Integrative multi-omics analysis depicts the methylome and hydroxymethylome in recurrent bladder cancers and identifies biomarkers for predicting PD-L1 expression.

BACKGROUND: Urinary bladder cancer (UBC) is a common malignancy of the urinary tract; however, the mechanism underlying its high recurrence and responses to immunotherapy remains unclear, making clinical outcome predictions difficult. Epigenetic alterations, especially DNA methylation, play important roles in bladder cancer development and are increasingly being investigated as biomarkers for diagnostic or prognostic predictions. However, little is known about hydroxymethylation since previous studies based on bisulfite-sequencing approaches could not differentiate between 5mC and 5hmC signals, resulting in entangled methylation results. METHODS: Tissue samples of bladder cancer patients who underwent laparoscopic radical cystectomy (LRC), partial cystectomy (PC), or transurethral resection of bladder tumor (TURBT) were collected. We utilized a multi-omics approach to analyze both primary and recurrent bladder cancer samples. By integrating various techniques including RNA sequencing, oxidative reduced-representation bisulfite sequencing (oxRRBS), reduced-representation bisulfite sequencing (RRBS), and whole exome sequencing, a comprehensive analysis of the genome, transcriptome, methylome, and hydroxymethylome landscape of these cancers was possible. RESULTS: By whole exome sequencing, we identified driver mutations involved in the development of UBC, including those in FGFR3, KDMTA, and KDMT2C. However, few of these driver mutations were associated with the down-regulation of programmed death-ligand 1 (PD-L1) or recurrence in UBC. By integrating RRBS and oxRRBS data, we identified fatty acid oxidation-related genes significantly enriched in 5hmC-associated transcription alterations in recurrent bladder cancers. We also observed a series of 5mC hypo differentially methylated regions (DMRs) in the gene body of NFATC1, which is highly involved in T-cell immune responses in bladder cancer samples with high expression of PD-L1. Since 5mC and 5hmC alternations are globally anti-correlated, RRBS-seq-based markers that combine the 5mC and 5hmC signals, attenuate cancer-related signals, and therefore, are not optimal as clinical biomarkers. CONCLUSIONS: By multi-omics profiling of UBC samples, we showed that epigenetic alternations are more involved compared to genetic mutations in the PD-L1 regulation and recurrence of UBC. As proof of principle, we demonstrated that the combined measurement of 5mC and 5hmC levels by the bisulfite-based method compromises the prediction accuracy of epigenetic biomarkers.

Acute myocardial infarction due to Kounis syndrome: A case report.

BACKGROUND: Acute myocardial infarction (AMI) can be induced by several factors. However, AMI induced by Kounis syndrome (an allergic reaction) is extremely rare and is highly susceptible to misdiagnosis. CASE SUMMARY: A 70-year-old man presented after suffering an allergic reaction that caused chest pain triggered upon eating ice cream. Troponin I was found to be elevated, and an electrocardiogram showed ST-segment elevation. The diagnosis was AMI. He underwent two coronary angiographies, with one intravascular ultrasound during hospitalization showing no evidence of atherosclerotic coronary artery disease. The final diagnosis was vasospastic myocardial infarction due to Kounis syndrome. The patient was then treated with hydrocortisone and intravenous antihistamines, and his chest pain symptoms resolved. CONCLUSION: Allergic reactions (such as Kounis syndrome) can cause serious damage to the heart. Physicians should be alert to the consequences and avoid misdiagnosis.

G-quadruplex induced chirality of methylazacalix[6]pyridine via unprecedented binding stoichiometry: en route to multiplex controlled molecular switch.

Nucleic acid based molecular device is a developing research field which attracts great interests in material for building machinelike nanodevices. G-quadruplex, as a new type of DNA secondary structures, can be harnessed to construct molecular device owing to its rich structural polymorphism. Herein, we developed a switching system based on G-quadruplexes and methylazacalix[6]pyridine (MACP6). The induced circular dichroism (CD) signal of MACP6 was used to monitor the switch controlled by temperature or pH value. Furthermore, the CD titration, Job-plot, variable temperature CD and (1)H-NMR experiments not only confirmed the binding mode between MACP6 and G-quadruplex, but also explained the difference switching effect of MACP6 and various G-quadruplexes. The established strategy has the potential to be used as the chiral probe for specific G-quadruplex recognition.

Regulating the Conformation of Methylazacalix[6]pyridine by Oligonucleotide BCL-2 2345 and Its Recognition of Hydroxymethylpiperazinofullerene.

Calixaromatics have attracted much attention on molecular recognition owing to their flexible conformations, cavity structures, versatile recognition properties, and functions. However, conformational control of calixaromatics is still a challenging topic in the field of calixaromatics. Therefore, we explore the possibility to control the chirality of achiral calixaromatics, methylazacalix[6]pyridine (abbreviated as MACP6), by templating of DNA. We have found that MACP6 with opposite chirality can be achieved by controlling the secondary structure of bcl-2 2345 DNA. Furthermore, MACP6 with different chirality has been used to recognize fullerene derivatives in aqueous solution. Our results have provided a possible approach to construct chiral calixaromatics.

[Construction of a dps mutant and its functional analysis in Deinococcus radiodurans].

Dps (DNA protection during starvation) is a member of the iron-binding protein family in prokaryotes. It has been shown previously that Dps possesses ferroxidase activity and the ability to sequester iron that seems to protect DNA from oxidative damage. Based on the method of Polymerase Chain Reaction and homologous genetic recombination in vivo, the gene (DRB0092) encoding a Dps protein homology in the extremely radioresistant bacterium Deinococcus radiodurans was deleted from the wild type strain R1 genome. The obtained mutant was designated as Kdps and further verified by PCR and sequencing. Survival rates of the mutant and wild type strain were investigated after challenged with different doses of hydrogen peroxide (H2O2). Results showed that the survival rate of dps mutant reduced rapidly under the low concentration of H2O2 (< or = 10mmol/L), while the wild type strain showed no sudden decrease. When the H2O2 concentration was higher than 30mmol/L, the difference of the survival rates between the mutant and wild type was more than 50-folds. The result demonstrated that the loss of dps gene in D. radiodurans made cells become more sensitive to oxidative damage. An iron staining method was used to determinate catalase activity in native polyacrylamide electrophoresis gels. The result displayed that two catalases in dps mutant were enhanced about 2-folds than that of wild type. The soluble Dps protein was obtained after construction of expression plasmid and inducement in E. coli transformant. The Dps protein showed the capacity of DNA binding and protected DNA from hydroxyl free radical cleavage in vitro. This study demonstrates that Dps protein of D. radiodurans plays an important role in its antioxidant system, which may contribute to its extreme resistance of this bacterium.

[Construction and functional analysis of the crtl gene disruptant in Deinococcus radiodurans].

With the method of Polymerase Chain Reaction and homologous genetic recombination in vivo, the key gene encoding bacterial-type phytoene desaturase (Crtl) which controls the carotenoids biosynthesis pathway in the non-photosynthetic and extremely radioresistant bacterium Deinococcus radiodurans was deleted from the genome. The colorless mutant obtained was designated as M61. Survival rates of mutant strain and wild type strain were investigated under different doses of gamma-radiation and hydrogen peroxide. The results showed that the radioresistant activity of M61 reduced rapidly under ionization radiation, and it became more sensitive to the treatment of hydrogen peroxide especially to high concentration of hydrogen peroxide compared to that of wild type R1. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) was used to investigate the carotenoid composition of wild type R1 and mutant M61. HPLC results exhibited that the deficient of crtl gene had important effect on pigment biosynthesis pathway, leading to inhibition of the biosynthesis of lycopene and other carotenoids in D. radiodurans. All the results indicated that crtl gene was a key gene controlling the biosynthesis of red carotenoid including lycopene in D. radiodurans. The roles of carotenoids in protecting the bacterial cell from damage by ionization radiation and hydrogen peroxide suggest that the carotenoids contribute to the defense system in D. radiodurans. This study is important for elucidating the radioresistant and antioxidant mechanism in which carotenoids are involved, and it will supply some ideas to the further investigation on the biosynthesis pathway and functions of carotenoids in D. radiodurans.