Loss of heterozygosity for chromosomes 16q in Wilms tumors predicts outcomes:A meta-analysis

BACKGROUND The research findings suggest that the prognosis of children with Wilms tumor(WT)is affected by various factors.Some scholars have indicated that loss of heterozygosity(LOH)on chromosome 16q is associated with a poor prognosis in patients with WT.AIM To further elucidate this relationship,we conducted a meta-analysis.METHODS This meta-analysis was registered in INPLASY(INPLASY2023100060).We systematically searched databases including Embase,PubMed,Web of Science,Cochrane,and Google Scholar up to May 31,2020,for randomized trials reporting any intrapartum fetal surveillance approach.The meta-analysis was performed within a frequentist framework,and the quality and network inconsistency of trials were assessed.Odds ratios and 95%CIs were calculated to report the relationship between event-free survival and 16q LOH in patients with WT.RESULTS Eleven cohort studies were included in this meta-analysis to estimate the relationship between event-free survival and 16q LOH in patients with WT(I^(2)=25%,P<0.001).As expected,16q LOH can serve as an effective predictor of eventfree survival in patients with WT(risk ratio=1.95,95%CI:1.52–2.49,P<0.001).CONCLUSION In pediatric patients with WT,there exists a partial correlation between 16q LOH and an unfavorable treatment prognosis.Clinical detection of 16q chromosome LOH warrants increased attention to the patient’s prognosis.

Knock-in of exogenous sequences based on CRISPR/Cas9 targeting autosomal genes and sex chromosomes in the diamondback moth,Plutella xylostella

Genetic pest control strategies based on precise sex separation and only releasing sterile males can be accomplished by site-specific genome editing.In the current study,we showed that the mutation of single-allele Pxfl(2)d can significantly impair the normal mating behavior and testis development in male adults of the notorious cruciferous insect pest Plutella xylostella,in addition to its known functions in the ovarian development in female adults and egg hatching.Subsequent CRISPR/Cas9-based knock-in experiments revealed that site-specific integration of an exogenous green fluorescent protein(GFP)gene into autosomal Pxfl(2)d for labelling mutants could be achieved.However,this gene is not a suitable target for GFP insertion to establish a genetically stable knock-in strain because of the severe decline in reproductive capacity.We further screened for the W-chromosome-linked and Z-chromosome-linked regions to test the knock-in efficiency mediated by CRISPR/Cas9.The results verified that both types of chromosomes can be targeted for the site-specific insertion of exogenous sequences.We ultimately obtained a homozygous knock-in strain with the integration of both Cas9 and cyan fluorescent protein(CFP)expression cassettes on a Z-linked region in P.xylostella,which can also be used for early sex detection.By injecting the sgRNA targeting Pxfl(2)d alone into the eggs laid by female adults of the Z-Cas9-CFP strain,the gene editing efficiency reached 29.73%,confirming the success of expressing a functional Cas9 gene.Taken together,we demonstrated the feasibility of the knock-in of an exogenous gene to different genomic regions in P.xylostella,while the establishment of a heritable strain required the positioning of appropriate sites.This study provides an important working basis and technical support for further developing genetic strategies for insect pest control.

Rapid identification of Psathyrostachys huashanica Keng chromosomes in wheat background based on ND-FISH and SNP array methods

Psathyrostachys huashanica Keng(2n=2x=14,NsNs)is regarded as a valuable wild relative species for common wheat cultivar improvement because of its abundant beneficial agronomic traits.However,although the development of many wheat–P.huashanica-derived lines provides a germplasm base for the transfer of excellent traits,the lag in the identification of P.huashanica chromosomes in the wheat background has limited the study of these lines.In this study,three novel nondenaturing fluorescence in situ hybridization(ND-FISH)-positive oligo probes were developed.Among them,HS-TZ3 and HS-TZ4 could specifically hybridize with P.huashanica chromosomes,mainly in the telomere area,and HS-CHTZ5 could hybridize with the chromosomal centromere area.We sequentially constructed a P.huashanica FISH karyotype and idiogram that helped identify the homologous groups of introduced P.huashanica chromosomes.In detail,1Ns and 2Ns had opposite signals on the short and long arms,3Ns,4Ns,and 7Ns had superposed two-color signals,5Ns and 6Ns had fluorescent signals only on their short arms,and 7Ns had signals on the intercalary of the long arm.In addition,we evaluated different ways to identify alien introgression lines by using low-density single nucleotide polymorphism(SNP)arrays and recommended the SNP homozygosity rate in each chromosome as a statistical pattern.The 15K SNP array is widely applicable for addition,substitution,and translocation lines,and the 40K SNP array is the most accurate for recognizing transposed intervals between wheat and alien chromosomes.Our research provided convenient methods to distinguish the homologous group of P.huashanica chromosomes in a common wheat background based on ND-FISH and SNP arrays,which is of great significance for efficiently identifying wheat–P.huashanica-derived lines and the further application of Ns chromosomes.

Nannochloropsis artificial chromosomes(Nanno ACs)loom on the horizon

Species in genus Nannochloropsis,especially N.oceanica and N.gaditana,have been evolving as the model microalgae for both application and theory studies.The position effect of genome integration,the carrying capability limitation of integrative vectors and the instability of non-integrative vectors have hindered Nannochloropsis genetic modification with concatenate genes and extremely long DNA fragments.The molecular tools including genetic transformation,homologous recombination,gene edition,gene stacking and episome vectors for transient gene expression and diverse reporters and selection markers have been rapidly developing in Nannochloropsis species.The construction of animal and plant artificial chromosomes with“top down”strategy has set fine examples for the construction of Nannochloropsis artificial chromosomes(NannoACs).It seems that the methods and materials to set the foundation for constructing NannoACs are at hands.In this review,we outlined the current status of transgenes in Nannochloropsis species,summarized the limitations of both integrative and non-integrative vectors,and proposed a tentative approach to construct NannoACs by doubling and stabilizing the genome first,and then truncating the natural chromosomes.NannoACs once constructed will facilitate transferring the desired traits and concatenate genes into Nannochloropsis genetic backgrounds,thus contributing towards its genetic improvement and synthetic biological studies.

Studies on the integration of hepatitis B virus DNA sequence in human sperm chromosomes

Aim: To study the integration of hepatitis B virus (HBV) DNA into sperm chromosomes in hepatitis B patients and the features of its integration. Methods: Sperm chromosomes of 14 subjects (5 healthy controls and 9 HB patients, including 1 acute hepatitis B, 2 chronic active hepatitis B, 4 chronic persistent hepatitis B, 2 HBsAg chronic carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free hamster oocytes and human spermatozoa. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes. Results: Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis B. In 9(9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots and the others 2 to 4 signals. The fluorescence intensity showed significant difference among the signal spots. The distribution of signal sites among chromosomes seems to be random. Conclusion: HBV could integrate into human sperm chromosomes. Results suggest that the possibility of vertical transmission of HBV via the germ line to the next generation is present.

Effects of hepatitis B virus infection on human sperm chromosomes

AIM: To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients.METHODS: Sperm chromosomes of 14 tested subjects (5healthy controls, 9 patients with HBV infection, including 1with acute hepatitis B, 2 with chronic active hepatitis B, 4with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes.RESULTS: The total frequency of sperm chromosome aberrations in HBV infection group (14.8%, 33/223) was significantly higher than that in the control group (4.3%,5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random.CONCLUSION: HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.

Development and genetic analysis of wheat double substitution lines carrying Hordeum vulgare 2H and Thinopyrum intermedium 2Ai#2 chromosomes

Thinopyrum intermedium and barley are two close relatives of wheat and carry many genes that are potentially valuable for the improvement of various wheat traits. In this study we created wheat double substitution lines by hybridizing different wheat–Th. intermedium and wheat–barley disomic alien substitution lines, with the aim of using genes in Th. intermedium and barley for wheat breeding and investigating the genetic behavior of alien chromosomes and their wheat homoeologs. As expected, we obtained two types of wheat double substitution lines,2D2Ai#2(2B)2H( A) and 2A2 Ai#2(2B)2H(2D), in which different group 2 wheat chromosomes were replaced by barley chromosome 2 H and Th. intermedium chromosome 2Ai#2. The new materials were characterized using molecular markers, genomic in situ hybridization(GISH), and fluorescent in situ hybridization(FISH). GISH and FISH experiments revealed that the double substitution lines harbor 42 chromosomes including 38 wheat chromosomes, a pair of barley chromosomes, and a pair of Th. intermedium chromosomes. Analysis using specific DNA markers showed that two pairs of wheat homoeologous group 2 chromosomes in the new lines were substituted by a pair of 2H and a pair of 2Ai#2 chromosomes. Chromosome 2H showed a higher transmission rate than 2Ai#2, and both chromosomes were preferentially transmitted between generations via female gametes. Evaluation of botanic and agronomic traits demonstrated that,compared with their parents, the new lines showed similar growth habits and plant type but differences in plant height, flowering date, and self-fertility. Cytological observations using different probes suggested that the double substitution lines showed nearly normal genetic behavior before and during meiosis. The novel substitution lines can potentially be used in wheat meiosis research and breeding programs.

Immunolcocalization of actin in intact and DNA-and histone-depleted nuclei and chromosomes of allium cepa

The presence of actin in eukaryotic nuclei and chromosomes, and especially in higher plant nuclei and chromosomes, has not been well established. We detected actin in meristematic cells of Allium cepa with indirect immunofluorescence technique and observed bright fluorescence in the intact nuclei and chromosomes, indicating that actin is present in the nuclei and chromosomes of the higher plant. We labeled sections of the meristematic cells of A. cepa with immunogold technique, gold particles were found over the whole nuclei and a number of gold particles were concentrated in condensed chromatin and nucleoli, confirming the results of the immunofluoresence observations. We treated the nuclei and chromosomes of A.cepa with DNase I and 2M NaCl and obtained DNA- and histone-depleted nuclei and chromosomes. Indirect immunofluorescence tests showed that the DNA- and histonedepleted nuclei and chromosomes reacted positively with the anti-actin antibodies. These results demonstrate that actin exists not only in intact nuclei and chromosomes,but also in DNA- and histone-depleted nuclei and chrmosomes of the plant. In addition, our immuno-fluorescence tests indicate that tropomyosin is present in the nuclei and chromosomes of A. cepa.

An Integrated Genetic,Physical and Transcript Map of Homoeologous Chromosomes 12 and 26 in Upland Cotton(Gossypium hirsutum L.)

While Upland cotton(Gossypium hirsutum L.) represents 95% of the world production,its genetic improvement is hindered by the shortage of effective genomic tools and resources.The

Technique to Obtain Mitotic Chromosomes of <i>Conyza bonariensis</i>L. Cronquist (Asteraceae)

Cytogenetic analysis requires cytological preparations that unequivocally reveal the chromosome number and permit optimal visualizations of chromosome morphology for the construction of karyotypes and ideograms. Chromosomal characterization is possible only by establishing these two parameters. To cytogenetically characterize the weed Conyza bonariensis (L.) Cronquist (Asteraceae), it was necessary to improve cytological analysis techniques to obtain optimal results. This species belongs to a genus whose plants have wide phenotypic plasticity. These plants can be morphologically differentiated by other types of analysis, and thus the application of this technique will serve as a reference for cytogenetical analysis of other groups of plants that have cytogenetic characteristics similar to those of C. bonariensis. The methodology described here highlights three main protocol steps: 1) root tip collection from newly germinated seed radicles and from young root tips of mature plants, 2) pretreatment of meristems with antimitotic agents, both isolated and combined, and 3) acid and enzymatic hydrolytic processes.

Chromosomes and Karyotypes of Three Seagrass Species of Hydrocharitaceae

[Objective] This study aimed to investigate the chromosomes and karyotypes of three seagrass species of hydrocharitaceae-Enhalus acoroides, Thalassia hemprichii and Halophila minor collected from Li’an Lagoon, Hainan Province, China. [Method] The root-tips of E. acoroides, T. hemprichii and the axillary buds of H. minor were selected as the materials in this study. The conventional crushing method was used to prepare the slice specimens of chromosomes, and the karyotypes of the three species were analyzed. [Result] The chromosome numbers of E. acoroides, T. hemprichii and H. minor were 2n=18, 18, 28, respectively. The karyotype formulas were K （2n）=18= 12m＋6sm, K（2n）=18=12m＋6sm and K（2n）=28=16m＋8sm＋4st, respectively. According to the standard of Stebbins, the karyotypes of E. acoroides and T. hemprichii were 2A, and that of H. minor was 2B. There was no B chromosome and satellite in these three species. [Conclusion] According to the comparison on the karyotypes of this three species, E. acoroides and T. hemprichii are similar in karyotypes to each other, indicating that there is close, inter-relationship between E. acoroides and T. hemprichii.

XCAP-C-like Protein Existing in Nuclei and Chromosomes of Allium sativa

In studying the XCAP-C-like protein in the root meristematic cells of Allium sativa L., the nuclei were isolated from the cells and the nuclear matrices prepared. A 165 kD polypeptide, which is equivalent to XCAP-C in molecular weight, was demonstrated in the nuclei by SDS-PAGE, and was then proved to be an XCAP-C-like protein by Western blot using an anti-XCAP-C antiserum, but neither the polypeptide nor the XCAP-C-like protein was detected in die nuclear matrix. The nuclei, Chromosomes and chromosome scaffolds were observed to emanate strong, specific fluorescence after labeled with the anti-XCAP-C antiserum and an FITC-conjugated secondary antibody, indicating their containment of the XCAP-C-like protein. It was confirmed by viewing with immunoelectron microscopy that the gold particles representing the localization of the XCAP-C-like protein were found to be mainly distributed in the condensed chromatin regions of the nuclei and chromosomes.

SOFT X-RAY IMAGES OF CHROMOSOMES WITHIN INTACT TUMOR CELLS TREATED WITH COLCHICINE

In order to avoid any possible effect of separating procedures from intact cell on morphologic structure of the chromosomes, cultivated Hep-2 tumor cells were treated with colchicine and observed by soft X-ray contact microscopy for the first time. The fine structures of chromosomes are more clear with stereo features. The thread-like and coarse granular structures twine and tangle up together within chromosome masses which can not easily be revealed in Wright’s stained sample by light microscope or osmium stained sample by transmission electron

Spontaneous and Bleomycin-Induced <i>&gamma;</i>H2AX Signals in CHO9 Metaphase Chromosomes

In eukaryotes, a cascade of events named DNA damage response (DDR) has evolved to handle DNA lesions. DDR engages the recruitment of signaling, checkpoint control, repair and chromatin remodeling protein complexes, allowing cell cycle delay, DNA repair or induction of apoptosis. An early DDR event involves the phosphorylation of the histone variant γH2AX on serine 139 (H2AX139 phosphorylation) originating the so-called γH2AX. DDR-related H2AX139 phosphorylation have been extensively studied in interphase nuclei. More recently, γH2AX signals on mitotic chromosomes of asynchronously growing cell cultures were observed. We performed a quantitative analysis of γH2AX signals on γH2AX immunolabeled cytocentrifuged metaphase spreads, analyzing the γH2AX signal distributions of CHO9 chromosomes harboring homologous regions both in control and bleomycin (BLM)-treated cultures. We detected γH2AX signals in CHO9 chromosomes of controls which significantly increase after BLM-exposure. γH2AX signals were uniformly distributed in chromosomes of controls. However, the γH2AX signal distribution in BLM exposed cells was significantly different between chromosomes and among chromosome regions, with few signals near the centromeres and a tendency to increase towards the telomeres. Interestingly, both basal and BLM-induced γH2AX signal distribution were statistically equal between CHO9 homologous chromosome regions. Our results suggest that BLM exerts an effect on H2AX139 phosphorylation, prevailing towards acetylated and gene-rich distal chromosome segments. The comparable H2AX139 phosphorylation of homologous regions puts forward its dependence on chromatin structure or function and its independence of the position in the karyotype.

Multiple Chromosomes in Bacteria: Low Level of Evolutionary Constraint Drives the Rapid Genetic Divergence of Chromosome II

Multiple chromosomes in bacteria are designated as a larger primary chromosome (CI) and smaller accessory chromosomes (CII and CIII). Although previous studies examined multiple chromosomes in several bacterial species, the evolutionary mechanisms for the origin of CIIs still remain unclear. In this study, the four following hypotheses were tested. 1) CIIs exhibit lower sequence conservation and sequence divergence compared to their corresponding CIs across species of Proteobacteria. 2) The differential sequence divergence of CI and CII depends on pathogenic and non-pathogenic lifestyles. 3) CIIs harbor a higher level of horizontal gene transfers (HGTs) than CIs. 4) Orthologs located on CIIs experience less purifying selection than their corresponding orthologs on CIs. Results reveal a higher level of sequence conservation of CIs than the sequence conservation of CIIs. There is no significant difference in HGT estimates between CIs and CIIs. A majority of orthologous genes of CIs and CIIs experience purifying selection;however, genes on CIIs were significantly less constrained than the corresponding ones on CIs. This finding is true for both pathogenic and non-pathogenic bacteria, but the selective constraints for non-pathogenic bacteria are relatively less constrained. It was concluded that the differential selective constraint is a potent driving force for the rapid evolution of CII. Therefore, gene expression analysis at the transcriptome and proteome levels may shed light on the gene regulation mechanisms that might affect the sequence divergence between CI and CII.

Completely Distinguishing Individual A-genome Chromosomes and Their Karyotyping Analysis by Multiple BAC-FISH

Multiple BAC-FISH is a powerful tool for modern cytogenetic researching in both animals and plants.But in cotton,this technique is unavailable due to the high percentage of repetitive sequences.

Observation on Root Tip Chromosomes of Double－ear Wheat Induced by Heavy Ion Irradiation

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Telomeric associations of chromosomes in patients with esophageal squamous cell carcinomas

AIM To investigate the role of telomeric association in the development of esophageal cancer. METHODS Using chromosome R banding technique, telomeric association of chromosome in peripheral blood lymphocytes from 16 untreated patients with esophageal squamous cell carcinoma were observed and 16 healthy adults served as controls. RESULTS The telomeric association frequencies of cell and chromosomes were significantly higher than those of controls (x 2=9.56,P<0.01), but its distribution on the chromosome showed no significant difference (x 2=1.01, P>0.05) between the two groups. CONCLUSION Chromosomal instability can be initiated by telomeric associations, and sequential chromosome analysis can aid the understanding of the tumor occurrence and progression.

Experimental Study on Meiotic Spindles and Chromosomes of Mouse Mature (MⅡ) Stage Oocytes under Laser Scanning Confocal Microscopy

Taking the mouse as a model, the experimental method of observing the morphology of meiotic spindles and chromosomes in mature oocytes were investigated in order to evaluate the effects of various interventions on the quality of oocytes accurately and rapidly. Laser scanning confocal microscope （LSCM） was used to examine the meiotic spindles and chromosomes by the technologies of optical section and three-dimensional （3D） image reconstruction. The results showed that the configurations of meiotic spindles and chromosomes could be observed clearly by LSCM. The normal rate of meiotic spindles and chromosomes was 82% and 86% respectively. It was concluded that the LSCM was a valid instrument to observe the meiotic spindles and chromosomes of mature oocytes and could be used as a valid method to evaluate the quality of MⅡocytes.

Designations for individual genomes and chromosomes in Gossypium

Gossypium, as the one of the biggest genera, the most diversity, and the highest economic value in field crops, is assuming an increasingly important role in studies on plant taxonomy, polyploidization, phylogeny, cytogenetics, and genomics. Here we update and provide a brief summary of the emerging picture of species relationships and diversification, and a set of the designations for individual genomes and chromosomes in Gossypium. This cytogenetic and genomic nomenclature will facilitate comparative studies worldwide, which range from basic taxonomic exploration to breeding and germplasm introgression.