First Insight into Fetal Exposure to Legacy and Emerging Plasticizers Revealed by Infant Hair and Meconium: Occurrence, Biotransformation, and Accumulation.

Epidemiological studies have demonstrated the embryonic and developmental toxicity of plasticizers. Thus, understanding the in utero biotransformation and accumulation of plasticizers is essential to assessing their fate and potential toxicity in early life. In the present study, 311 infant hair samples and 271 paired meconium samples were collected at birth in Guangzhou, China, to characterize fetal exposure to legacy and emerging plasticizers and their metabolites. Results showed that most of the target plasticizers were detected in infant hair, with medians of 9.30, 27.6, and 0.145 ng/g for phthalate esters (PAEs), organic phosphate ester (OPEs), and alternative plasticizers (APs), and 1.44, 0.313, and 0.066 ng/g for the metabolites of PAEs, OPEs, and APs, respectively. Positive correlations between plasticizers and their corresponding primary metabolites, as well as correlations among the oxidative metabolites of bis(2-ethylhexyl) phthalate (DEHP) and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH), were observed, indicating that infant hair retained the major phase-I metabolism of the target plasticizers. While no positive correlations were found in parent compounds or their primary metabolites between paired infant hair and meconium, significant positive correlations were observed among secondary oxidative metabolites of DEHP and DINCH in hair and meconium, suggesting that the primary metabolites in meconium come from hydrolysis of plasticizers in the fetus but most of the oxidative metabolites come from maternal-fetal transmission. The parent compound/metabolite ratios in infant hair showed a decreasing trend across pregnancy, suggesting in utero accumulation and deposition of plasticizers. To the best of our knowledge, this study is the first to report in utero exposure to both parent compounds and metabolites of plasticizers by using paired infant hair and meconium as noninvasive biomonitoring matrices and provides novel insights into the fetal biotransformation and accumulation of plasticizers across pregnancy.

Sparse2Noise: Low-dose synchrotron X-ray tomography without high-quality reference data.

BACKGROUND: Synchrotron radiation computed tomography (SR-CT) holds promise for high-resolution in vivo imaging. Notably, the reconstruction of SR-CT images necessitates a large set of data to be captured with sufficient photons from multiple angles, resulting in high radiation dose received by the object. Reducing the number of projections and/or photon flux is a straightforward means to lessen the radiation dose, however, compromises data completeness, thus introducing noises and artifacts. Deep learning (DL)-based supervised methods effectively denoise and remove artifacts, but they heavily depend on high-quality paired data acquired at high doses. Although algorithms exist for training without high-quality references, they struggle to effectively eliminate persistent artifacts present in real-world data. METHODS: This work presents a novel low-dose imaging strategy namely Sparse2Noise, which combines the reconstruction data from paired sparse-view CT scan (normal-flux) and full-view CT scan (low-flux) using a convolutional neural network (CNN). Sparse2Noise does not require high-quality reconstructed data as references and allows for fresh training on data with very small size. Sparse2Noise was evaluated by both simulated and experimental data. RESULTS: Sparse2Noise effectively reduces noise and ring artifacts while maintaining high image quality, outperforming state-of-the-art image denoising methods at same dose levels. Furthermore, Sparse2Noise produces impressive high image quality for ex vivo rat hindlimb imaging with the acceptable low radiation dose (i.e., 0.5 Gy with the isotropic voxel size of 26 µm). CONCLUSIONS: This work represents a significant advance towards in vivo SR-CT imaging. It is noteworthy that Sparse2Noise can also be used for denoising in conventional CT and/or phase-contrast CT.

Low-density tissue scaffold imaging by synchrotron radiation propagation-based imaging computed tomography with helical acquisition mode.

Visualization of low-density tissue scaffolds made from hydrogels is important yet challenging in tissue engineering and regenerative medicine (TERM). For this, synchrotron radiation propagation-based imaging computed tomography (SR-PBI-CT) has great potential, but is limited due to the ring artifacts commonly observed in SR-PBI-CT images. To address this issue, this study focuses on the integration of SR-PBI-CT and helical acquisition mode (i.e. SR-PBI-HCT) to visualize hydrogel scaffolds. The influence of key imaging parameters on the image quality of hydrogel scaffolds was investigated, including the helical pitch (p), photon energy (E) and the number of acquisition projections per rotation/revolution (Np), and, on this basis, those parameters were optimized to improve image quality and to reduce noise level and artifacts. The results illustrate that SR-PBI-HCT imaging shows impressive advantages in avoiding ring artifacts with p = 1.5, E = 30 keV and Np = 500 for the visualization of hydrogel scaffolds in vitro. Furthermore, the results also demonstrate that hydrogel scaffolds can be visualized using SR-PBI-HCT with good contrast while at a low radiation dose, i.e. 342 mGy (voxel size of 26 µm, suitable for in vivo imaging). This paper presents a systematic study on hydrogel scaffold imaging using SR-PBI-HCT and the results reveal that SR-PBI-HCT is a powerful tool for visualizing and characterizing low-density scaffolds with a high image quality in vitro. This work represents a significant advance toward the non-invasive in vivo visualization and characterization of hydrogel scaffolds at a suitable radiation dose.

In situ wet pharmaceutical granulation captured using synchrotron radiation based dynamic micro-CT.

Synchrotron radiation based dynamic micro-computed tomography (micro-CT) is a powerful technique available at synchrotron light sources for investigating evolving microstructures. Wet granulation is the most widely used method of producing pharmaceutical granules, precursors to products like capsules and tablets. Granule microstructures are known to influence product performance, so this is an area for potential application of dynamic CT. Here, lactose monohydrate (LMH) was used as a representative powder to demonstrate dynamic CT capabilities. Wet granulation of LMH has been observed to occur on the order of several seconds, which is too fast for lab-based CT scanners to capture the changing internal structures. The superior X-ray photon flux from synchrotron light sources makes sub-second data acquisition possible and well suited for analysis of the wet-granulation process. Moreover, synchrotron radiation based imaging is non-destructive, does not require altering the sample in any way, and can enhance image contrast with phase-retrieval algorithms. Dynamic CT can bring insights to wet granulation, an area of research previously only studied via 2D and/or ex situ techniques. Through efficient data-processing strategies, dynamic CT can provide quantitative analysis of how the internal microstructure of an LMH granule evolves during the earliest moments of wet granulation. Here, the results revealed granule consolidation, the evolving porosity, and the influence of aggregates on granule porosity.

4D study of liquid binder penetration dynamics in pharmaceutical powders using synchrotron X-ray micro computed tomography.

The properties of pharmaceutical powders, and the liquid binder, directly influence the penetration behavior in the wet granulation process of the pharmaceutical industry. Conventional methods encounter challenges in understanding this fast process. In this work, an emerging synchrotron-based X-ray imaging technique (having fast imaging capability) was employed to investigate the internal process from 2D and 3D to real-time (in-situ with ms time intervals) 3D (also considered 4D) perspectives. Two commonly used excipients (lactose monohydrate (LMH) and microcrystalline cellulose (MCC)) were used to make binary mixtures with acetaminophen (APAP) as the active pharmaceutical ingredient (API). Isopropanol and water were employed as liquid binders in the single droplet impact method. Results showed that for most of the mixtures, the porosity increased at higher fractions of APAP. MCC mixtures experienced less agglomeration and more uniform pore distribution than LMH ones, resulting in a faster droplet penetration with isopropanol. Moreover, the imbibition-spreading studies showed that isopropanol penetration in MCC powders followed more unidirectional vertical movement than horizontal spreading. Our results also demonstrated that simultaneous granulation of LMH with water resulted in much slower penetration. This study revealed that synchrotron X-ray imaging can investigate 3D internal pore structures and how they affect the quantitively real-time internal penetration dynamics.

Long-Read RNA Sequencing Identifies Alternative Splice Variants in Hepatocellular Carcinoma and Tumor-Specific Isoforms.

Alternative splicing (AS) allows generation of cell type-specific mRNA transcripts and contributes to hallmarks of cancer. Genome-wide analysis for AS in human hepatocellular carcinoma (HCC), however, is limited. We sought to obtain a comprehensive AS landscape in HCC and define tumor-associated variants. Single-molecule real-time long-read RNA sequencing was performed on patient-derived HCC cells, and presence of splice junctions was defined by SpliceMap-LSC-IDP algorithm. We obtained an all-inclusive map of annotated AS variants and further discovered 362 alternative spliced variants that are not previously reported in any database (neither RefSeq nor GENCODE). They were mostly derived from intron retention and early termination codon with an in-frame open reading frame in 81.5%. We corroborated many of these predicted unannotated and annotated variants to be tumor specific in an independent cohort of primary HCC tumors and matching nontumoral liver. Using the combined Sanger sequencing and TaqMan junction assays, unique and common expressions of spliced variants including enzyme regulators (ARHGEF2, SERPINH1), chromatin modifiers (DEK, CDK9, RBBP7), RNA-binding proteins (SRSF3, RBM27, MATR3, YBX1), and receptors (ADRM1, CD44v8-10, vitamin D receptor, ROR1) were determined in HCC tumors. We further focused functional investigations on ARHGEF2 variants (v1 and v3) that arise from the common amplified site chr.1q22 of HCC. Their biological significance underscores two major cancer hallmarks, namely cancer stemness and epithelial-to-mesenchymal transition-mediated cell invasion and migration, although v3 is consistently more potent than v1. Conclusion: Alternative isoforms and tumor-specific isoforms that arise from aberrant splicing are common during the liver tumorigenesis. Our results highlight insights gained from the analysis of AS in HCC.

The inhibitory effect of gypenoside stereoisomers, gypenoside L and gypenoside LI, isolated from Gynostemma pentaphyllum on the growth of human lung cancer A549 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Gypenosides are major constituents in Gynostemma pentaphyllum (Thunb.) Makino. Previous studies have shown that gypenosides isolated from G. pentaphyllum possess inhibitory effect on the growth of cancer cells, especially A549 cells, with structure-activity relationship (SAR). However, the underlying mechanism of gypenoside-induced A549 cell death remains to be clarified. AIM OF THE STUDY: To further investigate SAR and the underlying mechanism of gypenosides in A549 cells. MATERIALS AND METHODS: Gypenosides were isolated from G. pentaphyllum using chromatography methods and identified using MS and NMR data. The cytotoxicity was determined with CCK-8 assay. The effects of gypenosides on apoptosis, cell cycle and migration were investigated through cell morphology observation, flow cytometry analysis and key proteins detection. RESULTS: Three gypenosides, 2α,3ß,12ß,20(S)-tetrahydroxydammar-24-ene-3-O-ß-D-glucopyranoside-20-O-ß-D-glucopyranoside, gypenoside L and gypenoside LI were isolated from G. pentaphyllum. Gypenoside stereoisomers, gypenoside L (S configuration at C20) and gypenoside LI (R configuration at C20) showed stronger activity against A549 cells. Furthermore, both induced A549 cell apoptosis through intrinsic and extrinsic pathways evidenced by reducing mitochondrial membrane potential (MMP), generating reactive oxygen species (ROS), releasing more cytochrome c and down-regulating procaspase 8. However, gypenoside L blocked A549 cells in G0/G1, while gypenoside LI induced G2/M arrest, which was further verified by different expression of CDK1, CDK2 and CDK4. In addition, both inhibited A549 cell migration, which was evidenced by down-regulation of MMP-2/9 as well as scratch wound assay and transwell assay. CONCLUSION: C20 of gypenoside played an important role in A549 cell cytotoxicity and gypenoside stereoisomers could be used as potential multi-target chemopreventive agents for cancer.

Copy number variation analysis based on AluScan sequences.

BACKGROUND: AluScan combines inter-Alu PCR using multiple Alu-based primers with opposite orientations and next-generation sequencing to capture a huge number of Alu-proximal genomic sequences for investigation. Its requirement of only sub-microgram quantities of DNA facilitates the examination of large numbers of samples. However, the special features of AluScan data rendered difficult the calling of copy number variation (CNV) directly using the calling algorithms designed for whole genome sequencing (WGS) or exome sequencing. RESULTS: In this study, an AluScanCNV package has been assembled for efficient CNV calling from AluScan sequencing data employing a Geary-Hinkley transformation (GHT) of read-depth ratios between either paired test-control samples, or between test samples and a reference template constructed from reference samples, to call the localized CNVs, followed by use of a GISTIC-like algorithm to identify recurrent CNVs and circular binary segmentation (CBS) to reveal large extended CNVs. To evaluate the utility of CNVs called from AluScan data, the AluScans from 23 non-cancer and 38 cancer genomes were analyzed in this study. The glioma samples analyzed yielded the familiar extended copy-number losses on chromosomes 1p and 9. Also, the recurrent somatic CNVs identified from liver cancer samples were similar to those reported for liver cancer WGS with respect to a striking enrichment of copy-number gains in chromosomes 1q and 8q. When localized or recurrent CNV-features capable of distinguishing between liver and non-liver cancer samples were selected by correlation-based machine learning, a highly accurate separation of the liver and non-liver cancer classes was attained. CONCLUSIONS: The results obtained from non-cancer and cancerous tissues indicated that the AluScanCNV package can be employed to call localized, recurrent and extended CNVs from AluScan sequences. Moreover, both the localized and recurrent CNVs identified by this method could be subjected to machine-learning selection to yield distinguishing CNV-features that were capable of separating between liver cancers and other types of cancers. Since the method is applicable to any human DNA sample with or without the availability of a paired control, it can also be employed to analyze the constitutional CNVs of individuals.

[Effects of local carbon ion irradiation on survival and peripheral blood leukocyte and platelet counts of tumor-bearing mice].

OBJECTIVE: To investigate the effects of local carbon ion irradiation on the length of survival and peripheral blood leukocyte and platelet counts of mice inoculated with pulmonary tumor cells. METHODS: Thirty tumor-bearing mice were randomly divided into control group (tumor-bearing but without carbon ion irradiation, n=10), 12 Gy group (n=10) and 24 Gy group (n=10). The right hind limbs of the mice, where the tumor cells were inoculated, were irradiated with carbon-ion beams at a single dose in 12 Gy and 24 Gy groups, and those of the control group received no irradiation. The peripheral blood leukocytes and platelets of the mice in all the 3 groups were counted immediately before and 7 and 14 days after irradiation, respectively, with the survival time of the mice recorded. RESULTS: There was no significant difference in the survival time of the mice between the 3 groups. The peripheral blood leukocyte counts in all groups increased after irradiation, no significant difference was noted between the two irradiation the groups and the control group. Irradiation at 24 Gy resulted in significant increase in peripheral blood platelet count on the 14th day (P=0.032, F=4.062), but no such increment was observed in the other 2 groups or on day 7 in the 24 Gy group. CONCLUSION: Local carbon ion irradiation may not produce significant effects on the length of survival and hemogram of the tumor-bearing mice.