Global chromosome rearrangement induced by CRISPR-Cas9 reshapes the genome and transcriptome of human cells.

Chromosome rearrangement plays important roles in development, carcinogenesis and evolution. However, its mechanism and subsequent effects are not fully understood. Large-scale chromosome rearrangement has been performed in the simple eukaryote, wine yeast, but the relative research in mammalian cells remains at the level of individual chromosome rearrangement due to technical limitations. In this study, we used CRISPR-Cas9 to target the highly repetitive human endogenous retrotransposons, LINE-1 and Alu, resulting in a large number of DNA double-strand breaks in the chromosomes. While this operation killed the majority of the cells, we eventually obtained live cell groups. Karyotype analysis and genome re-sequencing proved that we have achieved global chromosome rearrangement (GCR) in human cells. The copy number variations of the GCR genomes showed typical patterns observed in tumor genomes. The ATAC-seq and RNA-seq further revealed that the epigenetic and transcriptomic landscapes were deeply reshaped by GCR. Gene expressions related to p53 pathway, DNA repair, cell cycle and apoptosis were greatly altered to facilitate the cell survival. Our study provided a new application of CRISPR-Cas9 and a practical approach for GCR in complex mammalian genomes.

Phase-type diffractive micro-optics elements in sulfur-based polymeric glass by femtosecond laser direct writing.

Sulfur-based polymeric glasses are promising alternative low-cost IR materials due to their profoundly high IR transparency. In this Letter, femtosecond-laser-induced refractive index change (RIC) was investigated in one typical sulfur-based polymeric glass material, poly(S-r-DIB), for the first time, to the best of our knowledge. The RIC in the laser-engineered region was quantitively characterized, which laid a foundation for phase-type optical element design. By the integration of RIC traces, embedded phase-type micro-optics elements, including Fresnel zone plates, and a Dammann grating were fabricated in bulk poly(S-r-DIB) polymeric glass substrate via the femtosecond laser direct writing technique. The imaging and beam shaping performance were demoed in the near-infrared (NIR) region.

Comparative performance of growth, vertebral structure and muscle composition in diploid and triploid Paralichthys olivaceus.

Growth, skeletal structure and muscle composition of cold-shock-induced triploid olive flounder Paralichthys olivaceus were investigated. The average values of total length and total weight of triploids were higher than those of diploids from 5 to 11 months posthatch (mph). The growth difference disappeared after 11 mph. The skeletal structure of flounder at 11 mph was observed by X-ray imaging method. There are four kinds of vertebral deformity including vertebrae fusion, one-sided compression, two-sided compression and vertically shifted. The trunk region (V8-18) and tailing end of the vertebral column were the predominant locations of deformity. In general, the frequencies of vertebral deformities in triploids (60.0%) were higher than those in diploids (33.3%, p < 0.05). Both the number of fish with deformed vertebrae and the average frequencies of deformed vertebrae in triploids were significantly higher than those in diploids (p < 0.05). The muscle tissues of diploid and triploid flounder at 11 mph contain the same types of fatty acid and free amino acid profiles. The number of fatty acids with significant higher contents in diploids and triploids was one and ten, respectively (p < 0.05). The contents of free amino acids showed no difference between triploid and diploid fish.

Identification of a Novel Prognostic Signature Based on N-Linked Glycosylation and Its Correlation with Immunotherapy Response in Hepatocellular Carcinoma.

Background: The complex tumor microenvironment of hepatocellular carcinoma (HCC) has led to a low response to immune checkpoints inhibitors (ICIs) and a poor prognosis. PD-L1, as one of the indications for ICIs, is rich in glycosylation modifications, which result in untimely ICIs. Our study constructed a prognostic model based on N-linked glycosylation related genes for predicting the prognosis and the response to ICIs. Methods: The list of N-linked glycosylation related genes is from the AmiGO2 database. The patients in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts were enrolled. The Cox regression was performed to develop a prognostic model and patients were divided into a low- and high-risk subgroups. The role of signature in HCC was well investigated by prognostic analysis, gene set enrichment analysis, and immune infiltration analysis. 21 recurrent HCC patients who received postoperative adjuvant ICIs were recruited to evaluate the relationship between immunotherapy response and the signature. In vitro studies were conducted to investigate the oncogenic effects of DDOST, STT3A and TMEM165 in HCC. Results: 59 N-linked glycosylation related differentially expressed genes were screened from HCC and normal tissues in the TCGA cohort. The prognostic model was developed with DDOST, STT3A and TMEM165. The risk score could be an independent prognostic factor. Patients in the high-risk subgroup showed a worse prognosis than patients in the low-risk one. ssGSEA showed that patients in the low-risk subgroup tended to be in the immune-activated state, with higher levels of B cell and macrophage cell infiltrations and lower levels of regulatory T cell (Treg) infiltrations in both TCGC and GEO cohorts. Immunohistochemistry studies showed that DDOST, STT3A and TMEM165 are highly expressed in tumor tissues and patients with a high-risk score correlated with poor progression free survival and worse immunotherapeutic response. Furthermore, the proliferation of HCC cells was reduced after the knockdown of DDOST, as well as upon the knockdown of STT3A and TMEM165. Conclusion: In this study, we establish that the risk model based on N-linked glycosylation related genes could efficiently predict the prognosis and tumor microenvironment immune state of HCC patients, and the risk score could serve as a novel indicator of immunotherapy.

Effect of Initiation Time of Hydrostatic Pressure Shock on Chromosome Set Doubling of Tetraploidization in Turbot Scophthalmus maximus.

The objective of the study was to clarify the effects of initiation time on chromosome set doubling induced by hydrostatic pressure shock through nuclear phase fluorescent microscopy in turbot Scophthalmus maximus. The ratio of developmentally delayed embryo and chromosome counting was used to assess induction efficiency. For the embryos subjected to a pressure of 67.5 MPa for 6 min at prometaphase (A group), chromosomes recovered to the pre-treatment condition after 11-min recovering. The first nuclear division and cytokinesis proceeded normally. During the second cell cycle, chromosomes did not enter into metaphase after prometaphase, but spread around for about 13 min, then assembled together and formed a large nucleus without anaphase separation; the second nuclear division and cytokinesis was inhibited. The ratio of developmentally delayed embryo showed that the second mitosis of 78% A group embryo was inhibited. The result of chromosome counting showed that the tetraploidization rate of A group was 72%. For the embryos subjected to a pressure of 67.5 MPa for 6 min at anaphase (B group), chromosomes recovered to the pre-treatment condition after about 31-min recovering. Afterwards, one telophase nucleus formed without anaphase separation; the first nuclear division was inhibited. The time of the first cleavage furrow occurrence of B group embryos delayed 27 min compared with that of A group embryos. With the first cytokinesis proceeding normally, 81.3% B group embryos were at two-cell stage around the middle of the second cell cycle after treatment. Those embryos were one of the two blastomeres containing DNA and the other without DNA. The first nuclear division of those embryos was inhibited. During the third cell cycle after treatment, 65.2% of those abovementioned embryos were at four-cell stage, cytokinesis occurred in both blastomeres, and nuclear division only occurred in the blastomere containing DNA. Of those abovementioned embryos, 14.0% were at three-cell stage and cytokinesis only occurred in the blastomere containing DNA. The result of chromosome counting showed that the tetraploidization rate of B group was only 7%. To summarize what had been mentioned above, mechanisms on chromosome set doubling of tetraploid induction would be different with different initiation time of hydrostatic pressure treatment. Chromosome set doubling was mainly due to inhibition of the second mitosis when hydrostatic pressure treatment was performed at prometaphase. Otherwise, chromosome set doubling was mainly due to inhibition of the first nuclear division when hydrostatic pressure treatment was performed at anaphase. Induction efficiency of tetraploidization resulted from inhibition of the second cleavage was higher than which resulted from inhibition of the first nuclear division. This study was the first to reveal biological mechanisms on the two viewpoints of chromosome set doubling through effect of initiation time of hydrostatic pressure treatment on chromosome set doubling in tetraploid induction.

Effects of cold shock on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus).

Cytological changes and subsequent mitotic processes were studied in gynogenetically activated eggs of olive flounder subjected to cold-shock treatment using indirect immunofluorescence staining of isolated blastodisks. Obvious differences between controls and treated eggs were detected during early cell division. The developmental process of haploid control was similar to that of the diploid control except several minutes delayed. Spindles disassembled by the cold-shock treatment regenerated soon after treatment, resulting in the occurrence of the first mitosis. The immature daughter centriole was easily depolymerized by cold-shock treatment, leading to the formation of the bipolar spindle in the first cell cycle and the formation of the monopolar spindle in the second cell cycle, resulting in chromosome set doubling. Some two-cell stage eggs had a monopolar spindle in one blastomere and a bipolar spindle in another during the second mitosis. These eggs had a high potency developing into haploid-diploid mosaics. To the best of our knowledge, this study is the first to clarify the mechanism of chromosome set doubling in marine fishes and provides a preliminary cytological basis for developing a reliable and efficient protocol for mitotic gynogenesis induction by cold-shock treatment in olive flounder.

Effects of hydrostatic pressure on microtubule organization and nucleus changes in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus).

Fluorescent double-labeled technique was used to investigate the effects of hydrostatic pressure on microtubule organization and nucleus in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus). The parameter of hydrostatic pressure treatment was 600 kg/cm(2) for 6 minutes at prometaphase of the first mitosis. The data showed that nucleus and microtubule changes of the diploid control were basically similar to those of the haploid one (5 minutes behind those of the diploid control). Nuclear diameter of the haploid embryo was significantly smaller than that of the diploid one (P < 0.01). The ploidy of chromosome set could be determined basing on nuclear diameter. The results of nuclear diameter measurement and the ratio of developmentally delayed embryo showed that the chromosome set was not doubled during the second cell cycle, the first cleavage proceeded normally; but that of about 80% treated embryo was doubled during the third cell cycle, the second cleavage was inhibited. Microtubules were disassembled, and nucleation capacity of centrosome was just temporarily inhibited by pressure treatment. Centrosome renucleated microtubule, and a bipolar spindle reassembled 15 minutes after treatment, leading to occurrence of the first cleavage. During the second cell cycle, about 80% treated embryo had a single centrosome and formed a unipolar spindle in both blastomeres. After prometaphase, chromosomes spread around for about 20 minutes instead of aligning on the equatorial plane, then assembled and formed one large nucleus without anaphase separation. The second cleavage was inhibited, and the chromosome set was doubled. The data indicated that the chromosome set doubling of mitogynogenetic diploid induced by hydrostatic pressure treatment, which performed at prometaphase of the first mitosis, mainly resulted from the inhibition of the second cleavage rather than the first one. This study is the first to adapt fluorescent double-labeled technique to investigate the mechanism on chromosome set doubling of mitotic gynogenesis induction. This study will offer theoretical support for mitogynogenetic diploid induction in marine fish.

Nuclei fluorescence microscopic observation on early embryonic development of mitogynogenetic diploid induced by hydrostatic pressure treatment in olive flounder (Paralichthys olivaceus).

Sperm genetic material of olive flounder (Paralichthys olivaceus) was inactivated by ultraviolet irradiation. The nuclear phase changes during early embryonic development of diploid, haploid, and mitogynogenetic diploid induced by hydrostatic pressure treatment were observed under fluorescent microscope with 4',6-diamidino-2-phenylindole staining. The parameters of hydrostatic pressure treatment were 600 kg/cm(2) for 6 minutes at prometaphase stage. The data showed that developmental timing sequence of diploid and haploid fertilized eggs was similar. The cell cycle was about 48 minutes, including interphase (about 21 minutes), prophase (about 3 minutes), prometaphase (about 6 minutes), metaphase (about 6 minutes), anaphase (around 9 minutes), and telophase (about 3 minutes). After entering the fertilized egg, ultraviolet-inactivated sperm formed a male pronucleus and became a dense chromatin body in the cytoplasm. Dense chromatin body did not participate in nuclear division and unchanged all the time. For hydrostatic pressure-treated embryos, the first nuclear division and cytokinesis after treatment proceeded normally after about 15 minutes recovery. During the second mitosis, having undergone interphase, prophase, and prometaphase stage, chromosomes began to slowly spread around and scattered in the cell but not entered into metaphase and anaphase. The second nuclear division and cytokinesis was inhibited. The occurrence frequency of developmentally delayed embryos also showed that the second cleavage of about 80% treated eggs was inhibited. The inhibition of the second cleavage resulted to chromosome set doubling. So chromosome set doubling for mitogynogenetic flounder diploid induced by hydrostatic pressure treatment, performed at prometaphase stage, was mainly due to inhibition of the second mitosis rather than the first one.

Improvement in the detection of low concentration protein digests on a MALDI TOF/TOF workstation by reducing alpha-cyano-4-hydroxycinnamic acid adduct ions.

Alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA) as a matrix facilitates the ionization of proteins and peptides in a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer. The matrix itself also ionizes and so do its sodium and potassium adducts. Matrix clusters and metal ion adducts interfere with peptide ionization and peptide mass spectrum interpretation. These matrix adducts are significantly reduced with addition of ammonium monobasic phosphate or ammonium dibasic citrate to the matrix and sample deposited onto the MALDI target. The reduction of matrix adducts results in the increase of peptide intensity and signal-to-noise ratio as well as in improvement of peptide ionization for samples deposited onto the target at levels of 10 fmol or below. These improvements were particularly significant in the detection of peptides at amol levels when reduced amounts of matrix were also used.

The estimation of charge footprint size of ultraviolet photon counting imaging detector with induction readout.

The structure and work principle of an ultraviolet photon counting imaging detector based on wedge and strip anode with induction readout mode are introduced. Two methods of estimating the charge footprint size are presented. One way is theoretical calculation and simulation. The physical course of electrons is simulated from the microchannel plate output side to the readout anode. The calculated results show that the final charge footprint size is sensitive to the thickness of ceramic and not sensitive to the charge footprint size on the Ge layer. The other way is experimental image estimation. The final charge footprint size can be estimated according to the position where the light line of resolution board image begins to bend. Both methods show that the charge footprint size is sensitive to the ceramic substrate. The two methods are simple and effective for estimation of charge footprint size of photon counting imaging detector with induction readout.