In situ detection of silk fibroin using a dual recognition strategy with a flexible pressure immunosensor.

Silk is a symbol of ancient Chinese civilization that has made an indelible contribution to the development of world civilization. However, because ancient artifacts are often contaminated or degraded, it is difficult to detect the presence of silk therein, and the true origin of silk thus remains a mystery. Therefore, this work presents a flexible pressure immunosensor that was designed based on 3D polypyrrole (PPy) foams for the trace detection of silk fibroin at archaeological sites. Initially, silk fibroin (SF) was conjugated with antibody-functionalized copper oxide nanoparticles (CuO NPs) and carboxylated magnetic beads (MBs) to form a sandwich immune complex. Then, the sandwich immune complex was added to hydrogen peroxide (H2O2) by magnetic separation to catalyse the generation of oxygen (O2), which converted the antigen-antibody specific recognition signal to gas pressure. As the pressure within the device increases, the 3D PPy foam, as the sensing layer resistance was 150 Ω, undergoes extrusion and deformation. This deformation leads to alterations in the foam resistance. The flexible pressure immunosensor can sensitively monitor the change in electrical resistance in the system and quantitatively detect silk fibroin. With optimization, the flexible pressure immunosensor demonstrates a dynamic range of operation spanning from 10 ng mL-1 to 100 µg mL-1, exhibiting a remarkable detection limit of 10.58 ng mL-1 specifically for silk fibroin. Notably, this immunosensor surpasses enzyme-linked immunosorbent assay (ELISA) in terms of superior reproducibility, specificity, and accuracy. Therefore, this application provides a new method and technical support for silk 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.

Geographical origin traceability of mulberry leaves using stable hydrogen, oxygen, and carbon isotope ratios.

Geographical discrimination of mulberry leaves is very important for their efficacy and quality as a traditional Chinese medicine. Stable hydrogen, oxygen, and carbon isotope ratios were measured in 292 mulberry leaves collected at 2 growth stages in 2 seasons from 8 regions of China. A stepwise linear discriminant analysis (LDA) approach were proposed to combine with stable isotope technology to tracing the origin of mulberry leaves. The results showed that leaves sampled in autumn were extremely depleted in 2H and 18O and slightly enriched in 13C compared with leaves sampled in summer, correlated with the effect of season, transpiration and photorespiration on stable isotopes. δ2H and δ18O of the leaves were enriched during the growth process. The overall discrimination accuracy of the autumn tender model was 81%, demonstrating that analysis of δ2H, δ18O, and δ13C is a promising technique for tracing the geographical origin of mulberry leaves, although season, growth stage and number of samples affect the accuracy of discrimination.

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.

Identification, Fine Mapping and Application of Quantitative Trait Loci for Grain Shape Using Single-Segment Substitution Lines in Rice (Oryza sativa L.).

Quantitative trait loci (QTLs) and HQTL (heterosis QTLs) for grain shape are two major genetic factors of grain yield and quality in rice (Oryza sativa L.). Although many QTLs for grain shape have been reported, only a few are applied in production. In this study, 54 QTLs for grain shape were detected on 10 chromosomes using 33 SSSLs (single-segment substitution lines) and methods of statistical genetics. Among these, 23 exhibited significant positive additive genetic effects, including some novel QTLs, among which qTGW4-(1,2), qTGW10-2, and qTGW10-3 were three QTLs newly found in this study and should be paid more attention. Moreover, 26 HQTLs for grain shape were probed. Eighteen of these exhibited significant positive dominant genetic effects. Thirty-three QTLs for grain shape were further mapped using linkage analysis. Most of the QTLs for grain shape produced pleiotropic effects, which simultaneously controlled multiple appearance traits of grain shape. Linkage mapping of the F2 population derived from sub-single-segment substitution lines further narrowed the interval harbouring qTGW10-3 to 75.124 kb between PSM169 and RM25753. The candidate gene was identified and could be applied to breeding applications by molecular marker-assisted selection. These identified QTLs for grain shape will offer additional insights for improving grain yield and quality in rice breeding.

Pilot study on provenance tracing of cocoons via strontium isotopes.

Isotopic analysis has seen significant use in archaeological sciences to date objects, determine their origin, and depict ancient human dietary habits. However, the potential of this technique for provenance studies of ancient silks remains underdeveloped. In this study, we applied Sr isotopic ratios to the provenance tracing of silkworm cocoons. We investigated the 87Sr/86Sr ratios of cocoons from seven Chinese provinces to determine their regional differences. The 87Sr/86Sr ratios of mulberry leaves and cocoons in Shandong and Sichuan were analyzed and silkworms were cultured at four sampling locations in Hangzhou, Zhejiang, to determine isotopic signature relationships between mulberry leaves and cocoons. Those results showed that the 87Sr/86Sr signature of cocoons not only reflected regional differences, but also was related to the type of soil in each sampling location from which the samples were collected. It is suggested that the Sr isotope ratios was able to be an effective tool for the provenance tracing of cocoons. The Mann-Whitney test result indicated no significant differences in isotopic ratios between mulberry leaves and cocoons. In other words, mulberry leaves may predict mean isotopic values in the cocoons. No clear evidence of Sr isotopic fractionation was found in our control experiments. However, mulberry leaves and cocoons from Sichuan did not show significant correlation between them, overall reducing the predictive power of the 87Sr/86Sr ratios of mulberry leaf for provenance studies of cocoons. Finally, in order to improve the accuracy of Sr isotope ratios for the provenance tracing of cocoons, more 87Sr/86Sr data should be complemented and the relationship needs to be established between Sr isotope information in more kinds of proxies and cocoons.

Dynamic analysis of QTLs on plant height with single segment substitution lines in rice.

Dynamic regulation of QTLs remains mysterious. Single segment substitution lines (SSSLs) and conditional QTL mapping and functional QTL mappings are ideal materials and methods to explore dynamics of QTLs for complex traits. This paper analyzed the dynamics of QTLs on plant height with SSSLs in rice. Five SSSLs were verified with plant height QTLs first. All five QTLs had significant positive effects at one or more developmental stages except QTL1. They interacted each other, with negative effects before 49 d after transplanting and positive effects since then. The five QTLs selectively expressed in specific periods, mainly in the periods from 35 to 42 d and from 49 to 56 d after transplanting. Expressions of epistasis were dispersedly in various periods, negative effects appearing mainly before 35 d. The five QTLs brought the inflexion point ahead of schedule, accelerated growth and degradation, and changed the peak plant height, while their interactions had the opposite effects. The information will be helpful to understand the genetic mechanism for developmental traits.

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.

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.

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.

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.

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.

Nonsteroidal Anti-inflammatory Drugs Potently Inhibit the Replication of Zika Viruses by Inducing the Degradation of AXL.

Zika virus (ZIKV) is genetically and biologically related to other Flaviviridae family members and has disseminated to many countries. It is associated with severe consequences, including the abnormal development of the neural system in fetuses and neurological diseases in adults. Therefore, the development of anti-ZIKV drugs is of paramount importance. Screening of generic drugs revealed that several nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin, ibuprofen, naproxen, acetaminophen, and lornoxicam, potently inhibited the entry of Zika virus Env/HIV-1-pseudotyped viruses. They also significantly inhibited the replication of wild-type ZIKV both in cell lines and in primary human fetal endothelial cells. Interestingly, the NSAIDs exerted this inhibitory effect by potently reducing the expression of AXL, the entry cofactor of ZIKV. Further studies showed that the NSAIDs downregulated the prostaglandin E2/prostaglandin E receptor 2 (EP2)/cAMP/protein kinase A (PKA) signaling pathway and reduced PKA-dependent CDC37 phosphorylation and the interaction between CDC37 and HSP90, which subsequently facilitated CHIP/ubiquitination/proteasome-mediated AXL degradation. Taken together, our results highlight a new mechanism of action of antiviral agents which may assist in designing a convenient strategy for treating ZIKV-infected patients.IMPORTANCE Zika virus (ZIKV) infection, which causes congenital malformations, including microcephaly and other neurological disorders, has attracted global attention. We observed that several NSAIDs significantly inhibited ZIKV infection. Based on our observations, we propose a novel mechanism of action of antiviral compounds which involves the blockade of virus entry via degradation of the entry cofactor. Furthermore, NSAIDs can be practically used for preventing ZIKV infection in pregnant women, as certain NSAIDs, including ibuprofen and acetaminophen, are considered clinically safe.

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.

MoS2 quantum dots@TiO2 nanotube composites with enhanced photoexcited charge separation and high-efficiency visible-light driven photocatalysis.

MoS2 quantum dots (QDs) that are 5 nm in size were deposited on the surface of ultrathin TiO2 nanotubes (TNTs) with 5 nm wall thickness by using an improved hydrothermal method to form a MoS2 QDs@TNT visible-light photocatalyst. The ultrathin TNTs with high percentage of photocatalytic reactive facets were fabricated by the commercially available TiO2 nanoparticles (P25) through an improved hydrothermal method, and the MoS2 QDs were acquired by using a surfactant-assisted technique. The novel MoS2 QDs@TNT photocatalysts showed excellent photocatalytic activity with a decolorization rate of 92% or approximately 3.5 times more than that of pure TNTs for the high initial concentration of methylene blue solution (20 mg l-1) within 40 min under visible-light irradiation. MoS2 as the co-catalysts favored the broadening of TNTs into the visible-light absorption scope. The quantum confinement and edge effects of the MoS2 QDs and the heterojunction formed between the MoS2 QDs and TNTs efficiently extended the lifetime of photoinduced charges, impeded the recombination of photoexcited electron-hole pairs, and improved the visible-light-driven high-efficiency photocatalysis.

Application of Electron Paramagnetic Resonance and Solid-state 13C Nuclear Magnetic Resonance of Cross-polarization/Magic Angle Spinning to Study Enzymatic Degradation of Silk Fabrics.

The enzymatic degradation of silk by protease XIV has been investigated by using scanning electron microscopy (SEM), Fourier transfer infrared spectroscopy (FTIR), solid-state 13C nuclear magnetic resonance of cross-polarization/magic angle spinning (13C CP/MAS solid state NMR) and electron paramagnetic resonance (EPR). Micro-morphology of protease XIV aged samples showed that microfilaments were stripped out from the surface of silk fibers. The results of FTIR and 13C CP/MAS solid-state NMR indicated that the enzymatic degradation process could be divided into two stages. The EPR spectra indicated that the enzymatic degradation process was related to the free radical with the g-factor value of 2.0043. We also proposed that at the first degradation stage, the free radicals were apt to lose activities due to the loose structure of the non-crystalline region; at the second degradation stage, the free radicals produced in the crystalline region tended to be stored.

Mapping quantitative trait loci for heat tolerance at anthesis in rice using chromosomal segment substitution lines.

To study the genetic basis of heat tolerance at anthesis, a set of chromosome segment substitution lines (CSSLs) derived from Sasanishiki (japonica ssp. heat susceptible) and Habataki (indica spp. heat tolerant) were used for analysis across three high temperature environments. Spikelet fertility (SF), daily flowering time (DFT) and pollen shedding level (PSL) under high temperature (HT) were assessed. Eleven related QTLs were detected, of which, two QTLs qSF (ht) 2 and qSF (ht) 4.2 for spikelet fertility were identified on chromosomes 2 and 4. Four QTLs qDFT3, qDFT8, qDFT10.1 and qDFT11 for daily flowering time were detected on chromosomes 3, 8, 10 and 11. The other five QTLs qPSL (ht) 1, qPSL (ht) 4.1, qPSL (ht) 5, qPSL (ht) 7 and qPSL (ht) 10.2 on chromosomes 1, 4, 5, 7 and 10, respectively, were found had effects both on spikelet fertility and pollen shedding level. Of the 11 QTLs, 8 were overlapped with QTLs reported by others, 3 QTLs qPSL (ht) 4.1, qPSL (ht) 7 and qPSL (ht) 10.2 identified in this study were novel. The stability of qPSL (ht) 4.1 was further verified at different temperatures, which could be used to improve the pollen shedding and pollen growth on stigma for rice heat-tolerance breeding.

Characterization and fine mapping of a female fertility associated gene Ff1(t) in rice.

Female-sterile line can be used as a pollinator which has a great potential for hybrid seeds production. However, reports on female fertility are fewer than male fertility. Here, we characterized a recessive female fertility weakening mutant ff1(t) from rice. The spikelet fertility was seriously affected in the mutant. Reciprocal crosses and pollen vitality assay suggest that the decreased fertility was caused by the defective female gametophytes. Further investigation indicated that the mutant ovary development was inhibited before fertilization and failed swelling after flowering. Genetic analysis and fine mapping showed that the mutant was controlled by a single recessive gene, residing on a 16.8 kb region on the long arm of chromosome 1. The gene annotation indicated that there was only one putative gene encoding lysine decarboxylase-like protein in this region, which was allelic to LOG. Further, the sequence analysis was carried out and a substitution at the splice site of intron 2 / exon 3 was revealed in ff1(t) mutant, resulting in the change of reading frame. The finding of novel allele of LOG locus will facilitate the understanding of the mechanisms of female gametophyte development.