Objective:To investigate the expression differences of small non-coding RNAs (sncRNAs) in sperm cells with different DNA fragmentation index (DFI).Methods:Male patients who visited the Center of Reproductive Medicine of the Changhai Hospital Affiliated to Naval Medical University from October 2021 to August 2022 were included in the study. According to the DFI value, they were divided into <15%, 15%-30% and >30%, which were denoted as normal DFI group ( n=48), critical DFI group ( n=40) and increased DFI group ( n=48). The relative expression levels of sncRNAs in sperm cells of 3 groups were compared. The correlation between differential sncRNAs and sperm motility was analyzed. Results:There were no significant differences in age and sperm concentration among the three groups (all P>0.05). The sperm motility in the increased DFI group [(30.36±4.75)%] was lower than that in the normal DFI group [(63.38±9.56)%, P<0.001] and the critical DFI group [(56.50±5.87)%, P=0.034]. Compared with the other two groups, the relative expression levels of Glu-CTC-40-10 and SeC-TCA-37-4 in the increased DFI group decreased, while the relative expression levels of Gly-GCC, iMet-CAT-18-18, and hsa-miR-151a-5p increased, with statistical significances (all P<0.001). Spearman correlation analysis showed that Glu-CTC-40-10, SeC-TCA-37-4 were positively correlated with sperm motility ( r=0.384, P<0.001; r=0.441, P<0.001), and Gly-GCC, iMet-CAT-18-18 and hsa-miR-151a-5p were negatively correlated with sperm motility ( r=-0.437, P<0.001; r=-0.423, P<0.001; r=-0.515, P<0.001). Conclusion:Compared with semen with normal DFI, sncRNAs are differentially expressed in semen with critical and elevated DFI, which may be related to the molecular mechanism of DFI formation. SncRNAs have the potential to be used as biological markers of male infertility.

Objective:To investigate the expression differences of small non-coding RNAs (sncRNAs) in sperm cells with different DNA fragmentation index (DFI).Methods:Male patients who visited the Center of Reproductive Medicine of the Changhai Hospital Affiliated to Naval Medical University from October 2021 to August 2022 were included in the study. According to the DFI value, they were divided into <15%, 15%-30% and >30%, which were denoted as normal DFI group ( n=48), critical DFI group ( n=40) and increased DFI group ( n=48). The relative expression levels of sncRNAs in sperm cells of 3 groups were compared. The correlation between differential sncRNAs and sperm motility was analyzed. Results:There were no significant differences in age and sperm concentration among the three groups (all P>0.05). The sperm motility in the increased DFI group [(30.36±4.75)%] was lower than that in the normal DFI group [(63.38±9.56)%, P<0.001] and the critical DFI group [(56.50±5.87)%, P=0.034]. Compared with the other two groups, the relative expression levels of Glu-CTC-40-10 and SeC-TCA-37-4 in the increased DFI group decreased, while the relative expression levels of Gly-GCC, iMet-CAT-18-18, and hsa-miR-151a-5p increased, with statistical significances (all P<0.001). Spearman correlation analysis showed that Glu-CTC-40-10, SeC-TCA-37-4 were positively correlated with sperm motility ( r=0.384, P<0.001; r=0.441, P<0.001), and Gly-GCC, iMet-CAT-18-18 and hsa-miR-151a-5p were negatively correlated with sperm motility ( r=-0.437, P<0.001; r=-0.423, P<0.001; r=-0.515, P<0.001). Conclusion:Compared with semen with normal DFI, sncRNAs are differentially expressed in semen with critical and elevated DFI, which may be related to the molecular mechanism of DFI formation. SncRNAs have the potential to be used as biological markers of male infertility.

Malus floribunda Siebold. (Malus) is widely cultivated all over the world, which is of high ornamental value and breeding significance. Comparative analysis of the chloroplast genome can help enrich the phylogenetic relationship and facilitate germplasm utilization of Malus. Based on the whole genome sequencing data, a complete chloroplast genome (M. floribunda) with tetrad structure was assembled. The chloroplast genome (160 037 bp) was composed of a large single-copy (LSC) region (88 142 bp), inverted repeat (IR) B (26 353 bp), a small single-copy (SSC) region (19 189 bp), and IRA (26 353 bp). A total of 111 genes were annotated: 78 protein-coding genes, 29 tRNA genes and 4 rRNA genes. In addition, a large number of repeat sequences were identified in the genome, which was slightly different from that of M. sieboldii and M. toringoides. As for the relative synonymous codon usage, 30 high-frequency codons were found, and the codons tended to end with A/T. The results of interspecific sequence alignment and boundary analysis suggested the sequence variation of the LSC region was large, and the expansion and contraction of the SC region and IR region of the eight Malus species were generally similar. According to the phylogenetic analysis of chloroplast genome sequences, M. floribunda, M. hupehensis, and M. toringoides were grouped into one clade. The findings in this study can provide data support for the development of genetic markers and utilization of germplasm resources in the future.
Genome, Chloroplast ; Malus ; Phylogeny ; Plant Breeding ; Codon

To describe the fractal feature of CGR (Chaos-game representation) graph of genomes sequences, a multifractal theory is presented in the analysis. By studying the effect of three probability sets on the scale invariance range, the probability set with the best scale invariance is chosen, and then the smooth general dimension spectrum and multifractal spectrum are calculated. The experimental result shows that the probability set composed of the relative probability has the best scale-invariance performance. The scale invariance has three different variance regions, which indicate that genomes sequence segments with different lengths have different distribution rules. It is concluded that the multifractal method is effective for describing the fractal feature of CGR graph of genomes sequences.
Algorithms ; Base Sequence ; Computer Simulation ; Game Theory ; Genome ; Humans ; Mathematical Computing ; Sequence Analysis, DNA ; statistics & numerical data

Biosequence analysis is the primary research field of bioinformatics. In this field, useful information can be extracted by comparison analysis methods. Among them, sequence alignment is the most common comparison method. However the sequence comparison by alignment, which assumes conservation of contiguity between homologous segments, is at odds with genetic recombination. Especially for the multisequence alignment, there exists the difficulty in the complexity of calculation. Therefore, alignment-free sequence comparison methods are required. In this paper, two main categories of alignment-free sequence comparison methods are reviewed. The first one is based on the word (oligomer) frequency and its distribution. The sequences are compared using the distances defined in a Cartesian space by the frequency vectors. In the second category, sequences are compared using Kolmogorov complexity and chaos theory.
Algorithms ; Computational Biology ; Sequence Alignment ; Sequence Analysis ; methods

OBJECTIVETo detect the mutation sites of exons 2, 20, 11A and 11B in Chinese patients with breast cancer.

METHODSA total of 86 patients with breast cancer without blood relationship were randomly selected. Polymerase chain reaction (PCR) and double-strand DNA direct sequencing were applied.

RESULTSNo mutations, especially deletions were found in exons 2, 20 and 11 with carefully checking the sequencing results, although they were reported frequently in Europe populations with breast cancer. We found one polymorphism in exon 11, with high frequency, and in the test of chi-square, the frequencies of two alleles had no significant difference between the patients and controls.

CONCLUSIONThe above results suggest this SNP may not be associated with the breast cancer in Chinese population, and indicates that the gene sequence of what we have studied doesn't account much for occurrence of the breast cancer in the population of China.

Asian Continental Ancestry Group ; genetics ; BRCA1 Protein ; genetics ; Breast Neoplasms ; genetics ; Exons ; Female ; Gene Frequency ; Humans ; Mutation ; Polymorphism, Genetic

Objective　To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. Methods　A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP+E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6×105 CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors. Results　Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. Conclusion　The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.

Retroviral vectors are wildly used as vehicles for gene transfer into hematopoietic cells based on its potency for efficient gene delivery and integration of transgene in host genome. The development of better transduction protocols depends on gene markers that allow a rapid detection and effective selection of genetically transduced cells. In this study, the enhanced green fluorescent protein (EGFP), a gene that is optimized for detection and expression in mammalian cells, was firstly amplified and cloned by high-fidelity PCR. The vector LGSN carrying EGFP gene was then constructed and the retroviral producer cell lines that yield high titers of LGSN vector in supernatants were developed by liposome-mediated transfection in combination with cross infection. Both GP + envAm12 murine fibroblasts and K562 leukemic cells transduced with EGFP virus demonstrated a stable green fluorescence signal readily detectable by flow cytometry or fluorescence microscopy in up to 97% and 86% of examined cells, respectively. The integration of LGSN provirus in transduced cells was confirmed by PCR analysis. These results indicate EGFP is a suitable reporter molecule for gene transfer and expression in hematopoietic cells. Therefore, the bright and long-term expression of EGFP in living cells will advance the study of gene therapy in vitro and in vivo, particularly for human applications.

Objective To establish the replicated-deficient recombinant adenovirus-mediated thymidine kinase/acyclovir (Adtk/ACV) system and to evaluate its suicide effect on rat C6 brain gliomas in vitro and in vivo.Methods The plasmid pAdtk and pJM17 were co-infected into 293 cells (adenovector packaging cells) and the results were identified by polymerase chain reaction (PCR) assay. After the glioma C6 cells were transduced by Adtk at different multiplicity of infection (MOI) and exposed to different concentrations of ACV or gancyclovir (GCV), the cell survival curves were studied, and the cell surface was observed with scanning electronic microscopy (SEM). C6 gliomas in vivo at different inoculation days were injected with Adtk intratumorally and ACV intraperitoneally daily, and the survival duration and histologic changes of the rats were observed.Results The infectious Adtk virions had a suicide effect which was enhanced with the increase in MOIs of Adtk and ACV doses along with bystander effect. Under scanning electronic microscope, special pathologic changes were observed. ACV had a similar effect as GCV but a higher dose was used. The survival duration in day 3, day 6 and day 8 groups exceeded 90 days, and the rats in day 10 group survived 28.5±4.6 days, but the survival duration in untreated C6 group and AdLacZ/ACV (adenovirus-mediated LacZ/ACV) treated group were 16.8±3.1 and 14.0±2.2 days respectively. Conclusion Adtk/ACV system can effectively kill the rat brain gliomas in vitro and in vivo.