FLED: a full-length eccDNA detector for long-reads sequencing data.

Reconstructing the full-length sequence of extrachromosomal circular DNA (eccDNA) from short sequencing reads has proved challenging given the similarity of eccDNAs and their corresponding linear DNAs. Previous sequencing methods were unable to achieve high-throughput detection of full-length eccDNAs. Herein, a novel algorithm was developed, called Full-Length eccDNA Detection (FLED), to reconstruct the sequence of eccDNAs based on the strategy that combined rolling circle amplification and nanopore long-reads sequencing technology. Seven human epithelial and cancer cell line samples were analyzed by FLED and over 5000 full-length eccDNAs were identified per sample. The structures of identified eccDNAs were validated by both Polymerase Chain Reaction (PCR) and Sanger sequencing. Compared to other published nanopore-based eccDNA detectors, FLED exhibited higher sensitivity. In cancer cell lines, the genes overlapped with eccDNA regions were enriched in cancer-related pathways and cis-regulatory elements can be predicted in the upstream or downstream of intact genes on eccDNA molecules, and the expressions of these cancer-related genes were dysregulated in tumor cell lines, indicating the regulatory potency of eccDNAs in biological processes. The proposed method takes advantage of nanopore long reads and enables unbiased reconstruction of full-length eccDNA sequences. FLED is implemented using Python3 which is freely available on GitHub (https://github.com/FuyuLi/FLED).

A comprehensive analysis of library preparation methods shows high heterogeneity of extrachromosomal circular DNA but distinct chromosomal amount levels reflecting different cell states.

Extrachromosomal circular DNA (eccDNA) was discovered several decades ago, but little is known about its function. With the development of sequencing technology, several library preparation methods have been developed to elucidate the biogenesis and function of eccDNA. However, different treatment methods have certain biases that can lead to their erroneous interpretation. To address these issues, we compared the performance of different library preparation methods. Our investigation revealed that the utilization of rolling-circle amplification (RCA) and restriction enzyme linearization of mitochondrial DNA (mtDNA) significantly enhanced the efficiency of enriching extrachromosomal circular DNA (eccDNA). However, it also introduced certain biases, such as an unclear peak in ∼160-200 bp periodicity and the absence of a typical motif pattern. Furthermore, given that RCA can lead to a disproportionate change in copy numbers, eccDNA quantification using split and discordant reads should be avoided. Analysis of the genomic and elements distribution of the overall population of eccDNA molecules revealed a high correlation between the replicates, and provided a possible stability signature for eccDNA, which could potentially reflect different cell lines or cell states. However, we found only a few eccDNA with identical junction sites in each replicate, showing a high degree of heterogeneity of eccDNA. The emergence of different motif patterns flanking junctional sites in eccDNAs of varying sizes suggests the involvement of multiple potential mechanisms in eccDNA generation. This study comprehensively compares and discusses various essential approaches for eccDNA library preparation, offering valuable insights and practical advice to researchers involved in characterizing eccDNA.

Unbiased and Signal-Weakening Photoelectrochemical Hexavalent Chromium Sensing via a CuO Film Photocathode.

Photoelectrochemical (PEC) sensors show great potential for the detection of heavy metal ions because of their low background noise, high sensitivity, and ease of integration. However, the detection limit is relatively high for hexavalent chromium (Cr(VI)) monitoring in addition to the requirement of an external bias. Herein, a CuO film is readily synthesized as the photoactive material via reactive sputtering and thermal annealing in the construction of a PEC sensing photocathode for Cr(VI) monitoring. A different mechanism (i.e., Signal-Weakening PEC sensing) is confirmed by examining the electrochemical impedance and photocurrent response of different CuO film photoelectrodes prepared with the same conditions in contact with various solutions containing concentration-varying Cr(VI) for different durations. The detection of Cr(VI) is successfully achieved with the Signal-Weakening PEC response; a drop of photocathode signal with an increasing Cr(VI) concentration from the steric hindrance effect of the in situ formed Cr(OH)3 precipitates. The photocurrent of the optimized CuO film photocathode linearly declines as the concentration of Cr(VI) increases from 0.08 to 20 µM, with a detection limit down to 2.8 nM (Signal/Noise = 3) and a fitted sensitivity of 4.22 µA·µM-1. Moreover, this proposed sensing route shows operation simplicity, satisfactory selectivity, and reproducibility.

Integrated analysis of mRNA and extrachromosomal circular DNA profiles to identify the potential mRNA biomarkers in breast cancer.

Extrachromosomal circular DNAs (eccDNAs) have been proved an inseparable relationship with cancer, based on the biological mechanisms of its biogenesis and impact on tumorigenesis, but still lacked of methods to analyze its function on the pathogenesis and progression of breast cancer (BC). The mRNA and eccDNA from BC cell samples (MDA-MB-453 and MCF-12A) were extracted with the removal of rRNA and linear DNA, respectively. High-throughput sequencing and bioinformatics analysis were performed to explore their expression level and molecular characterization of eccDNA. A total number of 161,062 eccDNA ranging from 33 bp to 54229 bp were detected with a median size of 1143 bp, distributed on all chromosomes and enriched on chromosome 20 the most. EccDNAs located in exons, upstream and downstream 2 kb regions were significantly increased compared with background. Analysis of eccDNA-related differentially expressed genes (eccDEGs) showed that FAT2 properly separated the two cells. CTNNB1, CACNA2D2 and CACNA1D were the hub genes with higher degrees in critical modules. All these four genes were significantly differentially expressed between breast invasive carcinoma (BRCA) tissues and normal ones. FAT2 and CTNNB1 correlated with significantly different overall survival (OS) when differentially expressed. The four genes showed a strong correlation with each other significantly and changed between tumor and normal samples. The results showed the potential of FAT2, CTNNB1, CACNA2D2 and CACNA1D as biomarkers with analysis of both DEGs and eccDEGs, which might assist in clinical medical treatment.

A comprehensive characterization of cell-free RNA in spent blastocyst medium and quality prediction for blastocyst.

The rate of pregnancy can be affected by many factors in assisted reproductive technology (ART), and one of which is the quality of embryos. Therefore, selecting the embryos with high potential is crucial for the outcome. Fifteen spent blastocyst medium (SBM) samples were collected from 14 patients who received in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), seven from high-grade embryos and eight from low-grade embryos. Cell-free RNA (cf-RNA) profile of SBM samples were analyzed by RNA sequencing in the present study. It was found that a large amount of cf-RNA were released into SBM, including protein-coding genes (68.9%) and long noncoding RNAs (lncRNAs) (17.26%). Furthermore, a high correlation was observed between blastocyst genes and SBM genes. And the cf-mRNAs of SBM were highly fragmented, and coding sequence (CDS) and untranslated (UTR) regions were released equally. Two hundred and thirty-two differentially expressed genes were identified in high-grade SBM (hSBM) and low-grade SBM (lSBM), which could be potential biomarker in distinguishing the embryos with different quality as an alternative or supplementary approach for subjective morphology criteria. Hence, cf-RNAs sequencing revealed the characterization of circulating transcriptomes of embryos with different quality. Based on the results, the genes related to blastocyst quality were screened, including the genes closely related to translation, immune-signaling pathway, and amino acid metabolism. Overall, the present study showed the types of SBM cf-RNAs, and the integrated analysis of cf-RNAs profiling with morphology grading displayed its potential in predicting blastocyst quality. The present study provided valuable scientific basis for noninvasive embryo selection in ART by RNA-profiling analysis.

Novel Method of Full-Length RNA-seq That Expands the Identification of Non-Polyadenylated RNAs Using Nanopore Sequencing.

The occurrence of diseases displayed transcriptome alteration, including both coding and non-coding transcripts. The third-generation sequencing (TGS) technologies allow for intensive and comprehensive research of the transcriptome. However, the present standard TGS RNA sequencing method is unable to detect many of the non-polyadenylated [non-poly(A)] RNAs. To obtain more complete transcriptome information, we presented a new comprehensive sequencing approach by performing conventional poly(A) RNA-sequencing combined with the sequencing of non-poly(A) RNA fraction which was tailed by poly(U) on HepG2 and HL-7702 cell lines, enabling the detection of multiple categories of non-poly(A) RNAs excluded by the existing standard approach. Moreover, the length distribution of the full-splice match transcripts was longer than that assembled by short-reads, which contributed to characterizing alternative splicing events and provided a comprehensive portrait of transcriptional complexity. Besides the detection of genes with differential expression patterns in the poly(A) library between HepG2 and HL-7702, we also found a cancer-related non-coding gene in the poly(U) data, that is, growth arrest special 5 (GAS5). Collectively, our results suggested that the novel method effectively captured both poly(A) and non-poly(A) transcripts in the tested cell lines and allowed a deeper exploration of the transcriptome.

Impact of RNA degradation on next-generation sequencing transcriptome data.

RNA sequencing is an innovative technology to study transcriptomes in both biological and clinical research. However, clinical specimens from patients undergoing surgical operations have a major challenge due to sample degradation. This study replicated the process of RNA degradation by maintaining cells at room temperature to achieve none, slight, middle, and high levels of RNA degradation with decreasing RNA integrity numbers (RIN) of approximately 9.8, 6.7, 4.4, and 2.5, respectively. Next, the differential expression of mRNA and long non-coding RNA (lncRNA) was analyzed in the four degradation groups along with pathway enrichment analysis. The results showed that the similarity of lncRNAs exhibited significant differences even for a slight level of RNA degradation compared with the non-degraded RNA sample. Also, the RNA degradation process was found to be universal, global, and random; the differentially expressed genes increased with an increase in degradation but the pathway enrichment phenomenon was not significantly observed.

Identification of full-length circular nucleic acids using long-read sequencing technologies.

Unlike the traditional perception in genomic DNA or linear RNA, circular nucleic acids are a class of functional biomolecules with a circular configuration and are often observed in nature. These circular molecules encompass the full spectrum of size and play an important role in organisms, making circular nucleic acids research worthy. Due to the low abundance of most types of circular nucleic acids and the disadvantages of short-read sequencing platforms, accurate and full-length circular nucleic acid sequencing and identification is difficult. In this review, we have provided insights into full-length circular nucleic acid detection methods using long-read sequencing technologies, with a focus on the experimental and bioinformatics strategies to obtain accurate sequences.

Formulation and Pharmacokinetic Evaluation of a Drug-in-Adhesive Patch for Transdermal Delivery of Koumine.

The aim of this study was to develop a suitable drug-in-adhesive patch for transdermal delivery of koumine. Acrylic polymer Duro-Tak® 87-4287, which contains hydroxyl groups, may significantly enhance the skin permeation of koumine from transdermal patches containing 0.93-3.72% koumine. Among permeation enhancers, 10% azone showed the greatest potential and increased the flux of koumine to 1.48-fold that of the control. Therefore, an optimized patch formulation containing 3.72% koumine and 10% azone in Duro-Tak® 87-4287 that offers good physical properties was selected for an in vivo pharmacokinetic study using rats. The maximal plasma drug concentration (Cmax) of koumine after transdermal administration (4 mg/patch) was 25.80 ± 1.51 ng/mL, which was in the range of those after oral administration (3 mg/kg and 15 mg/kg). The time to the maximal concentration (Tmax) and the half-life (t1/2) of the drug with transdermal administration were 3.96 ± 0.46 h and 21.10 ± 1.36 h, respectively, which were longer than those with oral administration. Furthermore, the area under the concentration-time curve (AUC0-72 h) of 898.20 ± 45.57 ng·h/mL for the transdermal patch was much higher than that for oral administration (15 mg/kg). In conclusion, the drug-in-adhesive patch containing koumine provides a steady plasma koumine level and sustained release in vivo and can be an effective means of transdermal delivery for koumine.

A novel approach to improving hybrid capture sequencing targeting efficiency.

At present, approaches to hybrid capture sequencing have many limitations, from their significant complexity and labor requirements, to their low enrichment efficiency, resulting in their limited utilization. In an effort to overcome these drawbacks, we have developed a novel method that relied upon direct genomic DNA hybridization and single-stranded DNA library preparation. Using this novel protocol, we were able to achieve a targeting efficiency as high as 75%, and we found this approach to overall be an efficient and simple approach to DNA library construction.

Whole genome sequencing identifies ANXA3 and MTHFR mutations in a large family with an unknown equinus deformity associated genetic disorder.

The aim of this study was to characterize a previously uncharacterized genetic disorder associated with equinus deformity in a large Chinese family at the genetic level. Blood samples were obtained and whole genome sequencing was performed. Differential gene variants were identified and potential impacts on protein structure were predicted. Based on the control sample, several diseases associated variants were identified and selected for further validation. One of the potential variants identified was a ANXA3 gene [chr4, c.C820T(p.R274*)] variant. Further bioinformatic analysis showed that the observed mutation could lead to a three-dimensional conformational change. Moreover, a MTHFR variant that is different from variants associated with clubfoot was also identified. Bioinformatic analysis showed that this mutation could alter the protein binding region. These findings imply that this uncharacterized genetic disorder is not clubfoot, despite sharing some similar symptoms. Furthermore, specific CNV profiles were identified in association with the diseased samples, thus further speaking to the complexity of this multigenerational disorder. This study examined a previously uncharacterized genetic disorder appearing similar to clubfoot and yet having distinct features. Following whole genome sequencing and comparative analysis, several differential gene variants were identified to enable a further distinction from clubfoot. It is hoped that these findings will provide further insight into this disorder and other similar disorders.

Synthesis and tumor cytotoxicity of novel amide derivatives of ß-hederin.

Thirteen novel triterpenoid saponins, designed as amide derivatives of the natural cytotoxic saponin ß-hederin, were synthesized by a stepwise glycosylation strategy. The in vitro cytotoxic activity of these compounds was evaluated against five different tumor cell lines. Most of the evaluated compounds showed effective inhibitory activity against at least one tumor cell line at micromolar concentrations. The preliminary structure-activity relationships (SAR) indicate that mide derivatization at C-28 resulted in highly cytotoxic derivatives on specific tumor cell lines, and also resulted in an increase in the antitumor selectivity of ß-hederin. 

Synthesis of kaempferol 3-O-(3'',6''-di-O-E-p-coumaroyl)-ß-D-glucopyranoside, efficient glycosylation of flavonol 3-OH with glycosyl o-alkynylbenzoates as donors.

Kaempferol 3-O-(3'',6''-di-O-E-p-coumaroyl)-ß-D-glucopyranoside (1), an optimal metabolite of Scots pine seedlings for protection of deep-lying tissue against damaging UV-B, represents a typical acylated flavonol 3-O-glycoside. This compound was synthesized for the first time via two approaches. The first approach, starting with kaempferol, featured formation of the flavonol 3-O-glycosidic linkage with a glycosyl bromide under conventional PTC conditions. In the second approach, 5,7,4'-tri-O-benzyl-kaempferol was readily prepared from 2',4',6'-trihydroxyacetophenone and p-hydroxybenzoic acid, which was coupled with a glucopyranosyl o-hexynylbenzoate under the catalysis of a gold(I) complex to provide the desired 3-O-glycoside in excellent yield. A variety of the glycosyl o-hexanylbenzoates equipped with the 2-O-benzoyl group were also proven to be highly efficient donors for construction of the flavonol 3-O-glycosidic linkages.

Facile synthesis and cytotoxicity of triterpenoid saponins bearing a unique disaccharide moiety: hederacolchiside A1 and its analogues.

An improved synthetic approach toward hederacolchiside A1, an antitumor triterpenoid saponin bearing a unique disaccharide moiety, was established. This approach began from a partially protected intermediate and avoided tedious protection-deprotection manipulation. An abnormal ring conformation (1C4) of the center arabinose residue was found in the intermediate, which may account for the unusual regioselectivity between 3-OH and 4-OH of arabinose. Two analogues of hederacolchiside A1 were then facilely prepared by this approach and exhibited significant cytotoxicity in preliminary in vitro assay.