Genome dosage alteration caused by chromosome pyramiding and shuffling effects on karyotypic heterogeneity, reproductive diversity, and phenotypic variation in Zea-Tripsacum allopolyploids.

KEY MESSAGE: We developed an array of Zea-Tripsacum tri-hybrid allopolyploids with multiple ploidies. We unveiled that changes in genome dosage due to the chromosomes pyramiding and shuffling of three species effects karyotypic heterogeneity, reproductive diversity, and phenotypic variation in Zea-Tripsacum allopolyploids. Polyploidy, or whole genome duplication, has played a major role in evolution and speciation. The genomic consequences of polyploidy have been extensively studied in many plants; however, the extent of chromosomal variation, genome dosage, phenotypic diversity, and heterosis in allopolyploids derived from multiple species remains largely unknown. To address this question, we synthesized an allohexaploid involving Zea mays, Tripsacum dactyloides, and Z. perennis by chromosomal pyramiding. Subsequently, an allooctoploid and an allopentaploid were obtained by hybridization of the allohexaploid with Z. perennis. Moreover, we constructed three populations with different ploidy by chromosomal shuffling (allopentaploid × Z. perennis, allohexaploid × Z. perennis, and allooctoploid × Z. perennis). We have observed 3 types of sexual reproductive modes and 2 types of asexual reproduction modes in the tri-species hybrids, including 2n gamete fusion (2n + n), haploid gamete fusion (n + n), polyspermy fertilization (n + n + n) or 2n gamete fusion (n + 2n), haploid gametophyte apomixis, and asexual reproduction. The tri-hybrids library presents extremely rich karyotype heterogeneity. Chromosomal compensation appears to exist between maize and Z. perennis. A rise in the ploidy of the trihybrids was linked to a higher frequency of chromosomal translocation. Variation in the degree of phenotypic diversity observed in different segregating populations suggested that genome dosage effects phenotypic manifestation. These findings not only broaden our understanding of the mechanisms of polyploid formation and reproductive diversity but also provide a novel insight into genome pyramiding and shuffling driven genome dosage effects and phenotypic diversity.

Integrated single-molecule real-time sequencing and RNA sequencing reveal the molecular mechanisms of salt tolerance in a novel synthesized polyploid genetic bridge between maize and its wild relatives.

BACKGROUND: Tripsacum dactyloides (2n = 4x = 72) and Zea perennis (2n = 4x = 40) are tertiary gene pools of Zea mays L. and exhibit many abiotic adaptations absent in modern maize, especially salt tolerance. A previously reported allopolyploid (hereafter referred to as MTP, 2n = 74) synthesized using Zea mays, Tripsacum dactyloides, and Zea perennis has even stronger salt tolerance than Z. perennis and T. dactyloides. This allopolyploid will be a powerful genetic bridge for the genetic improvement of maize. However, the molecular mechanisms underlying its salt tolerance, as well as the key genes involved in regulating its salt tolerance, remain unclear. RESULTS: Single-molecule real-time sequencing and RNA sequencing were used to identify the genes involved in salt tolerance and reveal the underlying molecular mechanisms. Based on the SMRT-seq results, we obtained 227,375 reference unigenes with an average length of 2300 bp; most of the unigenes were annotated to Z. mays sequences (76.5%) in the NR database. Moreover, a total of 484 and 1053 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Functional enrichment analysis of DEGs revealed that multiple pathways responded to salt stress, including "Flavonoid biosynthesis," "Oxidoreductase activity," and "Plant hormone signal transduction" in the leaves and roots, and "Iron ion binding," "Acetyl-CoA carboxylase activity," and "Serine-type carboxypeptidase activity" in the roots. Transcription factors, such as those in the WRKY, B3-ARF, and bHLH families, and cytokinin negatively regulators negatively regulated the salt stress response. According to the results of the short time series-expression miner analysis, proteins involved in "Spliceosome" and "MAPK signal pathway" dynamically responded to salt stress as salinity changed. Protein-protein interaction analysis revealed that heat shock proteins play a role in the large interaction network regulating salt tolerance. CONCLUSIONS: Our results reveal the molecular mechanism underlying the regulation of MTP in the response to salt stress and abundant salt-tolerance-related unigenes. These findings will aid the retrieval of lost alleles in modern maize and provide a new approach for using T. dactyloides and Z. perennis to improve maize.

QTL-seq and transcriptomic integrative analyses reveal two positively regulated genes that control the low-temperature germination ability of MTP-maize introgression lines.

KEY MESSAGE: Two candidate genes (ZmbZIP113 and ZmTSAH1) controlling low-temperature germination ability were identified by QTL-seq and integrative transcriptomic analyses. The functional verification results showed that two candidate genes positively regulated the low-temperature germination ability of IB030. Low-temperature conditions cause slow maize (Zea mays L.) seed metabolism, resulting in slow seedling emergence and irregular seedling emergence, which can cause serious yield loss. Thus, improving a maize cultivar's low-temperature germination ability (LTGA) is vital for increasing yield production. Wild relatives of maize, such as Z. perennis and Tripsacum dactyloides, are strongly tolerant of cold stress and can thus be used to improve the LTGA of maize. In a previous study, the genetic bridge MTP was constructed (from maize, T. dactyloides, and Z. perennis) and used to obtain a highly LTGA maize introgression line (IB030) by backcross breeding. In this study, IB030 (Strong-LTGA) and Mo17 (Weak-LTGA) were selected as parents to construct an F2 offspring. Additionally, two major QTLs (qCS1-1 and qCS10-1) were mapped. Then, RNA-seq was performed using seeds of IB030 and the recurrent parent B73 treated at 10 °C for 27 days and 25 °C for 7 days, respectively, and two candidate genes (ZmbZIP113 and ZmTSAH1) controlling LTGA were located using QTL-seq and integrative transcriptomic analyses. The functional verification results showed that the two candidate genes positively regulated LTGA of IB030. Notably, homologous cloning showed that the source of variation in both candidate genes was the stable inheritance of introgressed alleles from Z. perennis. This study was thus able to analyze the LTGA mechanism of IB030 and identify resistance genes for genetic improvement in maize, and it proved that using MTP genetic bridge confers desirable traits or phenotypes of Z. perennis and tripsacum essential to maize breeding systems.

Physiological attributes and transcriptomics analyses reveal the mechanism response of Helictotrichon virescens to low temperature stress.

BACKGROUND: Helictotrichon virescens is a perennial grass that is primarily distributed in high altitude areas of 2000 ~ 4500 m. It is widely cultivated in the Qinghai-Tibet Plateau of China, strongly resistant to cold, and an essential part of the wild herbs in this region. However, the molecular mechanism of the response of H. virescens to low temperature stress and the key regulatory genes for specific biological processes are poorly understood. RESULTS: Physiological and transcriptome analyses were used to study the cold stress response mechanism in H virescens. During the low temperature stress period, the content of chlorophyll a and b decreased more and more with the delay of the treatment time. Among them, the difference between the controls was not significant, and the difference between the control and the treatment was significant. At the same time, the expression of related differential genes was up-regulated during low temperature treatment. In addition, the plant circadian pathway is crucial for their response to cold stress. The expression of differentially expressed genes that encode LHY and HY5 were strongly up-regulated during cold stress. CONCLUSIONS: This study should help to fully understand how H. virescens responds to low temperatures. It answers pertinent questions in the response of perennial herbs to cold stress, i.e., how light and low temperature signals integrate to regulate plant circadian rhythms and Decrease of content of chlorophylls (which can be also accompanied with decrease of total quantity of reaction centers) leads to an increase in photosynthetic damage.

Analysis of the genitor origin of an intergeneric hybrid clone between Zea and Tripsacum for forage production by McGISH.

In this study, the chromosome number and composition of a novel perennial forage crop, 'Yucao No. 6' (Yu6), was revealed by chromosome spread and McGISH (multicolor genomic in situ hybridization) techniques to clarify its genitor origin. Cytogenetic analysis showed that Yu6, which has 56 chromosomes, is an aneuploid representing 12, 17 and 27 chromosomes from Zea mays ssp. mays L. (Zm, 2n = 2x = 20), Tripsacum dactyloides L. (Td, 2n = 4x = 72), and Z. perennis (Hitchc.) Reeves & Mangelsd. (Zp, 2n = 4x = 40), respectively. This finding indicates that Yu6 is the product of a reduced egg (n = 36 = 12Zm + 17Td + 7Zp) of MTP (a near-allohexaploid hybrid, 2n = 74 = 20Zm + 34Td + 20Zp) fertilized by a haploid sperm nucleus (n = 20Zp) of Z. perennis. Moreover, 3 translocated chromosomes consisting of the maize-genome chromosome with the segment of Z. perennis were observed. These results suggest that it is practical to develop perennial forage maize by remodeling the chromosomal architecture of MTP offspring with Z. perennis as a pollen parent. Finally, the overview of forage breeding in the Zea and Tripsacum genera was discussed.

Allopolyploidization facilitates gene flow and speciation among corn, Zea perennis and Tripsacum dactyloides.

MAIN CONCLUSION: Tripsacum dactyloides is closely related to Zea mays since Zea perennis and the MTP tri- species hybrid have four possible reproductive modes. Eastern gamagrass (Tripsacum dactyloides L.) and tetraploid perennial teosinte (Zea perennis) are well known to possess genes conferring resistance against biotic and abiotic stresses as well as adaptation to flood and aluminum toxic soils. However, plant breeders have been hampered to utilize these and other beneficial traits for maize improvement due to sterility in their hybrids. By crossing a tetraploid maize-inbred line × T. dactyloides, a female fertile hybrid was produced that was crossed with Z. perennis to yield a tri-genomic female fertile hybrid, which was backcrossed with diploid maize to produce BC1 and BC2. The tri-genomic hybrid provided a new way to transfer genetic material from both species into maize by utilizing conventional plant breeding methods. On the basis of cytogenetic observations using multi-color genomic in situ hybridization, the progenies were classified into four groups, in which chromosomes could be scaled both up and down with ease to produce material for varying breeding and genetic purposes via apomixis or sexual reproduction. In the present study, pathways were found to recover maize and to obtain specific translocations as well as a speedy recovery of the T. dactyloides-maize addition line in a second backcross generation. However, phenotypes of the recovered maize were in most cases far from maize as a result of genetic load from T. dactyloides and Z. perennis, and could not be directly used as a maize-inbred line but could serve as an intermediate material for maize improvement. A series of hybrids was produced (having varying chromosome number, constitution, and translocations) with agronomic traits from all three parental species. The present study provides an application of overcoming the initial interspecific barriers among these species. Moreover, T. dactyloides is closely related to Z. mays L. ssp. mays since Z. perennis and the MTP tri- species hybrid have four possible reproductive modes.

TMPYP4 exerted antitumor effects in human cervical cancer cells through activation of p38 mitogen-activated protein kinase.

BACKGROUND: The aim of the present study was to investigate the potential effects of the 5,10,15,20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4) on the proliferation and apoptosis of human cervical cancer cells and the underlying mechanisms by which TMPyP4 exerted its actions. RESULTS: After human cervical cancer cells were treated with different doses of TMPyP4, cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method, the apoptosis was observed by flow cytometry (FCM), and the expression of p38 mitogen-activated protein kinase (MAPK), phosphated p38 MAPK (p-p38 MAPK), capase-3, MAPKAPK2 (MK-2) and poly ADP-ribose polymerase (PARP) was measured by Western blot analysis. The analysis revealed that TMPyP4 potently suppressed cell viability and induced the apoptosis of human cervical cancer cells in a dose-dependent manner. In addition, the up-regulation of p-p38 MAPK expression levels was detected in TMPyP4-treated human cervical cancer cells. However, followed by the block of p38 MAPK signaling pathway using the inhibitor SB203580, the effects of TMPyP4 on proliferation and apoptosis of human cervical cancer cells were significantly changed. CONCLUSIONS: It was indicated that TMPyP4-inhibited proliferation and -induced apoptosis in human cervical cancer cells was accompanied by activating the p38 MAPK signaling pathway. Taken together, our study demonstrates that TMPyP4 may represent a potential therapeutic method for the treatment of cervical carcinoma.

A teosinte-derived allele of ZmSC improves salt tolerance in maize.

Maize, a salt-sensitive crop, frequently suffers severe yield losses due to soil salinization. Enhancing salt tolerance in maize is crucial for maintaining yield stability. To address this, we developed an introgression line (IL76) through introgressive hybridization between maize wild relatives Zea perennis, Tripsacum dactyloides, and inbred Zheng58, utilizing the tri-species hybrid MTP as a genetic bridge. Previously, genetic variation analysis identified a polymorphic marker on Zm00001eb244520 (designated as ZmSC), which encodes a vesicle-sorting protein described as a salt-tolerant protein in the NCBI database. To characterize the identified polymorphic marker, we employed gene cloning and homologous cloning techniques. Gene cloning analysis revealed a non-synonymous mutation at the 1847th base of ZmSCIL76 , where a guanine-to-cytosine substitution resulted in the mutation of serine to threonine at the 119th amino acid sequence (using ZmSCZ58 as the reference sequence). Moreover, homologous cloning demonstrated that the variation site derived from Z. perennis. Functional analyses showed that transgenic Arabidopsis lines overexpressing ZmSCZ58 exhibited significant reductions in leaf number, root length, and pod number, alongside suppression of the expression of genes in the SOS and CDPK pathways associated with Ca2+ signaling. Similarly, fission yeast strains expressing ZmSCZ58 displayed inhibited growth. In contrast, the ZmSCIL76 allele from Z. perennis alleviated these negative effects in both Arabidopsis and yeast, with the lines overexpressing ZmSCIL76 exhibiting significantly higher abscisic acid (ABA) content compared to those overexpressing ZmSCZ58 . Our findings suggest that ZmSC negatively regulates salt tolerance in maize by suppressing downstream gene expression associated with Ca2+ signaling in the CDPK and SOS pathways. The ZmSCIL76 allele from Z. perennis, however, can mitigate this negative regulatory effect. These results provide valuable insights and genetic resources for future maize salt tolerance breeding programs.

Modified vaginal dilation rat model for postpartum stress urinary incontinence.

AIM: The aim of this study was to develop a modified rat model for postpartum stress urinary incontinence (SUI) which can be easily established and which will steadily evaluate urodynamic changes. MATERIAL AND METHODS: A urinary catheter was placed into the vaginas of rats (n=40) and the balloon was inflated for 8 h to simulate labor. A whisker was inserted into the nostril to induce sneezing. The urodynamic changes induced by vaginal dilation or sneezing were observed by measurement of leak point pressure (LPP), and maximum bladder volume. Serum lactose dehydrogenase (LDH) and creatine kinase (CK) were tested, and the mRNA expression of caspase-3 in pubococcygeus muscle tissue was measured to show the effect of muscle injury in SUI. RESULTS: After 8 h of balloon vaginal dilation, a rat model of postpartum SUI was established with a success rate of 72.5% (29/40). By sneeze assay, SUI was noted in 100% (5/5), 80% (4/5), 60% (3/5), 60% (3/5) and 40% (2/5) of experimental rats on day 1, 7, 14, 28 and 56, respectively. No SUI was found in the control group (n=10). The level of serum LDH and CK in these 29 experimental rats initially increased, peaked on day 7, and finally decreased toward normal values. Compared with control rats, the differences were significant (P<0.05). The expression of the caspase-3 gene in pubococcygeal muscle of experimental rats peaked on day 1 and decreased thereafter, while no such change was seen in the control group, and the difference was significant (P<0.05). CONCLUSION: Eight hours of vaginal balloon dilation resulted in SUI in this rat model. The alterations in serum LDH and CK, and in the muscle's apoptotic genes mimic those observed in human postpartum SUI patients. It can therefore be considered as a useful animal model for the study on the pathogenesis of postpartum SUI.

Multispecies polyploidization, chromosome shuffling, and genome extraction in Zea/Tripsacum hybrids.

By hybridization and special sexual reproduction, we sequentially aggregated Zea mays, Zea perennis, and Tripsacum dactyloides in an allohexaploid, backcrossed it with maize, derived self-fertile allotetraploids of maize and Z. perennis by natural genome extraction, extended their first six selfed generations, and finally constructed amphitetraploid maize using nascent allotetraploids as a genetic bridge. Transgenerational chromosome inheritance, subgenome stability, chromosome pairings and rearrangements, and their impacts on an organism's fitness were investigated by fertility phenotyping and molecular cytogenetic techniques genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). Results showed that diversified sexual reproductive methods produced highly differentiated progenies (2n = 35-84) with varying proportions of subgenomic chromosomes, of which one individual (2n = 54, MMMPT) overcame self-incompatibility barriers and produced a self-fertile nascent near-allotetraploid by preferentially eliminating Tripsacum chromosomes. Nascent near-allotetraploid progenies showed persistent chromosome changes, intergenomic translocations, and rDNA variations for at least up to the first six selfed generations; however, the mean chromosome number preferably maintained at the near-tetraploid level (2n = 40) with full integrity of 45S rDNA pairs, and a trend of decreasing variations by advancing generations with an average of 25.53, 14.14, and 0.37 for maize, Z. perennis, and T. dactyloides chromosomes, respectively. The mechanisms for three genome stabilities and karyotype evolution for formatting new polyploid species were discussed.

Transcriptomics integrated with metabolomics provides a new strategy for mining key genes in response to low temperature stress in Helictotrichon virescens.

H. virescens is a perennial herbaceous plant with highly tolerant to cold weather, but the key genes that respond to low temperature stress still remain unclear. Hence, RNA-seq was performed using leaves of H. virescens treated at 0 °C and 25 °C for 12 h, 36 h, and 60 h, respectively, and a total of 9416 DEGs were significantly enriched into seven KEGG pathways. The LC-QTRAP platform was performed using leaves of H. virescens leaves at 0 °C and 25 °C for 12 h, 36 h, and 60 h, respectively, and a total of 1075 metabolites were detected, which were divided into 10 categories. Additionally, 18 major metabolites, two key pathways, and six key genes were mined using a multi-omics analytical strategy. The RT-PCR results showed that with the extension of treatment time, the expression levels of key genes in the treatment group gradually increased, and the difference between the treatment group and the control group was extremely significant. Notably, the functional verification results showed that the key genes positively regulated cold tolerance of H. virescens. These results can lay a foundation for the in-depth analysis of the mechanism of response of perennial herbs to low temperature stress.

Evaluation of medical services from the perspective of COVID-19 vaccine demand satisfaction in Hangzhou, China.

The outbreak of COVID-19 has had a huge global impact, and it continues to test the resilience of medical services to emergencies worldwide. In the current post-epidemic era, vaccination has become a highly effective strategy to prevent the spread of COVID-19. However, using conventional mathematical models to evaluate the spatial distribution of medical resources, including vaccination, ignore people's behaviors and choices and make simplifications to the real world. In this study, we use an enhanced model based on the Theory of People Behavior (TPB) to perform a macro analysis of the satisfaction ability of medical resources for vaccination in Hangzhou, China, and attribute the city to a three-level structure. According to the allocation, the supply capacity of vaccination sites is calculated and divided into four categories (good, normal, not bad, and bad). Meanwhile, we raise an assumption based on the result and the general development law of the city and analyze the reasons for the impact of personal behavior on the spatial distribution of medical resources, as well as the relationship between the demand distribution and spatial distribution of medical resources and future development strategies. It is considered that the overall medical resources, especially vaccination in Hangzhou, feature the situation of central supply overflow, and are found to hardly meet the needs of population points in surrounding areas, requiring a more flexible strategy to allocate facilities in these areas.

Rare brain and pulmonary abscesses caused by oral pathogens started with acute gastroenteritis diagnosed by metagenome next-generation sequencing: A case report and literature review.

Odontogenic brain and pulmonary abscesses are extremely rare infectious diseases. It is mainly caused by the upward or downward transmission of local infection or blood-borne spread. In recent years, with the wide application of some novel testing methods in clinical practice, the diagnosis of unexplained infections such as odontogenic abscesses in different organs has gradually become clear. We report a case of a 21-year-old male who was healthy and had not received any oral treatment before onset. He started with acute gastroenteritis-related symptoms, then developed meningitis-related symptoms seven days later with septic shock. No obvious abscess lesions were found on head computed tomography (CT) at admission, and the etiology was not clear by routine examination, which was very easy to misdiagnose as a serious infection caused by intestinal pathogens. But odontogenic pathogens were found both in his blood and cerebrospinal fluid through metagenomic next-generation sequencing (mNGS) analysis. Subsequently, rechecked imaging examination displayed multiple brain and pulmonary abscesses. Finally, it was diagnosed as an odontogenic brain and pulmonary abscess. After an extremely lengthy anti-infection course (13 weeks of intravenous antibiotics plus 2 weeks of oral antibiotics) and surgery, the patient was improved and discharged from the hospital. From this case, we could see that the development of new diagnostic technologies such as mNGS plays an important role in the early and confirmed diagnosis of diseases previously difficult to diagnose such as odontogenic polymicrobial infections and ultimately helps to improve the prognosis of these patients.

RNA sequencing and weighted gene co-expression network analysis uncover the hub genes controlling cold tolerance in Helictotrichon virescens seedlings.

Helictotrichon virescens is a perennial herbaceous plant with a life expectancy of about 10 years. It has high cold and heat resistance and can successfully survive over winter in the habitats with a temperature range of -25 to 25°C. Therefore, this study aimed to identify the key genes regulating low-temperature stress responses in H. virescens and analyze cold tolerant at molecular level. This study used RNA sequencing (RNA-Seq) and weighted gene co-expression network analysis (WGCNA) to identify the hub genes associated with cold tolerance in H. virescens. RT-PCR was conducted, homologous genes were identified, and related bioinformatics were analyzed to verify the identified hub genes. Moreover, WGCNA analysis showed that only the brown module had the highest correlation with the active-oxygen scavenging enzymes [peroxide (POD), superoxide dismutase (SOD), and catalase (CAT)]. The expression levels of three hub genes in the brown module (Cluster-37118.47362, cluster-37118.47713, and cluster-37118.66740) were significantly higher under low-temperature stress than those under control conditions. Furthermore, gene ontology (GO) and KEGG annotations showed that the three hub genes were mainly enriched in the metabolism pathways of sphingolipids, selenocompounds, glyoxylate, and dicarboxylate, carotenoids biosynthesis, and other biological pathways. The results of this study also showed that the subcellular localization prediction results showed that the cold tolerance hub genes were all localized to the plasma membrane. By constructing a protein interaction network, it was found that the hub gene Cluster-37118.66740 interacted with Sb09g003460.1 and Sb04g020180.1 proteins in Sorghum bicolor. By constructing phylogenetic trees of the four species of H. virescens, Sorghum bicolo, Oryza sativa Japonica, and Arabidopsis thaliana, the results showed that, the hub gene Cluster 37118.66740 (of H. virescens) and Os03g0340500 (of Oryza sativa Japonica) belonged to the same ancestral branch and were in the same subfamily. Thus, this study provides methodology and guidance to identify the cold tolerance genes for other herbage and their cold tolerant molecular mechanisms at molecular level.

Secretion of oestrogen from murine-induced pluripotent stem cells co-cultured with ovarian granulosa cells in vitro.

POF (premature ovarian failure) is a distressing condition that is a common cause of infertility. No effective treatment is available to overcome the loss of fertility. A method to derive oestrogen from miPSCs (mouse-induced pluripotent stem cells) was explored as a potential treatment for POF. In this study, C57BL/6 female mice were injected with PMSG (pregnant mare's serum gonadotropin) to obtain ovarian GCs (granulosa cells) and then co-cultured with miPSCs. The morphological changes in the miPSCs co-cultured with GCs were observed by light microscopy. The expression of FSHR (follicle-stimulating hormone receptor) was detected by immunocytochemistry and flow cytometry. Radioimmunoassay was used to analyse the level of E2 (oestradiol) in culture supernatants. The results showed that the proportion of GCs expressing FSHR in GCs was over 90%. The E2 concentration of the culture supernatant of the GC group was 62.4 pg/ml on day 1 and decreased in a time-dependent manner. The opposite situation was observed in the miPSCs-GC co-cultured group with an E2 concentration of 87.9 pg/ml on day 1 that increased in a time-dependent manner to reach a concentration of 328.4 pg/ml on day 7. The data indicate that GC-like cells were effectively induced from miPSCs through indirect cell-to-cell contact. Our method provides a novel in vitro system to study miPSC differentiation, particularly the interactions between miPSCs and GCs. The ultimate goal of this approach would be to provide a treatment for POF in the future.

[Lung metastasis of human choriocarcinoma in mice: establishment of experimental metastatic model and its biological characteristics].

OBJECTIVE: To establish a satisfactory lung metastasis model of human choriocarcinoma using severe combined immunodeficient (SCID) mice and explore the appropriate cell concentration for the model. METHODS: Forty SCID mice aged between 5 - 6 weeks were randomly divided into four groups. 1 × 10(7) cells/ml × 0.1 ml, 5 × 10(6) cells/ml × 0.2 ml and 1 × 10(6) cells/ml × 0.1 ml of human choriocarcinoma cells JEG-3 were respectively injected in SCID mice of experimental groups by lateral tail vein, the remain group was assigned to the control group. The status and weight of mice were observed every three days. When these mice were being dying, the size and the number of the lesions of lung metastasis in every mouse were inspected with Micro CT. After Micro CT inspection, the SCID mice were executed dissected to note whether there were tumors on all organ surfaces with naked eyes, then made pathological sections from the metastatic foci of fresh lung tissues, and cultured primarily cells and purified cells and passaged cells isolated from the same metastastic foci. The pathological sections were observed under the microscope. The special antigen human chorionic gonadotropin-beta subunit (ß-hCG) of the choriocarcinoma cells was immunohistochemically detected in the pathological sections and the cells out of cultured primarily cells. The chromosomes of the cells out of cultured primarily cells were analysed. RESULTS: Of the group inoculated 1 × 10(7) cells/ml × 0.1 ml, all mice died when inoculating. In the group of 5 × 10(6) cells/ml × 0.2 ml, when inoculating, 3 mice died; the remain 7 mice were being dying on (18.0 ± 2.0) days after injection. 5 of them, there were 1 - 3 lesions of lung metastasis after Micro CT inspection in each mice, and the diameter of the tumors lesions reached 1.5 - 3.5 mm, which was choriocarcinoma confirmed by pathological sections. The special antigen ß-hCG was detected by immunohistochemical method in the pathological sections of pulmonary tissue with tumor and in the cells, which were purified and passaged from being cultured primarily cells isolated from metastastic foci of fresh lung tissues from the SCID mice. The chromosome numbers of these cells out of cultured primarily cells were variety from 19 to 128, and modal numbers were variety from 70 to 79. CONCLUSIONS: We successfully established the lung metastatic model of human choriocarcinoma in SCID mice by injecting JEG-3 cells into lateral tail vein, of which 5 × 10(6) cells/ml × 0.2 ml is the suitable concentration and volume for the model.

[Long-term and stable expression of transgene mediated by piggyBac transposon in gynecological malignant tumor cells].

OBJECTIVE: To investigate the expression of exogenous gene transferred by piggyBac (PB) transposon in various gynecological malignant cell lines and reveal its potential application of gene therapy in gynecological cancer. METHODS: Amplified herpes simplex virus thymidine kinase (HSV-tk) gene coding region by PCR and integrated it into PB expression vector, PB[Act-RFP]DS, for reconstructing PB[Act-RFP, HSV-tk]DS (pPB/TK). By using different transfection reagents: FuGENE HD, jetPEI, lipofectamine 2000, pPB/TK together with helper plasmid Act-PBase were cotransfected into four mostly common gynecological malignant tumor cell lines HeLa, JEG-3, SKOV3 and HEC-1B. The mRFP1 report gene expressions was observed and detected by fluorescence microscope and flow cytometry to analyze transfection efficiency. The expressions of HSV-tk and mRFP1 gene were detected by reverse transcription PCR (RT-PCR). The cytotoxic effect of various concentration of pro-drug ganciclovir (GCV) for transfected cells was detected by methyl thiazole tetrazolium assay. The transfected cells were positive sorted by flow cytometry and limiting diluted to obtain the stable transfected cell line. The insertion sites of foreign gene transferred by PB transposon in genome were analyzed by inverse PCR. RESULTS: (1) Double digests analysis and sequences test demonstrated that pPB/TK vector was reconstructed successfully. (2) Using three different transfective reagents, PB transposon transferred HSV-tk gene and mRFP1 gene into HeLa, HEC-1B, SKOV3 and JEG-3 cell efficiently, and the transfection efficiency of pPB/TK for the same cell was different by using different transfective reagents; in Hela cell, the transfection efficiency of FuGENE HD [(78.7+/-9.2)%] was higher than that of lipofectamine 2000 [(54.1+/-11.4)%] and jetPEI [(46.5+/-7.4)%, all P<0.05]; using the same transfective reagent, the transfection efficiency of pPB/TK was also different on various cell lines, using FuGENE HD, the transfection efficiency of pPB/TK on HeLa, JEG-3 and SKOV3 cell was (78.7+/-9.2)%, (74.4+/-8.9)% and (83.2+/-9.7)% respectively, which all were higher than that on HEC-1B [(39.5+/-8.7)%, P<0.05]. (3) RT-PCR showed that there were the mRNA expression of HSV-tk and mRFP1 in all cell lines. (4) 50% inhibitory concentration of GCV for transfected cells, HeLa, JEG-3, SKOV3 and HEC-1B, was 1.29, 3.35, 0.09 and 13.28 microg/ml respectively. Inhibitory effect of GCV (10 microg/ml) on SKOV3 transfected with pPB/TK was (86+/-9)%, which was superior to that transfected with pORF-HSVtk alone [(52+/-12)%, P<0.05]. (5) The insertion sites of PB transposon in the target cells genome were located at TTAA sites. mRFP1 expression still could be detected in three months after transfected. CONCLUSIONS: PB transposon could transfer exogenous gene into various gynecological malignant cells, which could integrated into genome and obtain a long-term and stable expression. It is expected that PB transposon may supply a more efficient and safer transgene technology platform for gene therapy in gynecological cancer.

Tripsazea, a Novel Trihybrid of Zea mays, Tripsacum dactyloides, and Zeaperennis.

A trispecific hybrid, MTP (hereafter called tripsazea), was developed from intergeneric crosses involving tetraploid Zea mays (2n = 4x = 40, genome: MMMM), tetraploid Tripsacum dactyloides (2n = 4x = 72, TTTT), and tetraploid Zperennis (2n = 4x = 40, PPPP). On crossing maize-Tripsacum (2n = 4x = 56, MMTT) with Zperennis, 37 progenies with varying chromosome numbers (36-74) were obtained, and a special one (i.e., tripsazea) possessing 2n = 74 chromosomes was generated. Tripsazea is perennial and expresses phenotypic characteristics affected by its progenitor parent. Flow cytometry analysis of tripsazea and its parents showed that tripsazea underwent DNA sequence elimination during allohexaploidization. Of all the chromosomes in diakinesis I, 18.42% participated in heterogenetic pairing, including 16.43% between the M- and P-genomes, 1.59% between the M- and T-genomes, and 0.39% in T- and P-genome pairing. Tripsazea is male sterile and partly female fertile. In comparison with previously synthesized trihybrids containing maize, Tripsacum and teosinte, tripsazea has a higher chromosome number, higher seed setting rate, and vegetative propagation ability of stand and stem. However, few trihybrids possess these valuable traits at the same time. The potential of tripsazea is discussed with respect to the deployment of the genetic bridge for maize improvement and forage breeding.

Serum containing Gengnianchun formula suppresses amyloid ß­induced inflammatory cytokines in BV­2 microglial cells by inhibiting the NF­κB and JNK signaling pathways.

As the resident macrophages of the brain's innate immune system, microglial cells are key modulators in the neurodegenerative disease Alzheimer's disease (AD). In particular, the activation and accumulation of microglial cells around amyloid plaques is considered to result in chronic neuroinflammation. Although the pathologic mechanism remains to be fully elucidated, inflammation has been shown to be critical in the pathogenesis of AD. The Gengnianchun (GNC) formula has long been used to treat perimenopausal syndrome clinically, and is particularly effective in improving learning ability and memory. Our previous study demonstrated that GNC formula had an anti­inflammatory effect and offered neuroprotection in animal experiments. In the present study, the anti­inflammatory properties of GNC and its underlying mechanism of action were examined in BV­2 microglial cells. Amyloid­ß peptide (Aß)­stimulated microglial cells were examined for the production of proinflammatory cytokines and the underlying signaling pathways. Compared with the normal control group, the protein expression levels of IL­1ß and TNF­α were significantly increased following treatment with Aß (P<0.01), but medicated rat serum containing GNC formula (MRS) could significantly attenuated the Aß­induced secretion of these pro­inflammatory cytokines. It was identified by CCK­8 assay that the viability of the BV­2 cells was not reduced following treatment with various concentrations of MRS. The phosphorylation of factor­κB (NF­κB) and c­Jun N­terminal kinase (JNK) was markedly increased following treatment with Aß, compared with the normal control group (P<0.01). However, treatment with MRS resulted in a significant reduction in the phosphorylation of NF­κB (P<0.05). These results suggested that MRS suppressed the Aß­induced inflammatory response of microglial cells by inhibiting the NF­κB and JNK signaling pathways. These novel findings provide insights into the development of GNC formula as a therapeutic agent for the treatment of neurodegenerative disorders.

Mysterious meiotic behavior of autopolyploid and allopolyploid maize.

This study was aimed to investigate the stability of chromosomes during meiosis in autopolyploid and allopolyploid maize, as well as to determine an association of chromosomes between maize (Zea mays ssp. mays Linnaeus, 1753) and Z. perennis (Hitchcock, 1922) Reeves & Mangelsdor, 1942, by producing a series of autopolyploid and allopolyploid maize hybrids. The intra-genomic and inter-genomic meiotic pairings in these polyploids were quantified and compared using dual-color genomic in-situ hybridization. The results demonstrated higher level of chromosome stability in allopolyploid maize during meiosis as compared to autopolyploid maize. In addition, the meiotic behavior of Z. perennis was relatively more stable as compared to the allopolyploid maize. Moreover, ten chromosomes of "A" subgenome in maize were homologous to twenty chromosomes of Z. perennis genome with a higher pairing frequency and little evolutionary differentiation. At the same time, little evolutionary differentiation has been shown by chromosomes of "A" subgenome in maize, while chromosomes of "B" subgenome, had a lower pairing frequency and higher evolutionary differentiation. Furthermore, 5IM + 5IIPP + 5IIIMPP and 5IIMM + 5IIPP + 5IVMMPP were observed in allotriploids and allotetraploids respectively, whereas homoeologous chromosomes were found between the "A" and "B" genome of maize and Z. perennis.