Reduced representative methylome profiling of cell-free DNA for breast cancer detection.

BACKGROUND: Whole-genome methylation sequencing of cfDNA is not cost-effective for tumor detection. Here, we introduce reduced representative methylome profiling (RRMP), which employs restriction enzyme for depletion of AT-rich sequence to achieve enrichment and deep sequencing of CG-rich sequences. METHODS: We first verified the ability of RRMP to enrich CG-rich sequences using tumor cell genomic DNA and analyzed differential methylation regions between tumor cells and normal whole blood cells. We then analyzed cfDNA from 29 breast cancer patients and 27 non-breast cancer individuals to detect breast cancer by building machine learning models. RESULTS: RRMP captured 81.9% CpG islands and 75.2% gene promoters when sequenced to 10 billion base pairs, with an enrichment efficiency being comparable to RRBS. RRMP allowed us to assess DNA methylation changes between tumor cells and whole blood cells. Applying our approach to cfDNA from 29 breast cancer patients and 27 non-breast cancer individuals, we developed machine learning models that could discriminate between breast cancer and non-breast cancer controls (AUC = 0.85), suggesting possibilities for truly non-invasive cancer detection. CONCLUSIONS: We developed a new method to achieve reduced representative methylome profiling of cell-free DNA for tumor detection.

Molecular Evidence of Structural Changes in Silk Using Unlimited Degradation Mass Spectrometry.

Proteomics has important uses in archeological science because it can distinguish species, reveal the evolution of paleontology, and provide biological evidence of historical events. However, this technique still has full potential in the study of silk aging mechanisms. In this work, we propose a strategy combining unlimited degradation with mass-spectrometry-based proteomics techniques, which interpret protein fragmentation propensity and secondary structure changes by detecting content changes of specific peptide groups in complex proteomes. This approach was employed to study the conformational changes in silk microscopic crystals after heat treatment. Combining conventional mechanics and crystallographic characterization, a thermal aging degradation mechanism model was proposed. At the same time, it explained the interesting problem that the crystallinity remained unchanged, but the mechanical properties decreased significantly. Focusing on the unlimited degradation process, this method will be widely applicable to the study of silk and wool aging processes and regenerated silk fibroin.

Effect of cocooning conditions on the structure, carbon and nitrogen isotope ratios of silks.

The stable isotope technique provides the possibility to trace ancient textiles because the technique is associated with advantages such as trace indication, fast detection, and accurate results. Since different cocooning conditions may impact cocoons even under identical habitats, it is important to investigate the effects of different cocooning temperatures and humidity on the isotope incorporation values in the cocoons. In this study, silk fibers were reeled under different conditions of temperature and humidity, followed by analysis of the secondary structure of cocoon proteins and isotope incorporation patterns. We found that the deviations in carbon isotope values of silk under different cocooning conditions could reach up to 0.76‰, while the deviation in carbon isotope values at different locations of a single silk was 2.75‰. Further, during the cocooning process, depletion of the 13C-isotope at different locations of the silk fibers was observed, reducing the δ13C values. We proposed that the changes in carbon isotopes in silk were related to the content of sericin and silk fibroin in silk. Finally, we did not observe a significant difference in isotope ratios in degummed cocoons. In summary, the 13C isotope was enriched in sericin, whereas 15N was enriched in fibroin, and these findings provide basic information for tracing the provenance of silks.

An ultrasensitive MXene-based electrochemical immunosensor for the detection and species identification of archaeological silk microtraces.

The origin and dissemination of silk have been hotly debated in the field of archaeology, and the key to resolving this controversy lies in the detection and species identification of ancient silk microtraces. Herein, a taxonomically specific anti-fibroin monoclonal antibody was successfully prepared and a layer-by-layer self assembly electrochemical immunosensor was innovatively proposed for detecting silk traces based on flexible carbon cloth. The immunosensor possessed a broad linear range of 10-2-103 ng mL-1 and a detection limit of 2.15 pg mL-1 for the ultrasensitive detection of Bombyx mori silk traces. In addition, the elaborate immunosensor exhibited satisfactory high specificity, storage stability and reproducibility. In particular, the qualitative and quantitative performance of the immunosensor was excellent in the analysis of archaeological samples. Therefore, this work demonstrates that the proposed method not only provides a reliable analytical tool for exploring the origin and spread of archaeological silk, but also improves our understanding of how to use emerging materials like two-dimensional titanium carbide to creat innovative biosensors.

A pilot study of stable isotope fractionation in Bombyx mori rearing.

Hydrogen, oxygen, carbon, and nitrogen isotopes derived from three different strains of silkworms at different life stages involved in silkworm rearing, were measured to understand the fractionation characteristics of stable isotopes at different stages of silkworm development, and to trace the movement of these isotopes from food to larva to excrement and finally to silk. We found that silkworm strain had little effect on δ2H, δ18O and δ13C values. However, a large difference was found in the δ15N levels of newly-hatched silkworms between Jingsong Haoyue and Hua Kang No. 3 orthogonal strains, suggesting that the mating and egg laying differences may result in an inconsistent kinetic nitrogen isotope fractionation. The δ13C values of silkworm pupae and silkworm cocoon also displayed significant differences, suggesting that heavy carbon isotopes are greatly fractionated from the larva to the silk during cocoon formation. Overall, these results may be used to clarify the relationship between isotope fractionation and the ecological process of the Bombyx mori and expand our ability to resolve stable isotope anomalies at a small regional-scale level.

Hepatocellular carcinoma detection via targeted enzymatic methyl sequencing of plasma cell-free DNA.

BACKGROUND: Epigenetic variants carried by circulating tumor DNA can be used as biomarkers for early detection of hepatocellular carcinoma (HCC) by noninvasive liquid biopsy. However, traditional methylation analysis method, bisulfite sequencing, with disadvantages of severe DNA damage, is limited in application of low-amount cfDNA analysis. RESULTS: Through mild enzyme-mediated conversion, enzymatic methyl sequencing (EM-seq) is ideal for precise determination of cell-free DNA methylation and provides an opportunity for HCC early detection. EM-seq of methylation control DNA showed that enzymatic conversion of unmethylated C to U was more efficient than bisulfite conversion. Moreover, a relatively large proportion of incomplete converted EM-seq reads contains more than 3 unconverted CH site (CH = CC, CT or CA), which can be removed by filtering to improve accuracy of methylation detection by EM-seq. A cohort of 241 HCC, 76 liver disease, and 279 normal plasma samples were analyzed for methylation value on 1595 CpGs using EM-seq and targeted capture. Model training identified 283 CpGs with significant differences in methylation levels between HCC and non-HCC samples. A HCC screening model based on these markers can efficiently distinguish HCC sample from non-HCC samples, with area under the curve of 0.957 (sensitivity = 90%, specificity = 97%) in the test set, performing well in different stages as well as in serum α-fetoprotein/protein induced by vitamin K absence-II negative samples. CONCLUSION: Filtering of reads with ≥ 3 CHs derived from incomplete conversion can significantly reduce the noise of EM-seq detection. Based on targeted EM-seq analysis of plasma cell-free DNA, our HCC screening model can efficiently distinguish HCC patients from non-HCC individuals with high sensitivity and specificity.

Ultrasensitive dual enhanced electrochemical immunosensor to detect ancient wool relics.

The identification of ancient wool is of great significance in archaeology; however, conventional methods are unwieldy or even ineffective when testing contaminated or severely degraded ancient samples. Therefore, it is highly desirable to develop an ultrasensitive detection method for ancient wool. In this study, an ultrasensitive electrochemical immunosensor is proposed and developed to detect ancient wool, where graphene oxide (GO), aldehyde-functionalized ionic liquid (AFIL) composites and gold nanoparticles (AuNPs) are synthesized as efficient signal amplifiers. With their large surface area and excellent electron transfer efficiency, the combination of GO-AFIL and AuNPs endows the immunosensor with excellent electrochemical properties. The fabricated immunosensor measures over a wide linear range of 0.01-100 ng mL-1 with a low detection limit of 0.9 ± 0.2 pg mL-1. Moreover, the immunosensor demonstrates excellent performance for detecting ancient wool. The identification of wool fabrics unearthed from Xinjiang, Tibet and Kazakhstan supports the historicity of prosperous sheepherding and wool trade in Central Asia during the Bronze Age.

Rapid Enrichment and Detection of Silk Residues from Tombs by Double-Antibody Sandwich ELISA Based on Immunomagnetic Beads.

The extraction and identification of silk residues in tombs is of great significance for studying the distribution and spread of early silk. However, the complex organic matter in the tomb hinders the accurate identification of silk. In this study, a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) based on immunomagnetic beads (IMBs) was developed for the rapid enrichment and detection of silk residues. The double-antibody sandwich ELISA method established by pairing the IMBs prepared by the silk fibroin monoclonal antibody SF-3 and the silk fibroin monoclonal-labeled antibody bio-SF-1 had the highest detection sensitivity, with a linear detection range of 10 to 104 ng mL-1 and a detection limit of 5.12 ng mL-1. This method was excellent in the extraction and analysis of silk residues from archaeological imprints and soil samples and successfully identified silk residues in samples at the final stage of silk degradation (physical invisible silk). The proteomics analysis results demonstrated the feasibility and practicability of this method.

Proteomics and Immunology Provide Insight into the Degradation Mechanism of Historic and Artificially Aged Silk.

The process and mechanism of silk degradation is still a bewildering mystery in the investigation and conservation of cultural relics, which rely on the development of accurate and tailored analysis technologies. Here, two advanced approaches, proteomics and immunology, were developed for determining the deterioration behavior of historic silk fabrics and artificially aged samples from the molecular to the holistic level. The surface morphology and secondary structure of silk were destroyed during degradation. Subsequently, the proteomics and immunology analysis demonstrated a new degradation model differing from previous reports. First, the amorphous region and the looser crystalline regions were destroyed together, and the macromolecular chains were broken randomly. Then, the tight ß-sheet blocks in the crystalline region were exposed and deteriorated, which expedited the degradation of tight ß-sheet blocks and relatively loose blocks in the crystalline domain as well as the amorphous domain, ultimately yielding small molecule polypeptides. However, the deterioration process of ancient fabrics could be accelerated by poor burial conditions, thus showing distinct destructive characteristics. Overall, the results gave us a more comprehensive and profound understanding of the degradation process of ancient silk.

Ultrasensitive Electrochemical Immunosensor Reveals the Existence of Silk Products on the Maritime Silk Road.

The Maritime Silk Road was the major trade route between eastern and western civilizations in the Middle Ages. However, hardly any silk products have been found along the transoceanic trade route. Thus, the extrasensitive detection of silk relic traces has tremendous importance in research regarding the Maritime Silk Road. In this study, an electrochemical immunosensor based on a tailored monoclonal antibody and gold nanoparticles using the layer-by-layer self-assembly method was devised. The fabricated immunosensor demonstrated preeminent performance in the analysis of silk fibroin, with a linear detection range of 0.01-100 ng mL-1 and a detection limit of 3.8 pg mL-1. In particular, the performance of the immunosensor was excellent in the analysis of ancient silk samples, especially in the qualitative and quantitative detection of soil samples extracted from Nanhai No. 1 shipwreck archeological sites. The proposed electrochemical immunosensor proves the existence of silk products on the Maritime Silk Road and demonstrates enormous potential for studying the formation and development of the ancient transoceanic trading route.

Tailored monoclonal antibody as recognition probe of immunosensor for ultrasensitive detection of silk fibroin and use in the study of archaeological samples.

The ultrasensitive detection of fibroin in unearthed silk relics has great significance for investigating the origin and transmission of silk. In this study, an anti-fibroin monoclonal antibody was successfully prepared through animal immunization. Next, a label-free electrochemical immunosensor was fabricated using layer-by-layer self-assembly technology, and an indirect enzyme-linked immunosorbent assay (ELISA) was proposed. The two methods exhibited excellent sensitivity and specificity in the detection of silk fibroin, while the immunosensor showed a wider quantitative detection range (0.1-100 ng mL-1) and a lower detection limit (0.051 ng mL-1) than ELISA (10-100 ng mL-1 and 8.71 ng mL-1). Furthermore, the performance of the immunosensor was superior in archaeological sample detection. Taking advantage of the well-prepared monoclonal antibody, the two proposed immunological assays demonstrate tremendous potential for the ultrasensitive detection of silk fibroin, which can make great contributions to exploring the origin and transmission routes of ancient silks.

Comparative analysis of proteins from Bombyx mori and Antheraea pernyi cocoons for the purpose of silk identification.

Achieving efficient identification of silk protein requires highly sensitive analytical techniques and favorable extraction methods, which is of great significance to the research of ancient silk, especially for the controversial issue of the silk origin. In this paper, proteomics and western blot were proposed to analyze the silk proteins of Bombyx mori (B. mori) and Antheraea pernyi (A. pernyi) dissolved by different methods. First, the differences in secondary structure were detected via spectroscopy. LC-MS/MS was then employed to characterize the peptides of silk proteins precisely. LiBr solution exhibited outstanding dissolution effect on B. mori cocoon, with 87 proteins detected; while copper-ethylenediamine solution (CED) was more appropriate for A. pernyi cocoon, and 16 proteins were identified in A. pernyi-CED. In addition to fibroin and sericin, abundant seroins, enzymes, protease inhibitors, other functional proteins and uncharacterized proteins were detected. Based on the LC-MS/MS data, diagnostic antibodies for the two species were prepared, and fibroin was successfully identified by western blot assay because both dissolution methods were gentle and did not destroy the antigenic epitopes in the protein molecule. Owing to their good specificity and high sensitivity, these diagnostic antibodies have good application prospects in immunoassays of different silk species. SIGNIFICANCE: This study presents the comprehensive analysis on silk identification of proteins from B. mori and A. pernyi extracted by different methods via the proteomic and immunology as well as the conventional approaches. Great coverage of two cocoon proteomes was accomplished, which demonstrated the outstanding difference in components and abundance. Based on the proteomics analysis, the diagnostic antibodies against two species were prepared and identified the corresponding fibroin successfully in the completed protein mixtures. To our knowledge, the proteomic and immunology procedures with high efficiency, sensitivity and specificity are novel analysis on the silk identification and has great potential in the field of ancient silk detection.

Degradation Behavior and Immunological Detection of Silk Fibroin Exposure to Enzymes.

The degradation behavior of silk fibroin (SF) is a significant and intriguing subject in the area of archaeological ancient silk research. In the present study, the immunological detection techniques combined with traditional characterization methods, jointly studied the degradation process of SF from Bombyx mori (B. mori) and Antheraea pernyi (A. pernyi) through exposure to alkaline proteinase, α-chymotrypsin, pepsin, and trypsin. Spectroscopic analysis revealed that different enzymes showed similar hydrolysis effects on the secondary structure, but the changes of B. mori SF and A. pernyi SF were mainly reflected in the decrease of ß-sheet and the reduction of α-helical structure, respectively. In further research of immunology, two diagnostic antibodies were prepared corresponding to SF of B. mori and A. pernyi, respectively. The enzyme-linked immunosorbent assay (ELISA) and western blot indicated the enzyme-treated SF proteins still exhibited higher immunoreactivity because the epitopes on the surface of SF molecules are retained. Although α-chymotrypsin possesses the most cleavage sites among these enzymes, the α-chymotrypsin-treated SF did not exhibit significant changes in secondary structures and high antibody binding capacity. The results deepen our understanding of the SF degradation process during enzymatic hydrolysis, and show far-reaching guiding significance in trace detection of SF.

Species identification of Bombyx mori and Antheraea pernyi silk via immunology and proteomics.

In recent years, increasing attention has been paid to the origin, transmission and communication of silk. However, this is still an unsolved mystery in archaeology. The identification of silk-producing species, especially silk produced by Bombyx mori (B. mori) and Antheraea pernyi (A. pernyi), is of key significance to address this challenge. In this study, two innovative methods, i.e. immunology and proteomics, were proposed and successfully established for the species identification of silks. ELISAs result demonstrated that the two prepared antibodies exhibited high sensitivity and specificity in distinguishing B. mori and A. pernyi silk. No cross-reactivity with each other was observed. Moreover, biomarkers were obtained for Bombyx and Antheraea through proteomic analysis. It was also confirmed that the biomarkers were suitable for identifying the species that produced a given silk sample. Compared with conventional methods for distinguishing silk species, immunological and proteomics techniques used in tandem can provide intact information and have the potential to provide accurate and reliable information for species identification.

Sesquiterpenes from Cyperus rotundus and 4α,5α-oxidoeudesm-11-en-3-one as a potential selective estrogen receptor modulator.

Estrogenic activity-oriented fractionation and purification of methanol extract from the rhizome of Cyperus rotundus, a well-known traditional herbal medicine, led to the isolation of six sesquiterpenes. 4α,5α-Oxidoeudesm-11-en-3-one (2) and cyper-11-ene-3,4-dione (3) together with four known sesquiterpenes, cyperotundone (1), caryophyllene α-oxide (4), α-cyperone (5), and isocyperol (6) were obtained from the hexane and dichloromethane fractions. Compounds 2 and 3 were newly isolated from natural resources in particular. To identify the possible use of isolated compounds as an alternative to hormone replacement therapy (HRT), estrogenic activity was evaluated by E-screen assay on MCF-7 BUS cells. Among the all isolated compounds from the rhizome of Cyperus rotundus, newly isolated from natural resource, 2 exhibited the most potent estrogenic activity. In an estrogen sensitive reporter gene assay, 2 significantly increased transcriptional activities. As a phytoestrogen, 2 was assessed by investigating dual action on ER-α and ER-ß in competitive binding assay. It was found that 2 exerted higher binding affinity to ER-ß than ER-α and it also showed both estrogenic and antiestrogenic effects depending on the E2 concentration. Our results indicate that newly isolated from Cyperus rotundus, 2 has biphasic activities on estrogen receptors which could be useful as an alternative HRT.

Species Identification of Silks from Bombyx mori, Eri Silkworm and Chestnut Silkworm Using Western Blot and Proteomics Analyses.

Species identification is of key significance for exploring the origin and transmission of ancient silks. In this study, two novel methods, i.e. western blot (WB) and proteomics analyses, were proposed and established to identify the differences between silks from Bombyx mori (B. mori) and two other distinctive species (Eri silkworm and Chestnut silkworm). Three diagnostic antibodies, a polyclonal anti-silk fibroin (anti-SF) antibody (pAb), a polyclonal anti-SF-specific peptide antibody (pAsb), and a monoclonal anti-SF antibody (mAb) were designed and prepared to distinguish silk species using the antibody-based WB technique. Proteomics analysis by liquid chromatography-tandem mass spectrometry was performed to further identify silk species at the protein level. WB results indicated that the three antibodies showed high specificity and affinity and could discern B. mori silk from Eri and Chestnut silks. Biomarkers for each SF were obtained using proteomics analysis, and they have the potential to serve as standards for identifying silk species. Thus, combining WB and proteomics analyses with conventional methods can provide more accurate silk information and may be suitable for identifying other proteinaceous materials in archaeological field.

Development of an Enzyme-Linked Immunosorbent Assay and Gold-Labelled Immunochromatographic Strip Assay for the Detection of Ancient Wool.

The identification of ancient wool is of great importance in archaeology. Despite lots of meaningful information can be achieved by conventional detection methods, that is, light and electron microscopy, spectroscopy, and chromatography, the efficacy is likely to be limited in the detection of ancient samples with contamination or severe degradation. In this work, an immunoassay was proposed and performed for the identification of ancient wool. First, a specific antibody, which has the benefits of low cost, easy operation, and extensive applicability, was developed directly through immunizing rabbits with complete antigen (keratin). Then, an enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-labelled immunochromatographic strip (ICS) were developed to qualitatively identify the corresponding protein in ancient wool samples unearthed from Kazakhstan and China. The anti-keratin antibody exhibited high sensitivity and specificity for the identification of modern and ancient wool. The limit of detection (LOD) of the ELISA method was 10 ng/mL, and no cross-reactions with other interfering antigens have been noted. It is concluded that the immunoassays are reliable methods for the identification of ancient wool.

Discerning Silk Produced by Bombyx mori from Those Produced by Wild Species Using an Enzyme-Linked Immunosorbent Assay Combined with Conventional Methods.

Recently, much interest has been paid to the separation of silk produced by Bombyx mori from silk produced by other species and tracing the beginnings of silk cultivation from wild silk exploitation. In this paper, significant differences between silks from Bombyx mori and other species were found by microscopy and spectroscopy, such as morphology, secondary structure, and amino acid composition. For further accurate identification, a diagnostic antibody was designed by comparing the peptide sequences of silks produced by Bombyx mori and other species. The results of the noncompetitive indirect enzyme-linked immunosorbent assay (ELISA) indicated that the antibody that showed good sensitivity and high specificity can definitely discern silk produced by Bombyx mori from silk produced by wild species. Thus, the antibody-based immunoassay has the potential to be a powerful tool for tracing the beginnings of silk cultivation. In addition, combining the sensitive, specific, and convenient ELISA technology with other conventional methods can provide more in-depth and accurate information for species identification.

Lanthanide-Labeled Immunochromatographic Strip Assay for the On-Site Identification of Ancient Silk.

The on-site identification of ancient silks has long been a key challenge in archeology. Therefore, a rapid, cost-effective, sensitive analytical approach is highly desirable. In this paper, a lanthanide-labeled immunochromatographic strip which is suitable for the on-site identification of ancient silks is described. Compared with the conventional colloidal gold-based immunochromatographic strip, this strip shows much higher analytical sensitivity and better quantitative discrimination. The limit of detection (LOD) of the strip for silk fibroin (SF) was calculated as 8.09 ng/mL, approximately 185 times lower than that of the colloidal gold-based immunochromatographic strip. No cross-reactions with other possible interfering antigens were observed. Moreover, the strip also shows good reproducibility, with a mean recovery of 94.15-102.55% and coefficient of variation of 5.22-17.57% in the repeated tests. Based on the advantages of portability and cost-effectiveness, as well as sensitivity, specificity, and reproducibility, the lanthanide-labeled immunochromatographic strip is a promising tool for on-site detection of ancient relics in archeological fieldwork.

An adaptive detection method for fetal chromosomal aneuploidy using cell-free DNA from 447 Korean women.

BACKGROUND: Noninvasive prenatal testing (NIPT) using massively parallel sequencing of cell-free DNA (cfDNA) is increasingly being used to predict fetal chromosomal abnormalities. However, concerns over erroneous predictions which occur while performing NIPT still exist in pregnant women at high risk for fetal aneuploidy. We performed the largest-scale clinical NIPT study in Korea to date to assess the risk of false negatives and false positives using next-generation sequencing. METHODS: A total of 447 pregnant women at high risk for fetal aneuploidy were enrolled at 12 hospitals in Korea. They underwent definitive diagnoses by full karyotyping by blind analysis and received aneuploidy screening at 11-22 weeks of gestation. Three steps were employed for cfDNA analyses. First, cfDNA was sequenced. Second, the effect of GC bias was corrected using normalization of samples as well as LOESS and linear regressions. Finally, statistical analysis was performed after selecting a set of reference samples optimally adapted to a test sample from the whole reference samples. We evaluated our approach by performing cfDNA testing to assess the risk of trisomies 13, 18, and 21 using the sets of extracted reference samples. RESULTS: The adaptive selection algorithm presented here was used to choose a more optimized reference sample, which was evaluated by the coefficient of variation (CV), demonstrated a lower CV and higher sensitivity than standard approaches. Our adaptive approach also showed that fetal aneuploidies could be detected correctly by clearly splitting the z scores obtained for positive and negative samples. CONCLUSIONS: We show that our adaptive reference selection algorithm for optimizing trisomy detection showed improved reliability and will further support practitioners in reducing both false negative and positive results.