New G-Triplex DNA Dramatically Activates the Fluorescence of Thioflavin T and Acts as a Turn-On Fluorescent Sensor for Uracil-DNA Glycosylase Activity Detection.

G-triplexes are G-rich oligonucleotides composed of three G-tracts and have absorbed much attention due to their potential biological functions and attractive performance in biosensing. Through the optimization of loop compositions, DNA lengths, and 5'-flanking bases of G-rich sequences, a new stable G-triplex sequence with 14 bases (G3-F15) was discovered to dramatically activate the fluorescence of Thioflavin T (ThT), a water-soluble fluorogenic dye. The fluorescence enhancement of ThT after binding with G3-F15 reached 3200 times, which was the strongest one by far among all of the G-rich sequences. The conformations of G3-F15 and G3-F15/ThT were studied by circular dichroism. The thermal stability measurements indicated that G3-F15 was a highly stable G-triplex structure. The conformations of G3-F15 and G3-F15/ThT in the presence of different metal cations were studied thoroughly by fluorescent spectroscopy, circular dichroism, and nuclear magnetic resonance. Furthermore, using the G3-F15/ThT complex as a fluorescent probe, a robust and simple turn-on fluorescent sensor for uracil-DNA glycosylase activity was developed. This study proposes a new systematic strategy to explore new functional G-rich sequences and their ligands, which will promote their applications in diagnosis, therapy, and biosensing.

Qualitative and Quantitative Analytical Techniques of Nucleic Acid Modification Based on Mass Spectrometry for Biomarker Discovery.

Nucleic acid modifications play important roles in biological activities and disease occurrences, and have been considered as cancer biomarkers. Due to the relatively low amount of nucleic acid modifications in biological samples, it is necessary to develop sensitive and reliable qualitative and quantitative methods to reveal the content of any modifications. In this review, the key processes affecting the qualitative and quantitative analyses are discussed, such as sample digestion, nucleoside extraction, chemical labeling, chromatographic separation, mass spectrometry detection, and data processing. The improvement of the detection sensitivity and specificity of analytical methods based on mass spectrometry makes it possible to study low-abundance modifications and their biological functions. Some typical nucleic acid modifications and their potential as biomarkers are displayed, and efforts to improve diagnostic accuracy are discussed. Future perspectives are raised for this research field.

Identification and Quantification of Locus-Specific 8-Oxo-7,8-dihydroguanine in DNA at Ultrahigh Resolution Based on G-Triplex-Assisted Rolling Circle Amplification.

Damage of reactive oxygen species to various molecules such as DNA has been related to many chronic and degenerative human diseases, aging, and even cancer. 8-Oxo-7,8-dihydroguanine (OG), the most significant oxidation product of guanine (G), has become a biomarker of oxidative stress as well as gene regulation. The positive effect of OG in activating transcription and the negative effect in inducing mutation are a double-edged sword; thus, site-specific quantification is helpful to quickly reveal the functional mechanism of OG at hotspots. Due to the possible biological effects of OG at extremely low abundance in the genome, the monitoring of OG is vulnerable to signal interference from a large amount of G. Herein, based on rolling circle amplification-induced G-triplex formation and Thioflavin T fluorescence enhancement, an ultrasensitive strategy for locus-specific OG quantification was constructed. Owing to the difference in the hydrogen-bonding pattern between OG and G, the nonspecific background signal of G sites was completely suppressed through enzymatic ligation of DNA probes and the triggered specificity of rolling circle amplification. After the signal amplification strategy was optimized, the high detection sensitivity of OG sites with an ultralow detection limit of 0.18 amol was achieved. Under the interference of G sites, as little as 0.05% of OG-containing DNA was first distinguished. This method was further used for qualitative and quantitative monitoring of locus-specific OG in genomic DNA under oxidative stress and identification of key OG sites with biological function.

Phytochemical Constituents from the Seeds of Capsella bursa-pastoris and Their Antioxidant Activities.

Phytochemical investigation of 70% EtOH extract of the seeds of Capsella bursa-pastoris led to the isolation of a new cyclobutane organic acid (1), and fourteen known compounds, including two organosulfur compounds (2, 3), two quinonoids (4, 5), five flavonoids (6-10), three sterols (11-13) and two other types (14, 15). The structures of the compounds were elucidated by extensive spectroscopic analyses as well as comparison of their spectroscopic data with those reported in the literature. The antioxidant capacities of all compounds and extractive fractions were evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging test and ferric reducing antioxidant power (FRAP) assay. Then the antioxidative substances were evaluated for their neuroprotective effects against H2O2-induced HT22 cell injury. The results indicated the strong scavenging ability to free radical of the extractive fractions and compounds 1-3, 8-10 and 13, and the ferric reducing antioxidant power of the extractive fractions and compounds 1-3, 8 and 10, which were close to or higher than that of the positive control trolox. The EtOAc fraction, n-BuOH fraction, and compounds 1, 3 and 8 can protect HT-22 cells from oxidative damage.

Glycerol monolaurate ameliorates DSS-induced acute colitis by inhibiting infiltration of Th17, neutrophils, macrophages and altering the gut microbiota.

Background and aims: Inflammatory bowel disease (IBD) places a heavy medical burden on countries and families due to repeated and prolonged attacks, and the incidence and prevalence of IBD are increasing worldwide. Therefore, finding an effective treatment is a matter of great urgency. Glycerol monolaurate (GML), which has a twelve-carbon chain, is a compound naturally found in human breast milk. Some studies have shown that GML has antibacterial and anti-inflammatory effects. However, the specific mechanism of action remains unclear. Methods: Acute colitis was established in mice using 3% DSS, and glycerol monolaurate (500 mg·kg-1) was administered for two weeks. QPCR and western blotting were performed to examine the inflammatory status. Mice described were subjected to flow cytometry analysis for immune cell activation. Results: GML treated alleviated macroscopic symptoms such as shortened colons, increased spleen weight, and caused weight loss in mice with DSS-induced colitis. In addition, GML decreased the expression of pro-inflammatory factors (NF-α, IL-1ß and IL-1α) and increased the expression of anti-inflammatory factors (IL-10 and TGF-ß). GML inhibited the activation of the MAPK and NF-κB signalling pathways, improved tissue damage, and increased the expression of intestinal tight junction proteins. In addition, LPMCs extracted from intestinal tissue via flow cytometry showed that GML treatment led to a decrease of Th17 cells, Neutrophils and Macrophages. 16S rDNA sequencing showed that GML increased the abundance of commensal bacterium such as Akkermansia and Lactobacillus murinus. Conclusions: We showed that oral administration of GML ameliorated DSS-induced colitis by inhibiting infiltration of Th17 cells, Neutrophils, and Macrophages, protecting the intestinal mucosal barrier and altered the abundance of commensal bacterium. This study provides new insights into the biological function and therapeutic potential of GML in the treatment of IBD.

Ultrasensitive and Single-Base Resolution Quantification of 8-Oxo-7,8-dihydroguanine in DNA by Extension and Ligation-Based qPCR.

Oxidative DNA damage is tightly linked to the development of multiple age-related diseases. The prominent oxidation product is 8-oxo-7,8-dihydroguanine (OG), which has been proved to be an important epigenetic-like biomarker. Quantification of the locus-specific OG frequency includes quantitative and locating information, which is of great significance for exploring the functional roles of OG in disease induction and gene regulation. Herein, an ultrasensitive quantification of OG at single-base resolution was established using real-time fluorescence quantitative polymerase chain reaction as an amplification tool. Based on the coding property of Bsu DNA polymerase that incorporates adenine on the opposite site of OG and the selectivity of the ligase for perfectly matched sequences, the difference between OG and G on the sequence could be enlarged. Well-performed Taq DNA ligase was selected out, and as low as 46.2 zmol of target DNA with an OG site and an OG frequency of 5% could be detected. G contents on a specific site were also detectable based on the similar principle, thus the OG frequency of this locus could be accurately determined by a standard addition method. This strategy was successfully applied to the evaluation of locus-specific OG in both model DNA and genomic DNA from human cervical carcinoma cell lines under multiple oxidative stress, showing the potential for functional research and dynamic monitoring of critical OG sites.

Tubular epithelial cell-to-macrophage communication forms a negative feedback loop via extracellular vesicle transfer to promote renal inflammation and apoptosis in diabetic nephropathy.

Background: Macrophage infiltration around lipotoxic tubular epithelial cells (TECs) is a hallmark of diabetic nephropathy (DN). However, how these two types of cells communicate remains obscure. We previously demonstrated that LRG1 was elevated in the process of kidney injury. Here, we demonstrated that macrophage-derived, LRG1-enriched extracellular vesicles (EVs) exacerbated DN. Methods: We induced an experimental T2DM mouse model with a HFD diet for four months. Renal primary epithelial cells and macrophage-derived EVs were isolated from T2D mice by differential ultracentrifugation. To investigate whether lipotoxic TEC-derived EV (EVe) activate macrophages, mouse bone marrow-derived macrophages (BMDMs) were incubated with EVe. To investigate whether activated macrophage-derived EVs (EVm) induce lipotoxic TEC apoptosis, EVm were cocultured with primary renal tubular epithelial cells. Subsequently, we evaluated the effect of LRG1 in EVe by investigating the apoptosis mechanism. Results: We demonstrated that incubation of primary TECs of DN or HK-2 mTECs with lysophosphatidyl choline (LPC) increased the release of EVe. Interestingly, TEC-derived EVe activated an inflammatory phenotype in macrophages and induced the release of macrophage-derived EVm. Furthermore, EVm could induce apoptosis in TECs injured by LPC. Importantly, we found that leucine-rich α-2-glycoprotein 1 (LRG1)-enriched EVe activated macrophages via a TGFßR1-dependent process and that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-enriched EVm induced apoptosis in injured TECs via a death receptor 5 (DR5)-dependent process. Conclusion: Our findings indicated a novel cell communication mechanism between tubular epithelial cells and macrophages in DN, which could be a potential therapeutic target.

Anatomical Study on the Safety of Anterior Cervical Craniovertebral Fusion with Clival Screw Placement in Children Aged 1-6 Years.

OBJECTIVE: This study aimed to investigate the safety of clival screw placement in children aged 1-6 years. METHODS: The cranial computed tomography data of 92 children aged 1-6 years were divided into three groups, according to age, for three-dimensional reconstruction. Three clival screw placement points were defined: (1) median point A at the middle and upper third of the tripartite distance from the spheno-occipital synchondrosis to the base of the skull; (2, 3) critical points B and C on the horizontal line with point A, where the screw placement passage was parallel to the sagittal plane. Parameters such as the passage length and angle were measured for horizontal, vertical facial, and extreme screw placement. RESULTS: The length parameters of the clival screw placement increased with age, and the screw passage length was the shortest for the vertical facial type in each age group. There were significant differences in all three groups between the length of screw placement in the vertical bone surface, the length of screw placement in the horizontal direction, and the length of screw placement on the limit at points A, B, and C (P < 0.05); the length of screw placement on the vertical bone surface was the shortest. There was no significant difference between the horizontal screw length at point A and the extreme screw length (P > 0.05). The difference between the horizontal screw length and the extreme screw length in the groups aged 1-2 years and 5-6 years was statistically significant (P < 0.05), and the horizontal screw length was longer. CONCLUSION: The cranial slope of children aged 1-6 years has the morphological basis for the placement of 3.5 mm screws, and each placement point has a safe angle range for screw placement.

Detection of average methylation level of specific genes by binary-probe hybridization.

We developed a simple and highly-selective method for 5-methylcytosine detection of specific gene sequence based on binary-probe DNA hybridization. The sequence complementary to the target was designed into two probes, and each fragment of binary probes bound to a relatively short sequence of the target, which made it sensitive to the base mismatches introduced by bisulfite treatment. The advantages of a low detection limit of methylation abundance of 0.1% for the fully methylated target and high sensitivity of 10 pM have been proved by the successful design of binary-probe hybridization. The successful design of the binary probes makes it possible to quantify the average methylation levels of five CpG sites. Thirty-two DNA strands containing 5, 4, 3, 2, 1 and 0 CpG sites were successfully analyzed with the same pair of binary probes. The higher the average methylation level of the target was, the higher the degree of the hybridization reaction. Based on the simple construction of the binary-probe hybridization, the developed biosensor exhibited signals proportional to the average methylation level of the vimentin gene and could evaluate the average methylation level of artificial mixtures. Furthermore, the method has been used to detect vimentin methylation in a genomic context with good specificity, which indicated its potential in the pre-diagnosis of methylation related disease.

Venous thromboembolism in patients with severe lung cancer: a narrative review.

OBJECTIVE: At present, there are several guidelines for cancer complicated with VTE, but there is no specific recommendation for the treatment of lung cancer complicated with VTE. Whether is necessary to explore treatment and prevention of VTE in lung cancer. BACKGROUND: Venous thromboembolism (VTE) is a common complication of severe lung cancer that can entail many adverse effects for patients. The incidence of VTE is higher in patients with lung cancer than in those with other kinds of solid tumors, and it is especially high among patients with lung adenocarcinoma, at advanced tumor-node-metastasis (TNM) stages, or with a history of central venous catheter (CVC) or chemotherapy. However, the clinical symptoms of VTE in patients with lung cancer are not typical and cannot be detected easily, and the clinical prevention rate is low. In the acute phase of VTE in lung cancer, the Eastern Cooperative Oncology Group (ECOG) performance status score of patients typically ranges from 2 to 4 points, leaving end-stage maintenance therapy as the only treatment option. METHODS: Here, we analyze the existing literature and discuss the current status (including epidemiology, clinical manifestations, and risk factors), risk assessment tools, and the treatment and prevention of VTE in severe lung cancer. We focus particularly on the use of low-molecular-weight heparin and new oral anticoagulants (including in the management of thrombocytopenia after antitumor therapy) in lung cancer patients with VTE. CONCLUSIONS: Large-scale prospective multicenter studies on the treatment and prevention of VTE in lung cancer are necessary.

Ultrasensitive Multiplex Detection of Single Nucleotide Polymorphisms Based on Short-Chain Hybridization Combined with Online Preconcentration of Capillary Electrophoresis.

Reliable multiple single nucleotide polymorphisms (SNPs) detection at low abundance is of great significance for disease diagnosis and biomedical research. Herein, we have developed a novel and simple method for multiple SNPs detection combining solid-phase capture by specific hybridization with online preconcentration of capillary gel electrophoresis-laser-induced fluorescence (CGE-LIF). The method presents an excellent performance due to its favorable traits: the solid-phase short-chain hybridization ensures the high specificity of SNP detection; the effective separation ability of CGE can easily achieve multiplex detection; the simple online preconcentration significantly improves the detection sensitivity of fluorescent probe by nearly 100-fold. For a single SNP target, the assay achieves a limit of detection as low as 0.01-0.02% for three different NRAS mutations in the same codon. For multiple SNP targets, as low as 0.05% abundance can be easily realized. Our method is simple, efficient, ultrasensitive, and universal for multiple SNPs detection without complex enzymatic or chemical ligation reaction, which shows great potential in early clinical diagnosis.

[Assessment of the value of acute physiology and chronic health evaluation II/IV prognostic models in elderly patients with sepsis].

OBJECTIVE: To assess and compare the performance of acute physiology and chronic health evaluation II/IV (APACHEII/IV) prognostic models in elderly patients with sepsis. METHODS: A totally of 82 elderly patients with sepsis were retrospectively assessed in geriatric intensive care unit of General Hospital of Guangzhou Military Command between July 2011 and December 2011. APACHEII/IV scores were recorded within 24 hours after admission. The prognosis accuracy of both scores was assessed by area under the receiver operating characteristic curve (AUC). Based on the best cutoff value corresponding with the highest accuracy, patients were divided into the low and high risk of hospital mortality group. The predictive power of APACHEII/IV in total population and subgroups was compared. RESULTS: Patients with severe sepsis constituted 57.3% (47/82) of all patients with sepsis, and hospital mortality was 61.0% (50/82). APACHEII/IV scores of the patients were 17.5±6.3 and 55.8±22.3, and mortality rate was 22.5% (18.4/82) and 17.9% (14.6/82) respectively, with significant differences compared with actual mortality (both P<0.01). Both APACHEII/IV scores showed underestimation of hospital mortality in total population [standardized mortality ratio (SMR) with APACHEII=2.71, 95% confidence interval (95%CI) 1.92-3.48 and SMR with APACHEIV=3.33, 95%CI 2.79-4.37]. APACHEII (AUC 0.664±0.066), and APACHEIV presented poor estimation(AUC 0.716±0.056). There was no difference in accuracy in prognosticating hospital death prognosis between the two APACHE models (Z=0.991, P=0.322). Cutoff values of APACHEII/IV were >11 and >59. According to the value, patients were divided into the low and high risk hospital mortality group. There was no significant difference between actual mortality and prognostic mortality in APACHEII low risk group [0-11, 20.0% (3/15) vs. 1.6% (0.2/15), Z=-1.023, P=0.306]. The actual mortality in high risk group with APACHEII (>11) was significantly higher than prognostic mortality [70.1% (47/67) vs. 27.2% (18.2/67), t=6.989, P=0.000]. In the high risk group, APACHEII underestimated mortality (SMR=2.58, 95%CI 2.22-3.51). The actual mortality of the low (0-59) and high (>59) risk group of APACHEIV were higher than prognostic mortality [lower risk group: 44.0% (22/50) vs. 7.5% (3.8/50), Z=-2.235, P=0.025; higher risk group: 87.5% (28/32) vs. 34.1% (10.9/32), Z=-4.712, P=0.000]. Two groups of patients with APACHEIV score, the mortality was underestimated (low risk group: SMR=5.90, 95%CI 5.19-7.07; high risk group: SMR=2.56, 95%CI 2.07-3.24). Mortality rate of the low risk group with APACHEIV score was prone to be underestimated. CONCLUSIONS: The accuracy of APACHEII/IV are not ideal in foretelling mortality rate. Hospital mortality was underestimated with APACHEII in high risk patients, and it was underestimated with APACHEIV both in low and high risk patients, and it is even more prone to be underestimated in low risk group of APACHEIV. More accurate prognostic modality is in need in elderly patients with sepsis.

Blastocyst injection of wild type embryonic stem cells induces global corrections in mdx mice.

Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology and function. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection increases the amount of fat in the chimeras to reach WT levels. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally in a dose-dependent manner.