated in different oncogenic processes. METHODS: RIP-seq and RIP-qPCR assays were performed to determine the most enriched lncRNAs (including circular RNAs) bound by p53, followed by bioinformatic assays to estimate the relevance of their expression with p53 signaling and gliomagenesis. Subsequently, the clinical significance of CDR1as was evaluated in the largest cohort of Chinese glioma patients from CGGA (n = 325), and its expression in human glioma tissues was further evaluated by RNA FISH and RT-qPCR, respectively. Assays combining RNA FISH with protein immunofluorescence were performed to determine co-localization of CDR1as and p53, followed by CHIRP assays to confirm RNA-protein interaction. Immunoblot assays were carried out to evaluate protein expression, p53/MDM2 interaction and p53 ubiquitination in cells in which CDR1as expression was manipulated. After AGO2 or Dicer was knocked-down to inhibit miRNA biogenesis, effects of CDR1as on p53 expression, stability and activity were determined by immunoblot, RT-qPCR and luciferase reporter assays. Meanwhile, impacts of CDR1as on DNA damage were evaluated by flow cytometric assays and immunohistochemistry. Tumorigenicity assays were performed to determine the effects of CDR1as on colony formation, cell proliferation, the cell cycle and apoptosis (in vitro), and on tumor volume/weight and survival of nude mice xenografted with GBM cells (in vivo). RESULTS: CDR1as is found to bind to p53 protein. CDR1as expression decreases with increasing glioma grade and it is a reliable independent predictor of overall survival in glioma, particularly in GBM. Through a mechanism independent of acting as a miRNA sponge, CDR1as stabilizes p53 protein by preventing it from ubiquitination. CDR1as directly interacts with the p53 DBD domain that is essential for MDM2 binding, thus disrupting the p53/MDM2 complex formation. Induced upon DNA damage, CDR1as may preserve p53 function and protect cells from DNA damage. Significantly, CDR1as inhibits tumor growth in vitro and in vivo, but has little impact in cells where p53 is absent or mutated. CONCLUSIONS: Rather than acting as a miRNA sponge, CDR1as functions as a tumor suppressor through binding directly to p53 at its DBD region to restrict MDM2 interaction. Thus, CDR1as binding disrupts the p53/MDM2 complex to prevent p53 from ubiquitination and degradation. CDR1as may also sense DNA damage signals and form a protective complex with p53 to preserve p53 function. Therefore, CDR1as depletion may play a potent role in promoting tumorigenesis through down-regulating p53 expression in glioma. Our results broaden further our understanding of the roles and mechanism of action of circular RNAs in general and CDR1as in particular, and can potentially open up novel therapeutic avenues for effective glioma treatment.

Differential Proteomics Analysis Unraveled Mechanisms of Arma chinensis Responding to Improved Artificial Diet.

The development of artificial diets could considerably simplify and reduce the cost of mass rearing of natural enemies compared to conventional rearing methods. However, improvement of artificial diets can be tedious, convoluted and often uncertain. For accelerating diet development, a better method that can offer informative feedback to target deficiencies in diet improvement is required. Our previous research demonstrated several biological characteristics were diminished in the insect predator, Arma chinensis Fallou, fed on an artificial diet formulated with the aid of transcriptomic methods compared to the Chinese oak silk moth pupae. The present study reports differential proteomic analysis by iTRAQ-PRM, which unravels the molecular mechanism of A. chinensis responding to improvements in the artificial diet. Our study provides multivariate proteomic data and provides comprehensive sequence information in studying A. chinensis. Further, the physiological roles of the differentially expressed proteins and pathways enable us to explain several biological differences between natural prey-fed and improved diet-fed A. chinensis, and subsequent proposed reformulation optimizations to artificial diets.

Effect of Prey Species and Prey Densities on the Performance of Adult Coenosia attenuata.

Mass production of Coenosia attenuata Stein at low cost is very important for their use as a biological control agent. The present study reports the performance of C. attenuata adults when reared on Drosophila melanogaster Meigen or Bradysia impatiens (Johannsem). Different densities (6, 9, 15, 24 and 36 adults per predator) of D. melanogaster or (6, 12, 24, 36 and 48 adults per predator) of B. impatiens were used at 26 ± 1 °C, 14:10 (L:D) and 70 ± 5% RH. The results concluded that C. attenuata adults had higher fecundity, longer longevity and less wing damage when reared on B. impatiens adults compared to D. melanogaster adults. Additionally, C. attenuata adults demonstrated greater difficulty catching and carrying heavier D. melanogaster adults than lighter B. impatiens adults. In this case, 12 to 24 adults of B. impatiens daily per predator were considered optimal prey density in the mass rearing of adult C. attenuata.

[Simultaneous Quantitative Detection of Thirteen Common Antibiotics in Leafy Vegetables by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry].

An analytical approach was developed to simultaneously determine 13 antibiotics in sulfonamides, quinolones, and macrolides in leafy vegetables by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS). After optimizing extracted solutions, purification methods, and eluents of antibiotics in vegetable substrates, and taking into account the influence of environmental changes and experimental conditions on the results, the optimal experimental scheme was determined. This involved ①weighing 500 mg of vegetable samples and adding 20 mL of methanol-Mcllvaine-Na2 EDTA solution; ② conducting ultrasonic and centrifugal extraction three times; ③ Allowing rotary evaporation to 20 mL to pass a HLB solid phase extraction column; ④ Eluting the extraction column using 6 mL of methanol, upon which the eluent was dried almost completely; ⑤ Re-dissolving the eluent with a mixed solution of acetonitrile:water (volume ratio of 2:8); ⑥ Detecting by UPLC-MS/MS after centrifugation and filtering. Phase A and B of UPLC-MS/MS used an aqueous solution of 1‰ formic acid and acetonitrile, respectively to conduct gradient elution. Results showed that when the pakchoi spiked at 300 ng·g-1, the spiked recoveries of 13 antibiotics were 38.05%-96.97%. At 150 ng·g-1, the spiked recoveries were 34.52%-111.10%. At 50 ng·g-1, the recoveries of standard addition were 41.75%-107.13%, and the relative deviation (RSD) values were all below 8.68%. The detection limit was 0.4-1 ng·g-1, and the limit of quantification was 1.5-3 ng·g-1. This demonstrated good extraction and recovery efficiency on different types of leafy vegetables, and presented a good analytical application effect. The antibiotic residues were detected in four kinds of leafy vegetables in found in markets. The total content ranged from 1.59 ng·g-1 to 32.01 ng·g-1, and the antibiotic content in samples was calculated by dry weight. The content of antibiotics in pakchoi was the highest, followed by Chinese cabbage, lettuce, and coriander. Among the antibiotics detected, sulfadimidine was the most abundant from the selected leafy vegetables. The content of antibiotics was very low, however the potential health risks caused by long-term consumption could not be ignored.

Nutrigenomics in Arma chinensis: transcriptome analysis of Arma chinensis fed on artificial diet and Chinese oak silk moth Antheraea pernyi pupae.

BACKGROUND: The insect predator, Arma chinensis, is capable of effectively controlling many pests, such as Colorado potato beetle, cotton bollworm, and mirid bugs. Our previous study demonstrated several life history parameters were diminished for A. chinensis reared on an artificial diet compared to a natural food source like the Chinese oak silk moth pupae. The molecular mechanisms underlying the nutritive impact of the artificial diet on A. chinensis health are unclear. So we utilized transcriptome information to better understand the impact of the artificial diet on A. chinensis at the molecular level. METHODOLOGY/PRINCIPAL FINDINGS: Illumina HiSeq2000 was used to sequence 4.79 and 4.70 Gb of the transcriptome from pupae-fed and artificial diet-fed A. chinensis libraries, respectively, and a de novo transcriptome assembly was performed (Trinity short read assembler). This resulted in 112,029 and 98,724 contigs, clustered into 54,083 and 54,169 unigenes for pupae-fed and diet-fed A. chinensis, respectively. Unigenes from each sample's assembly underwent sequence splicing and redundancy removal to acquire non-redundant unigenes. We obtained 55,189 unigenes of A. chinensis, including 12,046 distinct clusters and 43,143 distinct singletons. Unigene sequences were aligned by BLASTx to nr, Swiss-Prot, KEGG and COG (E-value <10(-5)), and further aligned by BLASTn to nt (E-value <10(-5)), retrieving proteins of highest sequence similarity with the given unigenes along with their protein functional annotations. Totally, 22,964, 7,898, 18,069, 15,416, 8,066 and 5,341 unigenes were annotated in nr, nt, Swiss-Prot, KEGG, COG and GO, respectively. We compared gene expression variations and found thousands of genes were differentially expressed between pupae-fed and diet-fed A. chinensis. CONCLUSIONS/SIGNIFICANCE: Our study provides abundant genomic data and offers comprehensive sequence information for studying A. chinensis. Additionally, the physiological roles of the differentially expressed genes enable us to predict effects of some dietary ingredients and subsequently propose formulation improvements to artificial diets.