Analysis of mixed DNA‐bisnaphthalimide interactions involving groove association and intercalation with surface‐based and solution methodologies

The bisnaphthalimide cytotoxic agent elinafide exhibits a mixed DNA binding mode including groove‐association and intercalation. We have compared the interaction of elinafide and two bisnaphthalimide analogues with various natural and modified DNA sequences using solution NMR and UV‐melting methods and surface plasmon resonance (SPR) experiments at different pH conditions. The combined data obtained with these techniques established a high‐affinity binding mode comprising intercalation and strong electrostatic contacts with guanine bases in the major groove, and a weaker interaction with A·T pairs likely involving groove association. However, the SPR binding constants and the NMR and UV‐melting binding parameters responded differently to variations in DNA bases and ligand intercalating moieties. The rates and equilibrium constants determined by SPR clearly responded to changes in pH and DNA groove composition, but were rather insensitive to alterations in drug rings and DNA bases affecting the intercalation process. Conversely, the intermolecular stacking interactions detected by NMR and the ligand‐induced thermal stabilizations measured by UV depended on both sets of factors and were controlled by the sequence‐dependent properties of the DNA helices, indicating that these data were modulated by naphthalimide stacking in addition to groove association. A two‐step binding process where a groove‐bound state is required prior to intercalation is proposed as an explanation for these observations. These findings may be useful for studying other classes of DNA‐ and RNA‐binding drugs, which frequently combine groove‐binding and stacking moieties. © 2012 Wiley Periodicals, Inc. Biopolymers 97:974–987, 2012.     

Distinguishing Anuran species by high‐resolution melting analysis of the COI barcode (COI‐HRM)

Taxonomic identification can be difficult when two or more species appear morphologically similar. DNA barcoding based on the sequence of the mitochondrial cytochrome c oxidase 1 gene (COI) is now widely used in identifying animal species. High‐resolution melting analysis (HRM) provides an alternative method for detecting sequence variations among amplicons without having to perform DNA sequencing. The purpose of this study was to determine whether HRM of the COI barcode can be used to distinguish animal species. Using anurans as a model, we found distinct COI melting profiles among three congeners of both Lithobates spp. and Hyla spp. Sequence variations within species shifted the melting temperature of one or more melting domains slightly but do not affect the distinctness of the melting profiles for each species. An NMDS ordination plot comparing melting peak profiles among eight Anuran species showed overlapping profiles for Lithobates sphenocephala and Gastrophryne carolinensis. The COI amplicon for both species contained two melting domains with melting temperatures that were similar between the two species. The two species belong to two different families, highlighting the fact that COI melting profiles do not reveal phylogenetic relationships but simply reflect DNA sequence differences among stretches of DNA within amplicons. This study suggests that high‐resolution melting analysis of COI barcodes (COI‐HRM) may be useful as a simple and rapid method to distinguish animal species that appear morphologically similar.     

Effects of phosphate and nitrate supply on productivity,agar content and physical properties of agar ofGracilaria strain G-16S

Gracilaria strain G-16S was cultured in various phosphorus (P) supply rates with low or high nitrogen (N) supply to determine the effects of nutrient supply on its productivity, agar content and physical properties of the agar. Productivity was reduced after four weeks of growth in zero P supply as plants reached 0.07% P tissue content (critical level), with fragmentation of these plants by six weeks (0.05% P; minimum viable level). Native agar content was higher in low P and high N, or low N conditions. Agar content appeared to increase with decreasing P under high N supply. This increase was not apparent with alkali treatment prior to extraction. Agar gel strength was greatly increased by alkali treatment. The highest gel strengths were obtained under high N supply at all P supply rates except zero P, and under low N supply at 12 M P week^–1. Native agar gel strengths showed a similar pattern on a lower scale. Melting temperatures were higher in agars with higher gel strengths. Dynamic gelling temperatures were generally high for alkali-treated agar, with agar from plants grown in zero P supply showing a slightly elevated gelling temperature. Melting and gelling temperatures of native agars with the highest gel strengths were in the same range as bacteriological agar. These results show that P and N supply affects productivity, agar content and agar physical properties, but the tradeoffs between a slightly higher agar quantity under nutrient limitation and higher agar quality under nutrient-replete conditions seem to favor the latter.     

Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: Identification of novel mutations

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene.     

Structural and thermodynamic signatures that define pseudotriloop RNA hairpins

Pseudotriloop (PTL) structures in RNAs have been recognized as essential elements in RNA folding and recognition of proteins. PTL structures are derived from hexaloops by formation of a cross–loop base pair leaving a triloop and 3′ bulged out residue. Despite their common presence and functional importance, insufficient structural and thermodynamic data are available that can be used to predict formation of PTLs from sequence alone. Using NMR spectroscopy and UV-melting data we established factors that contribute to the formation and stability of PTL structures derived from hepatitis B virus and human foamy virus. The NMR data show that, besides the cross–loop base pair, also a 3′ pyrimidine bulge and a G–C loop-closing base pair are primary determinants of PTL formation. By changing the G–C closing base pair into C–G, the PTL switches into a hexaloop. Comparison of these rules with regular triloop hairpins and PTLs from other sources is discussed as well as the conservation of a PTL in human foamy virus and other spumaretroviruses.     

雌激素受体α(ESR1)基因PvuⅡ和XbaI位点基因多态性与HBV慢性感染的相关性研究

摘要 目的：探讨雌激素受体ESR1(Estrogen Receptor alpha gene)基因的PvuⅡ(rs2234693)和XbaI (rs9340799)两个单核苷酸多态性(single nucleotide polymorphisms, SNPs)位点的基因多态性与乙型肝炎病毒HBV(Hepatitis B Virus)慢性感染的相关性,为控制HBV持续感染提供新的思路和科学依据。方法：选择107例慢性乙型病毒性肝炎患者为病例组及107例同期体检的健康人群为对照组,基于高分辨熔解曲线技术(High Resolution Melting,HRM)建立PCR-HRM分子诊断方法,检测其雌激素受体ESR1基因两个SNP位点rs2234693(T>C)和rs9340799(A>G)的基因多态性,并通过基因测序进一步验证,探讨上述两个SNP位点与HBV慢性感染的相关性。结果：病例组和健康对照组ESR1基因rs2234693(T>C)位点的基因型频率比较差异具有统计学意义(P<0.05),而两组间rs2234693位点等位基因频率比较差异没有统计学意义(P>0.05);病例组和健康对照组间ESR1基因rs9340799(A>G)位点的各基因型频率差异具有统计学意义(P<0.05),慢性乙肝病例组GG基因型明显升高,两组间rs9340799位点等位基因频率差异亦具有统计学意义(P<0.05)。Logistic回归分析显示rs9340799位点的G基因可增加HBV慢性感染的发病风险,A基因可降低HBV慢性感染的发病风险。结论：雌激素受体基因ESR1的rs9340799 (A>G)位点的GG基因型和G等位基因可能是HBV感染慢性化的遗传易感基因,GG基因型与HBV的慢性感染具有一定的相关性。     

The cornerstone of integrating circulating tumor DNA into cancer management

Recent circulating tumor DNA (ctDNA) research has demonstrated its potential as a non-invasive biomarker for cancer. However, the deployment of ctDNA assays in routine clinical practice remains challenging owing to variability in analytical approaches and the assessment of clinical significance. A well-developed, analytically valid ctDNA assay is a prerequisite for integrating ctDNA into cancer management, and an appropriate analytical technology is crucial for the development of a ctDNA assay. Other determinants including pre-analytical procedures, test validation, internal quality control (IQC), and continual proficiency testing (PT) are also important for the accuracy of ctDNA assays. In the present review, we will focus on the most widely used ctDNA detection technologies and the key quality management measures used to assure the accuracy of ctDNA assays. The aim of this review is to provide useful information for technology selection during ctDNA assay development and assure a reliable test result in clinical practice.     

IDH1 mutations is prognostic marker for primary glioblastoma multiforme but MGMT hypermethylation is not prognostic for primary glioblastoma multiforme

Purpose

To establish the frequency of IDH1 mutations and MGMT methylation in primary glioblastomas.

Experimental design

We screened primary glioblastoma multiforme (GBM) in a population-based study for IDH1 mutations and MGMT methylation and correlated them with clinical data.

Results

IDH1 mutations were detected in 5 of 40 primary glioblastomas (12,5%). Primary GBM patients carrying IDH1 mutations were significantly younger, mean age of 41 ± 5.06 years, than patients with wild-type IDH1, mean age of 57 ± 2,29 years, p = 0.011. The mean survival time of all GBM patients with and without IDH1 mutations was 19 months (5 cases) and 16 months (35 cases), respectively (p > 0,05). MGMT methylation was detected in 13 of the 40 patients (32,5%). MGMT-promoter methylation did not correlate with overall survival (OS; p > 0,05).

Conclusion

In summary, our study is the first study to investigate the IDH1 mutation status and MGMT methylation in primary GBMs in Turkish population and confirmed IDH1 mutation as a genetic marker for also primary GBMs. Our data are still insufficient for definite ascertainment; and our preliminary results suggest: IDH1 status shows an association with younger age and there is a lack of association between IDH1 mutation and survival time. Furthermore MGMT promoter methylation had no prognostic value and lower frequency in primary glioblastomas.     

Analysis of Streptomyces coelicolor membrane proteome using two-dimensional native/native and native/sodium dodecyl sulfate gel electrophoresis

Analysis of the oligomeric state of a protein may provide insights into its physiological functions. Because membrane proteins are considered to be the workhorses of energy generation and polypeptide and nutrient transportation, in this study we characterized the membrane-associated proteome of Streptomyces coelicolor by two-dimensional (2D) blue native/sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), high-resolution clear native/native PAGE, and native/SDS–PAGE. A total of 77 proteins were identified, and 20 proteins belonging to 15 complexes were characterized. Moreover, the resolution of high-resolution clear native/SDS–PAGE is much higher than that of blue native/SDS–PAGE. OBP (SCO5477) and BldKB (SCO5113) were identified as the main protein spots from the membrane fractions of S. coelicolor M145, suggesting that these two proteins are involved in extracellular peptide transportation. These two transporters exhibited multiple oligomeric states in the native PAGE system, which may suggest their multiple physiological functions in the development of S. coelicolor.