Colorectal cancers with aneuploids show high CD133 expression and poor prognosis

Objective： The aim of the study was to investigate the relationship of DNA ploidy status in colorectal cancers with patients＇ prognosis and also the relationship of DNA ploidy status with expression of the colorectal cancer stem cell marker CD133. Methods： The DNA ploidy status and CD 133 expression in colorectal cancers were detected by flow cytometry. The clinicopathological characteristics and progression-free survival analysis of patients was evaluated based on the clinical data. Results： DNA ploidy pattern did not correlated with gender, age, lesion diameter, histological type, depth of tumor invasion, lymphatic invasion and Dukes stage. Only primary lesion cite showed significant correlation with DNA ploidy pattern, more aneuploids were observed in colonic cancer than rectal cancer, P 〈 0.05. The 2-year progress/on-free survival rate and total progression-free time in patients with aneuploids were lower than that with diploids, P 〈 0.05. Tumors contained aneuploids showed higher expression of CD133 than tumors of only diploids, P 〈 0.05. Conclusion： Tumor DNA ploidy status is a significant prognostic factor in patients with colorectal cancer and also associated with the existence of CD133 positive colorectal cancer stem cells.

High efficiency production and genomic in situ hybridization analysis of Brassica aneuploids and homozygous plants

Interspecific and intergeneric hybridizations have been widely used in plant genetics and breeding to construct stocks for genetic analysis and to introduce into crops the desirable traits and genes from their relatives. The intergeneric crosses between Brassica juncea (L.) Czern. & Coss., B. carinata A. Braun and Orychophragmus violaceus (L.) O. E. Schulz were made and the plants produced were subjected to genomic in situ hybridization analysis. The mixoploids from the cross with B. juncea were divided into three groups. The partially fertile mixoploids in the first group (2n = 36—42) mainly contained the somatic cells and pollen mother cells (PMCs) with the 36 chromosomes of B. juncea and additional chromosomes of O. violaceus. The mixoploids (2n = 30—36) in the second and third groups were morphologically quite similar to the mother plants B. juncea and showed nearly normal fertility. The plants in the second group produced the majority of PMCs (2n = 36) with their chromosomes paired and segregated normally, but 1—4 pairs of the O. violaceus chromosomes were included in some PMCs. The plants in the third group produced only PMCs with the 36 B. juncea chromosomes, which were paired and segregated normally. The mixoploids (2n = 29—34) from the cross with B. carinata produced the majority of PMCs (2n = 34) with normal chromosome pairing and segregation, but some plants had some PMCs with 1—3 pairs of chromosomes from O. violaceus and other plants had only PMCs with the B. carinata chromosomes. The Brassica homozygous plants and aneuploids with complete or partial chromo-some complements of Brassica parents and various numbers of O. violaceus chromosomes were derived from these progeny plants. The results in this study provided the molecular cytogenetic evidence for the separation of parental genomes which was previously proposed to occur in the hybridizations of these two genera.

Disseminated tumor cells in bone marrow as predictive classifiers for small cell lung cancer patients

Background: Small cell lung cancer (SCLC) is a highly aggressive disease characterized by early metastasis. Ane- uploid CD31- disseminated tumor cells (DTCs) and CD31+ disseminated tumor endothelial cells (DTECs) residing in the bone marrow are generally considered as the initiators of metastatic process. However, the clinical signifi- cance of DTCs and DTECs in SCLC remains poorly understood. The aim of this study is to investigate the clinical implications of diverse subtypes of highly heterogeneous DTCs and DTECs in SCLC patients. Methods: Subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) was applied to enrich and perform comprehensive morphologic, karyotypic, and phenotypic characterization of aneuploid DTCs and DTECs in 30 patients. Additionally, co-detection of circulating tumor cells (CTCs) and circulating tumor endothelial cells (CTECs) was conducted on 24 of the enrolled patients. Proof-of-concept of the whole exon sequencings (WES) on precisely selected different subtypes of CTCs or DTCs, longitudinally detected from a representative case with pathologically confirmed bone marrow metastasis, was validated to feasibly reveal genetic mutations in these cells. Results: DTCs, DTECs and their subtypes were readily detectable in SCLC patients. Comparative analysis re- vealed that the number of DTCs and DTECs was significantly higher than that of their corresponding CTCs and CTECs ( P < 0.001 for both). Positive detection of disseminated tumor microemboli (DTM) or disseminated tumor endothelial microemboli (DTEM) was associated with inferior survival outcomes ( P = 0.046 and P = 0.048). Pa- tients with EpCAM+ DTCs detectable displayed significantly lower disease control rate (DCR) (16.67% vs 73.33%, P = 0.019), reduced median progression-free survival (mPFS) and median overall survival (mOS) compared with those with EpCAM- DTCs ( P = 0.028 and P = 0.002, respectively). WES analysis indicated that post-treatment DTCs isolated from bone marrow at the time of disease progression shared more homologous somatic gene mu- tations with pre-treatment CTCs compared with post-treatment CTCs. Conclusions: Our findings suggest that bone marrow sampling and characterization of DTC subtypes provided a valuable tool for predicting treatment response and the prognosis in SCLC. Moreover, DTCs inherit a greater amount of homologous somatic information from pre-treatment CTCs, indicating their potential role in disease progression and treatment resistance.

Regeneration and Characterization of Plants Derived from Asymmetric Protoplast Fusion in Citrus

Protoplasts of Valencia sweet orange ( Citrus sinensis Osb.),irradiated by X_ray with a dose rate of 3.8 krad/min for 45 min, were electrically fused with protoplasts of Murcott tangor ( C. reticulata×C. sinensis ) that were treated with 0.25 mmol/L iodoacetic acid for 15 min. It took nearly 15 months for the fusion_derived calli to develop into embryoids that were only originated in the medium of MT supplemented with 2% glycerol. The shoots were recalcitrant to rooting in the root_induction medium. In vitro grafting was employed to produce whole plants though one self_rooting plant was obtained. Cytological determination of root and shoot tips showed mainly diploid and aneuploid cells, together with few tetraploid cells in some plants. RAPD (random amplified polymorphism DNA) analysis with 10_mer primers demonstrated that bands specific to the fusion parents were detected in the regenerated plants, indicating that interspecific somatic hybrids have been obtained via protoplast asymmetric fusion in Citrus .

Production of aneuhaploid and euhaploid sporocytes by meiotic restitution in fertile hybrids between durum wheat Langdon chromosome substitution lines and Aegilops tauschii

Fertile F1 hybrids were obtained between durum wheat （Triticum durum Desf.） Langdon （LDN） and its 10 disomic substitution （LDN DS） lines with Aegilops tauschii accession AS60 without embryo rescue. Selfed seedset rates for hybrids of LDN with AS60 were 36.87% and 49.45% in 2005 and 2006, respectively. Similar or higher selfed seedset rates were observed in the hybrids of 1D （1A）, 1D （1B）, 3D （3A）, 4D （4B）, 7D （7A）, and 2D （2B） with AS60, while lower in hybrids of 3D （3B） ＋ 3BL, 5D （5A） ＋ 5AL, 5D （5B） ＋ 5B and 6D （6B） ＋ 6BS with AS60 compared with the hybrids of LDN with AS60. Observation of male gametogenesis showed that meiotic restitution, both first-division restitution （FDR） and single-division meiosis （SDM） resulted in the formation of functional unreduced gametes, which in turn produced seeds. Both euhaploid and aneuhaploid gametes were produced in F1 hybrids. This suggested a strategy to simultaneously transfer and locate major genes from the ancestral species T. turgidum or Ae. tauschii. Moreover, there was no significant difference in the aneuhaploid rates between the F1 hybrids of LDN and LDN DS lines with AS60, suggesting that meiotic pairing between the two D chromosomes in the hybrids of LDN DS lines with AS60 did not promote the formation of aneuhaploid gametes.

Mitotic Slippage Process Concealed Cancer-Sought Chromosome Instability Mechanism (S-CIN)

Official (NIH) cancer investigation is on identification of inherited cancer genes in you and me for early interventions, and for use of such knowledge in therapy. In this review the emphasis is on the unknown cancer initiation, and on the question of a mechanism for inherited CIN (chromosomal instability). Evidence for fitness increased cells from the mitotic slippage process (in vivo/in vitro) originated from genome damaged diploid cells in G2/M, skipping mitosis to G1, which illegitimately permitted S-phase re-replication of the chromatid cohesed-2n cells to 4n-tetraploidy. During which, down-load of genome-wide cohesin occurred, producing 4-chromatid diplochromosomes, evolutionary conserved in repair of DNA. This type of 4n cells divided 2-step meiotic-like, leading to diploid aneuploid cells with increased fitness, and expression of gross chromosomal anomalies in proliferation. The diploid cohesed chromatids during re-replication would hinder replication of sticky heterochromatic regions, resulting in their under-replication, and known from Drosophila. The human chromosomes are longitudinally differentiated into satellite DNA regions, folic acid sensitive sites and the primary constriction (centromere);they are breakage sensitive regions and being heterochromatic. This strongly suggests, multiple, chromosomal regional under-replication-cites, translated to origin of slippage, S-CIN, a genome inherited destabilization mechanism. Logically, S-CIN would affect genes differentially depending on chromosome location, for example, the high frequency in cancers of mutated p53 on the small 17p-arm, which with centromere breakage would be preferentially lost in mitosis. This likely S-CIN mechanism in cancer evolution can be studied in vivo for APC mutated crypt cells with demonstrated mitotic slippage process.

Improved Detection of Cervical Cancer and High Grade Neoplastic Lesions by a Combination of Conventional Cytology and DNA Automated Image Cytometer

OBJECTIVE: To reduce false-negative rates of population based cervical screening programs employing conventional cytology in combination with automated DNA image cytometer. METHODS: Involved cervical samples from a total of 3603 women were taken by a cervix brush and then placed into a fixative solution. The cells were separated from mucus by mechanical and chemical treatment after which they were deposited onto microscope slides by a cytospin. Two slides were prepared from each case;one slide was stained by Papanicolaou stain for conventional cytology examination, while the other slide was stained by a DNA specific and stoichiometric stain. The latter slide was used to determine the relative amount of DNA in the cell nuclei in order to assess the ploidy status of the epithelial cells. Enrolled in the study, 157 women were followed by colposcopy examination where punch biopsies were taken from the visible lesions or from suspicious areas. The results of the conventional cytology were then compared to the DNA image cytometer for all samples. RESULTS: Histopathology diagnosed 51 lesions from the 132 biopsied cases as CIN2 or higher, including 27 CIN2, 16 CIN3 and 8 invasive cancers. Conventional cytology correctly identified 29 of the 51 high grade CIN and in-vasive cancer, while DNA image cytometer correctly identified 38 high grade CIN and invasive cancer using the crite-rion that at least three cells were found on the slide that contained DNA amount in excess of 5c. 42 out of 51 high grade CIN and invasive cancer were found by conventional cytology in combination with DNA image cytometer. Sensitivities were 56.8%, 74.5% and 82.4%, while specificities were 86.2%, 81.5% and 81.5% in conventional cytology, DNA image cytometer and combination both cytology and DNA image cytometer respectively. CONCLUSION: The study demon-strated that screening for high grade neoplastic lesions and cervical cancer by DNA image cytometer or combination of conventional cytology and DNA image cytometer is more sensitive than conventional screening approach.

Production of Aneuploid Pinctada martensii Dunker in Tetraploid Induction

Aneuploidy embryos of Pinctada martensii Dunker are produced during tetraploid induction by inhibiting the first polar body in eggs from triploid fertilized with haploid sperms with cytochalasin B treatment. Chromosome analysis reveals that there are 88.18 ±6.79% aneuploidy embryos, and 28.70% aneuploids in pearl oysters of one-year age These aneuploids have five chromosomal conditions, such as 2n + 1(29), 2n + 2 (30), 3n-2 (40), 3n-1(41) and 3n + 1 (43). Results of growth measurement show that there is no significant difference between aneuploids (as a group) and diploids in body size and weight (p ＞ 0.10), but the aneuploide is obviously different from triploid (p ＜ 0.01). The mean body size and weight of aneuploids in diploid condition (2n ± 1 and 2n ± 2) are significantly smaller than those of diploids (p ＜ 0.01),but aneuploids within triploid condition (3n ± 1 and 3n ± 2) are not smaller than diploids in body size and weight (p ＞ 0.1).This study indicates Pinctada martensii Dunker could tolerate aneuploidy by 7 ～ 14% of the haploid genome, and that aneuploids of this species are viable under certain conditions.

Non-invasive chromosome screening for embryo preimplantation using cell-free DNA

Preimplantation genetic testing (PGT) is a widely adopted screening method that can be performed to identify and select embryos with normal ploidy;however, PGT relies on embryo biopsy, that is, polar body, embryo cells, or trophectoderm biopsy, to obtain embryonic DNA, increase its technical limitations. Studies have indicated that biopsy may have an influence on the quality and development of embryos, and increase the chance of abnormal epigenetic modifications. Therefore, non-invasive PGT (niPGT) detection of cell-free DNA (cfDNA) has gradually become a hot research topic in the field of assisted reproduction. Studies showed cfDNA could be detected in blastocyst fluid and spent culture medium (SCM) in vitro cultured embryos. The cfDNA collection requires less skill and makes lower risk to embryos. Some studies have been conducted to evaluate the feasibility of SCM-based niPGT approaches. When comparing the ploidy consistency of cfDNA in SCM, its consistency to the conventional PGT for aneuploidies results fluctuated widely, it is critical to recognize the factors influencing accuracy. These contradictory results may be related to factors such as the difference in SCM sampling methods and sampling time, and the definition of consistency. In this review, we aimed to comprehensively summarize how researchers use embryonic cfDNA to conduct niPGT detection. It also systematically reviews the factors affecting the accuracy of the test and its underlying issues, as well as prospective applications. We hope to provide a basis for future niPGT research and a useful reference for the standardized operation of niPGT that can be widely applied in clinical practice.