Construction of 3-Methyl-2-Substituted Benzo[b]furans and 3-Methyl-2-Substituted Benzo[b]thiophenes Using Solid Calcium Carbide as a Substitute for Gaseous Acetylene.

A concise method for the facile construction of 3-methyl-2-substituted benzo[b]furans and 3-methyl-2-substituted benzo[b]thiophenes using low-cost, abundant, and easy-to-use solid calcium carbide instead of flammable and explosive gaseous acetylene as an original alkyne source, o-bromophenyl ethers or o-bromophenyl thioethers as substrates through an intramolecular carbanion-yne cyclization in a 5-exo-dig manner, and subsequent double-bond isomerization is described. The simultaneous formation of two C-C bonds is realized in a one-step route. The wide substrate scope, high yield, and simple workup manipulations are also merits of this method. The synthetic strategy can also be suitable for the gram scale.

Identification of molecular characteristics of hepatocellular carcinoma with microvascular invasion based on deep targeted sequencing.

BACKGROUND: As an indicator of tumor invasiveness, microvascular invasion (MVI) is a crucial risk factor for postoperative relapse, metastasis, and unfavorable prognosis in hepatocellular carcinoma (HCC). Nevertheless, the genetic mechanisms underlying MVI, particularly for Chinese patients, remain mostly uncharted. METHODS: We applied deep targeted sequencing on 66 Chinese HCC samples. Focusing on the telomerase reverse transcriptase (TERT) promoter (TERTp) and TP53 co-mutation (TERTp+/TP53+) group, gene set enrichment analysis (GSEA) was used to explore the potential molecular mechanisms of the TERTp+/TP53+ group on tumor progression and metastasis. Additionally, we evaluated the tumor immune microenvironment of the TERTp+/TP53+ group in HCC using multiplex immunofluorescence (mIF) staining. RESULTS: Among the 66 HCC samples, the mutated genes that mostly appeared were TERT, TP53, and CTNNB1. Of note, we found 10 cases with TERTp+/TP53+, of which nine were MVI-positive and one was MVI-negative, and there was a co-occurrence of TERTp and TP53 (p < 0.05). Survival analysis demonstrated that patients with the TERTp+/TP53+ group had lower the disease-free survival (DFS) (p = 0.028). GSEA results indicated that telomere organization, telomere maintenance, DNA replication, positive regulation of cell cycle, and negative regulation of immune response were significantly enriched in the TERTp+/TP53+ group (all adjusted p-values (p.adj) < 0.05). mIF revealed that the TERTp+/TP53+ group decreased CD8+ T cells infiltration (p = 0.25) and enhanced PDL1 expression (p = 0.55). CONCLUSIONS: TERTp+/TP53+ was significantly enriched in MVI-positive patients, leading to poor prognosis for HCC patients by promoting proliferation of HCC cell and inhibiting infiltration of immune cell surrounding HCC. TERTp+/TP53+ can be utilized as a potential indicator for predicting MVI-positive patients and poor prognosis, laying a preliminary foundation for further exploration of co-mutation in HCC with MVI and clinical treatment.

AIE luminogen labeled polymeric micelles for biological imaging and chemotherapy.

In this study, an amphiphilic polymer FA-CS-DBA-CHO with aggregation-induced emission (AIE) feature was prepared by introducing 4-(diphenylamino)benzaldehyde derivative (DBA-CHO), imine bond and folic acid (FA) to the molecular structure of chitosan (CS). The amphiphilicity drove the polymer to self-assemble into micelles, and paclitaxel (PTX) could be solubilized in the hydrophobic core. Due to the excellent AIE effect, FA-CS-DBA-CHO exhibited strong cellular imaging capability. The pH-sensitive imine bond in the polymer allowed for accurate drug release in acidic environment. Both in vitro and in vivo studies demonstrated that the PTX-loaded FA-CS-DBA-CHO micelles could significantly inhibit the growth of tumor cells but without any notable toxicity. This micellar system was excellent carrier for bioimaging and chemotherapeutic drug delivery.

Optimization of cold plasma processing conditions for fresh-cut kiwifruit slices by using response surface methodology.

The work aims to optimize the process of cold plasma for fresh-cut kiwifruit. The effects of discharge times, treatment voltages, and slice thickness as well as the interaction between them were investigated. Factor analysis was used to screen out the characteristic indices of fresh-cut kiwifruit. Design-Expert software was used to design three-factor response surface tests and find the optimal parameters. The results revealed that the quality indices of fresh-cut kiwifruit were the color difference, brittleness, and solid-acid ratio, the established binomial regression equations were significant (P < 0.05). At the optimal level: 26 kV treatment voltage, 120 s discharge times, and 10 mm slice thickness, the optimized test values for the color difference, brittleness, solid-acid ratio and decreased logarithm value of total plate count were 2.25, 128.96 g·s, 18.03 and 2.30 lg(CFU·g-1), respectively. Cold plasma could significantly improve the inactivation of bacteria in fresh-cut kiwifruit.

High spindle and kinetochore-associated complex subunit-3 expression predicts poor prognosis and correlates with adverse immune infiltration in hepatocellular carcinoma.

BACKGROUND: Spindle and kinetochore-associated complex subunit 3 (SKA3) is a malignancy-associated gene that plays a critical role in the regulation of chromosome separation and cell division. However, the molecular mechanism through which SKA3 regulates tumor cell proliferation in hepatocellular carcinoma (HCC) has not been fully elucidated. AIM: To investigate the molecular mechanisms underlying the role of SKA3 in HCC. METHODS: SKA3 expression, clinicopathological, and survival analyses were performed using multiple public database platforms, and the results were verified by Western blot and immunohistochemistry staining using collected clinical samples. Functional enrichment analyses were performed to evaluate the biological functions and molecular mechanisms of SKA3 in HCC. Furthermore, the Tumor Immune Estimation Resource and single-sample Gene Set Enrichment Analysis (ssGSEA) algorithms were utilized to investigate the abundance of tumor-infiltrating immune cells in HCC. The response to chemotherapeutic drugs was evaluated by the R package "pRRophetic". RESULTS: We found that upregulated SKA3 expression was significantly correlated with poor prognosis in patients with HCC. Multivariable Cox regression analysis indicated that SKA3 was an independent risk factor for survival. GSEA revealed that SKA3 expression may facilitate proliferation and migratory processes by regulating the cell cycle and DNA repair. Moreover, patients with high SKA3 expression had significantly decreased ratios of CD8+ T cells, natural killer cells, and dendritic cells. Drug sensitivity analysis showed that the high SKA3 group was more sensitive to sorafenib, sunitinib, paclitaxel, doxorubicin, gemcitabine, and vx-680. CONCLUSION: High SKA3 expression led to poor prognosis in patients with HCC by enhancing HCC proliferation and repressing immune cell infiltration surrounding HCC. SKA3 may be used as a biomarker for poor prognosis and as a therapeutic target in HCC.

Correlation between diabetic retinopathy and Helicobacter pylori infection: a cross-sectional retrospective study.

AIM: To explore the correlation between diabetic retinopathy (DR) and Helicobacter pylori (Hp) infection, based on data from a physical examination population. METHODS: This cross-sectional retrospective analysis included data of 73 824 health examination participants from December 2018 to December 2019. Participants were divided into the diabetic group and non-diabetic group, non-diabetic retinopathy (NDR) group, non-proliferative diabetic retinopathy (NPDR) group, proliferative diabetic retinopathy (PDR) group, and Hp infection group. Gender, age, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting plasma glucose (FPG), glycated hemoglobin A1c (HbA1c), triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and Hp data were recorded to compare the degree of DR lesions and Hp infection. Logistic regression analysis was used to evaluate the correlation between DR and Hp infection. RESULTS: There was a statistically significant difference between the diabetic and non-diabetic group (χ2=94.17, P<0.0001). Logistic regression analysis showed that male sex, age, BMI, SBP, TG, LDL-C, and Hp infection were independent risk factors for DR. There was no correlation between the degree of DR lesions and Hp infection (ρ=-0.00339, P=0.7753). Age [odds ratio (OR)=1.035, 95%CI: 1.024, 1.046, P<0.0001] and SBP (OR=1.009, 95%CI: 1.004, 1.015, P=0.0013) were independent risk factors for the degree of DR. CONCLUSION: There is a significant correlation between DR and Hp infection in the physical examination population. Hp infection is a risk factor for DR, and there is no significant difference between Hp infection and DR of different pathological degrees. Actively eradicating Hp may be of help to prevent DR.

One-Pot Synthesis of 1,2,3-Triarylindoles through Cascade Reactions Using Calcium Carbide, Iodoarenes, and Aromatic Amines.

An efficient method for the construction of 1,2,3-triarylindoles through one-pot multicomponent reactions using calcium carbide, 3 mol of iodoarenes, and aromatic amines as starting materials was described. A series of target products were obtained by the simultaneous formation of five bonds in one step. The main advantages for this protocol are the use of a convenient alkyne source, wide scope of substrates, and simple workup procedure. The method can be extended to the gram scale.

Study on the radiation and self-absorption characteristics of plasma under various background gases.

The self-absorption effect is a primary factor responsible for the decline in the precision of quantitative analysis techniques using plasma emission spectroscopy, such as laser-induced breakdown spectroscopy (LIBS). In this study, based on the thermal ablation and hydrodynamics models, the radiation characteristics and self-absorption of laser-induced plasmas under different background gases were theoretically simulated and experimentally verified to investigate ways of weakening the self-absorption effect in plasma. The results reveal that the plasma temperature and density increase with higher molecular weight and pressure of the background gas, leading to stronger species emission line intensity. To reduce the self-absorption effect in the later stages of plasma evolution, we can decrease the gas pressure or substitute the background gas with a lower molecular weight. As the excitation energy of the species increases, the impact of the background gas type on the spectral line intensity becomes more pronounced. Moreover, we accurately calculated the optically thin moments under various conditions using theoretical models, which are consistent with the experimental results. From the temporal evolution of the doublet intensity ratio of species, it is deduced that the optically thin moment appears later with higher molecular weight and pressure of the background gas and lower upper energy of the species. This theoretical research is essential in selecting the appropriate background gas type and pressure and doublets in self-absorption-free LIBS (SAF-LIBS) experiments to weaken the self-absorption effect.

Non-contact bacterial identification and decontamination based on laser-induced breakdown spectroscopy.

As a new kind of modern military biological weapon, bacterial agents pose a serious threat to the public health security of human beings. Existing bacterial identification requires manual sampling and testing, which is time-consuming, and may also introduce secondary contamination or radioactive hazards during decontamination. In this paper, a non-contact, nondestructive and "green" bacterial identification and decontamination technology based on laser-induced breakdown spectroscopy (LIBS) is proposed. The principal component analysis (PCA) combined with support vector machine (SVM) based on radial basis kernel function is used to establish the classification model of bacteria, and the two-dimensional decontamination test of bacteria is carried out using laser-induced low-temperature plasma combined with a vibration mirror. The experimental results show that the average identification rate of the seven types of bacteria, including Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, Bacillus megatherium, Pseudomonas aeruginosa, Bacillus thuringiensis and Enterococcus faecalis reaches 98.93%, and the corresponding true positive rate, precision, recall and F1-score reaches 0.9714, 0.9718, 0.9714 and 0.9716, respectively. The optimal decontamination parameters are laser defocusing amount of -50 mm, laser repetition rate of 15-20 kHz, scanning speed of 150 mm/s and number of scans of 10. In this way, the decontamination speed can reach 25.6 mm2/min, and the inactivation rates for both Escherichia coli and Bacillus subtilis are higher than 98%. In addition, it is confirmed that the inactivation rate of plasma is 4 times higher than that of thermal ablation, meaning that the decontamination ability of LIBS mainly relies on the plasma rather than the thermal ablation effect. The new non-contact bacterial identification and decontamination technology does not require sample pretreatment, and can quickly identify bacteria in situ and decontaminate the surfaces of precision instruments, sensitive materials, etc., which has potential application value in modern military, medical and public health fields.

Study on direct identification of bacteria by laser-induced breakdown spectroscopy.

Bacteria are everywhere in the natural environment. Although most of them are harmless, there are still some hazardous bacteria that will harm human health, so it is particularly important to identify bacteria quickly. Compared with traditional time-consuming and complicated identification methods, laser-induced breakdown spectroscopy (LIBS) is one of the potential technologies for rapid identification of bacteria. In this paper, six weakly active bacteria, including Escherichia coli, Enterococcus faecalis, Bacillus megaterium, Bacillus thuringiensis, Pseudomonas aeruginosa and Bacillus subtilis, are taken as analysis samples. The thawed bacteria are placed in deionized water, and then uniformly smeared on five kinds of substrates to verify the feasibility of using LIBS to identify these bacteria. Spectrum filtering, normalization and principal component analysis (PCA) are used to preprocess the spectra, and a multi-class identification method based on the one-against-all linear kernel function of support vector machine (SVM) is proposed to establish the prediction model. The identification performance is evaluated by using precision and recall. The experimental results show that high-purity graphite is the best substrate with the least interference to the LIBS spectrum of bacteria. The prediction precision of these six bacteria is 77.27%, 92.86%, 84.21%, 94.12%, 81.82% and 76.92%, respectively, recall is 85%, 100%, 94.12%, 80%, 81.82% and 75% respectively, and the identification rate is 84.17%. It can be seen that the direct identification of bacteria can be preliminarily realized by smearing bacteria on the graphite substrate and analyzing its LIBS spectra, which provides a feasible way for simple, rapid and on-site bacterial identification.

Elevated FOXA1 Expression Indicates Poor Prognosis in Liver Cancer due to Its Effects on Cell Proliferation and Metastasis.

Purpose: Studying the pathogenesis of liver cancer is conducive to the exploration of effective diagnostic and prognostic biomarkers. In this study, we investigated the expression of FOXA1 and its oncogenic role in hepatocellular carcinoma (HCC). Methods: Transcriptome data of HCC tissues were downloaded from The Cancer Genome Atlas (TCGA) and GEO databases and analyzed using R software. We also upregulated FOXA1 expression in HCC cells and investigated the role of FOXA1 in the proliferation and migration of HCC cells through proliferation, colony formation, wound healing, and Transwell assays. Results: An analysis of the transcriptome data in TCGA database revealed found that FOXA1 is highly expressed in HCC tissues and that patients with low FOXA1 expression have a better prognosis. High FOXA1 expression was mainly associated with extracellular matrix organization, cancer, and mitosis. The results of an immunohistochemistry (IHC) assay showed that FOXA1 protein was highly expressed in HCC tissues, and patients with low FOXA1 expression showed longer disease-specific survival times and progression-free intervals. The results from quantitative reverse transcription-PCR (RT-qPCR) and Western blot experiments showed that the expression of FOXA1 in liver cancer cell lines was higher than that in immortalized human liver cell lines. Proliferation, wound healing, and Transwell experiments showed that FOXA1 enhanced the proliferation and migration abilities of liver cancer and immortalized human cell lines. Conclusion: Our research suggests that FOXA1 plays an important role in promoting the recurrence and metastasis of HCC by increasing cell proliferation and metastasis.

Risk stratification of indeterminate thyroid nodules by novel multigene testing: a study of Asians with a high risk of malignancy.

Molecular testing of indeterminate thyroid nodules informs about the presence of point mutations, insertions/deletions, copy number variants, RNA fusions, transcript alterations and miRNA expression. American Thyroid Association (ATA) guidelines suggest molecular testing of indeterminate thyroid nodules may be considered to supplement risk of malignancy (ROM). Although these recommendations have been incorporated in clinical practices in the United States, molecular testing of indeterminate thyroid nodules is not common practice in Asia. Here, we performed molecular testing of 140 indeterminate nodules from Chinese patients using a novel molecular platform composed of RNA and DNA-RNA classifiers, which is similar to Afirma GEC and ThyroSeq v3. Compared with reports from North America, the new RNA and DNA-RNA classifiers had a higher positive predictive value (p1 = 0.000 and p2 = 0.020) but a lower negative predictive value (p1 = 0.004 and p2 = 0.098), with no significant differences in sensitivity (p1 = 0.625 and p2 = 0.179) or specificity (p1 = 0.391 and p2 = 0.264). Out of 58 resected nodules, 10 were borderline and 33 malignant, indicating a 74.1% ROM, which was higher than reports in North America (10-40% ROM). Our findings emphasize molecular testing with the newly reported RNA and DNA-RNA classifiers can be used as a 'rule-in' test when ROM is high.

Autophagy-related gene 7 deficiency caused by miR-154-5p overexpression suppresses the cell viability and tumorigenesis of retinoblastoma by increasing cell apoptosis.

BACKGROUND: Retinoblastoma is a rare cancer of the retina that accounts for 3% of all childhood cancers. The aim of this study was to illuminate the oncogenic role and potential molecular mechanisms of the microRNA miR-154-5p and autophagy-related gene 7 (ATG7) in retinoblastoma, and to establish a nude mouse model in order to explore new therapeutic horizons for the disease. METHODS: Quantitative reverse transcription-polymerase chain reaction and western blot were performed to detect the expression levels of miR-154-5p and ATG7. The targeting relationship between miR-154-5p and ATG7 was analyzed by employing the luciferase reporter assay. MiR-154-5p mimic and pcDNA-ATG7 were transfected, either alone or in combination, into Y79 cells. The subsequent in vitro experiments involved four groups: the control group, miR-154-5p group, ATG7 group, and miR-154-5p + ATG7 group. Orthotopic xenograft models were established by injecting BALB/c athymic nude mice with treated and untreated Y79 cells. RESULTS: Y79 cells were transfected with miR-NC or miR-154-5p. Compared to those in the control group, the mRNA expression levels of miR-154-5p were increased in the miR-154-5p mimic group; in contrast, decreases were observed in the mRNA and protein expression levels of ATG7. Y79 cells were transfected with PcDNA or pcDNA-ATG7. The mRNA expression level of ATG7 was increased in pcDNA-ATG7 group. MiR-154-5p was found to have an element complementary to the three prime untranslated region of ATG7. Overexpression of miR-154-5p inhibited Y79 cells proliferation and migration, and promoted Y79 cells apoptosis via targeting of ATG7. In the in vivo experiment, the tumors of the miR-154-5p group of mice were significantly reduced in weight. Tumor growth and the protein levels of Survivin were both suppressed when miR-154-5p was overexpressed in vivo; however, cell apoptosis and the protein levels of p21 were promoted. In the miR-154-5p group, the expression levels of miR-154-5p were upregulated compared to those in the control group, but the ATG7 expression level was downregulated. CONCLUSIONS: MiR-154-5p overexpression downregulated ATG7, which inhibited cell proliferation and apoptosis in vitro, as well as tumor formation in vivo.

Thyroid Hormone Changes in Euthyroid Patients with Diabetes.

BACKGROUND: Thyroid dysfunction is associated with diabetes, but it is unclear if the thyroid hormone levels change in euthyroid adults with diabetes. OBJECTIVE: To investigate the association between thyroid hormone levels and diabetes in euthyroid adults. METHODS: Among the euthyroid adults who underwent health examination in West China Hospital of Sichuan University in 2016, patients with diabetes were identified according to the medical history, fasting blood glucose and HbA1c. Age and sex matched controls were identified from the population. The patients with diabetes group was further divided into two subgroups: patients with newly diagnosed diabetes (NDD) and with previously diagnosed diabetes (PDD). Independent t-test and multivariate logistic regression models were used to investigate the difference in the levels of thyroid stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3) and the ratio of FT4/FT3 between groups. RESULTS: We included 32,557 participants, 2,271 with diabetes. Compared to the adults without diabetes, the odds ratios (ORs) per one unit elevation of TSH, FT4, FT4/FT3 ratio and FT3 in patients with diabetes were 0.88 [95% confidence interval (CI): 0.82-0.95], 1.11 (95% CI: 1.08-1.14), 2.05 (95% CI: 1.81-2.32) and 0.85 (95% CI: 0.78-0.93), respectively. Compared to the NDD group, the ORs per one unit elevation of TSH, FT4, FT4/FT3 ratio and FT3 of the PDD group were 0.81 (95% CI: 0.71-0.92), 1.08 (95% CI: 1.04-1.12), 1.76 (95% CI: 1.49-2.08) and 1.01 (95% CI: 0.92-1.12), respectively. CONCLUSION: In euthyroid adults, diabetes was associated with increased FT4/FT3 ratio, which is linked to the peripheral turnover of the thyroid hormones.

Direct Synthesis of Propen-2-yl Sulfones through Cascade Reactions Using Calcium Carbide as an Alkyne Source.

A simple method for the construction of propen-2-yl sulfones through cascade reactions of calcium carbide with arylsulfonylhydrazones using copper as a mediator is described. The salient features of this protocol are the use of readily available and easy-to-handle alkyne source, broad substrate scope, open-air condition, and simple operation procedure.

Automatic Corneal Ulcer Segmentation Combining Gaussian Mixture Modeling and Otsu Method.

In this paper, we proposed and validated a novel and accurate pipeline for automatically segmenting flaky corneal ulcer areas from fluorescein staining images. The ulcer area was segmented within the cornea by employing a joint method of Otsu and Gaussian Mixture Modeling (GMM). In the GMM based segmentation, the total number of Gaussians was determined intelligently using an information theory based algorithm. And the fluorescein staining images were processed in the HSV color model rather than the original RGB color model, aiming to improve the segmentation results' robustness and accuracy. In the Otsu based segmentation, the images were processed in the grayscale space with Gamma correction being conducted before the Otsu binarization. Afterwards, morphological operations and median filtering were employed to further improve the Otsu segmentation result. The GMM and Otsu segmentation results were then intersected, for which post-processing was conducted by identifying and filling holes through a fast algorithm using priority queues of pixels. The proposed pipeline has been validated on a total of 150 clinical images. Accurate ulcer segmentation results have been obtained, with the mean Dice Similarity Coefficient (DSC) being 0.88 when comparing the automatic segmentation result with the manually-delineated gold standard. For images in the RGB color space, the mean DSC was 0.83, being much lower than that of the images in the HSV color space.

Transcriptomic Analysis Reveals New Insights into High-Temperature-Dependent Glume-Unclosing in an Elite Rice Male Sterile Line.

Glume-unclosing after anthesis is a widespread phenomenon in hybrid rice and also a maternal hereditary trait. The character of Glume-unclosing in rice male sterile lines also seriously influences germination rate and the commercial quality of hybrid rice seeds. We validated that the type of glume-unclosing after anthesis in the elite rice thermo-sensitive genic male sterile (TGMS) line RGD-7S was caused by high temperature. Transcriptomic sequencing of rice panicles was performed to explore the change of transcript profiles under four conditions: pre- and post-anthesis under high temperature (HRGD0 and HRGD1), and pre- and post-anthesis under low temperature (LRGD0 and LRGD1). We identified a total of 14,540 differentially expressed genes (DEGs) including some heat shock factors (HSFs) across the four samples. We found that more genes were up-regulated than down-regulated in the sample pair HRGD1vsHRGD0. These up-regulated genes were significantly enriched in the three biological processes of carbohydrate metabolism, response to water and cell wall macromolecular metabolism. Simultaneously, we also found that the HSF gene OsHsfB1 was specially up-regulated in HRGD1vsHRGD0. However, the down-regulated DEGs in LRGD1vsLRGD0 were remarkably clustered in the biological process of carbohydrate metabolism. This suggests that carbohydrate metabolism may play a key role in regulation of glume-unclosing under high temperature in RGD-7S. We also analyzed the expression pattern of genes enriched in carbohydrate metabolism and several HSF genes under different conditions and provide new insights into the cause of rice glume-unclosing.

Genome-wide DNA polymorphism in the indica rice varieties RGD-7S and Taifeng B as revealed by whole genome re-sequencing.

Next-generation sequencing technologies provide opportunities to further understand genetic variation, even within closely related cultivars. We performed whole genome resequencing of two elite indica rice varieties, RGD-7S and Taifeng B, whose F1 progeny showed hybrid weakness and hybrid vigor when grown in the early- and late-cropping seasons, respectively. Approximately 150 million 100-bp pair-end reads were generated, which covered ∼86% of the rice (Oryza sativa L. japonica 'Nipponbare') reference genome. A total of 2,758,740 polymorphic sites including 2,408,845 SNPs and 349,895 InDels were detected in RGD-7S and Taifeng B, respectively. Applying stringent parameters, we identified 961,791 SNPs and 46,640 InDels between RGD-7S and Taifeng B (RGD-7S/Taifeng B). The density of DNA polymorphisms was 256.8 SNPs and 12.5 InDels per 100 kb for RGD-7S/Taifeng B. Copy number variations (CNVs) were also investigated. In RGD-7S, 1989 of 2727 CNVs were overlapped in 218 genes, and 1231 of 2010 CNVs were annotated in 175 genes in Taifeng B. In addition, we verified a subset of InDels in the interval of hybrid weakness genes, Hw3 and Hw4, and obtained some polymorphic InDel markers, which will provide a sound foundation for cloning hybrid weakness genes. Analysis of genomic variations will also contribute to understanding the genetic basis of hybrid weakness and heterosis.

Genetic and cytological analysis of a novel type of low temperature-dependent intrasubspecific hybrid weakness in rice.

Hybrid weakness (HW) is an important postzygotic isolation which occurs in both intra- and inter-specific crosses. In this study, we described a novel low temperature-dependent intrasubspecific hybrid weakness in the F1 plants derived from the cross between two indica rice varieties Taifeng A and V1134. HW plants showed growth retardation, reduced panicle number and pale green leaves with chlorotic spots. Cytological assay showed that there were reduced cell numbers, larger intercellular spaces, thicker cell walls, and abnormal development of chloroplast and mitochondria in the mature leaves from HW F1 plants in comparison with that from both of the parental lines. Genetic analysis revealed that HW was controlled by two complementary dominant genes Hw3 from V1134 and Hw4 from Taifeng A. Hw3 was mapped in a 136 kb interval between the markers Indel1118 and Indel1117 on chromosome 11, and Hw4 was mapped in the region of about 15 cM between RM182 and RM505 on chromosome 7, respectively. RT-PCR analysis revealed that only LOC_Os11g44310, encoding a putative calmodulin-binding protein (OsCaMBP), differentially expressed among Taifeng A, V1134 and their HW F1. No recombinant was detected using the markers designed based on the sequence of LOC_Os11g44310 in the BC1F2 (Taifeng A//Taifeng A/V1134) population. Hence, LOC_Os11g44310 was probably the candidate gene of Hw3. Gene amplification suggested that LOC_Os11g44310 was present in V1134 and absent in Taifeng A. BLAST search revealed that LOC_Os11g44310 had one copy in the japonica genomic sequence of Nipponbare, and no homologous sequence in the indica reference sequence of 9311. Our results indicate that Hw3 is a novel gene for inducing hybrid weakness in rice.

[The primary culture of rats cochlear sensory epithelial cell and the significance of expression of hair cell characteristic markers of CSEC].

OBJECTIVE: To further investigate the mechanisms of hair cell generation or regeneration, the primary culture systems of cochlear sensory epithelial cell (CSEC) of rats were established. METHOD: Cochlear sensory epithelial (containing the organ of Corti) of postnatal day 1 (P1) rats was isolated by mechanical dissociation. The explants were grown on sterilized disposable plastic dishes,cultured in Dulbecco modified Eagle medium(DMEM), and observed daily by inverted microscopy. The culture medium was changed twice a week. The pure CSEC was harvested by the limiting dilution method. CSEC was identified by immunocytochemical method with cytokeratin 18, vimentin, Brn3. a, Calretinin, BrdU and ultrastructural examination with transmission electron microscopy. The markers of epithelial cell and the markers of hair cell were used to identify the origin and character of CSEC. CSEC mitotic division was detected by BrdU staining of nuclear DNA. RESULT: The fresh explants were light yellow. The morphology of CSEC couldn't be seen clearly under the inverted microscope because of the complex structures of cochlear sensory epithelial. CSEC grew out of the explants usually at 2nd culture day. Fibroblast like cells (FLC) around the CSEC grew faster than CSEC, but could easily be excluded. Pure CSEC may grow into monolayer with 'cobblestone-like' appearance and show a large, flat, polygonal epithelial morphotype with big, round nuclei. Some cells showed 'Dome' formation, probably due to fluid collection underneath the cell monolayer. The culture CSEC coexpressed cytokeratin 18 and vimentin has rich microvilli and complex tight junction,which indicated the epithelial origination of CSEC. Coexpressed of the Brn3. a and Calretinin of the hair cell characteristic markers suggested the culture cell may represent rat progenitor hair cell. BrdU staining showed CSEC produced new hair cell-like cell (progenitor hair cell) by the mitotic division. CONCLUSION: The primary culture systems of cochlear sensory epithelial cell of rats were successfully established in vitro. CSEC coexpressed the characteristic markers of the immature hair cell,which identified the culture cell may represent progenitor cell of rats hair cell. It may be a suitable model for in-depth investigation the molecular mechanisms of hair cell generation or regeneration.