A tomato NAC transcription factor, SlNAM1, positively regulates ethylene biosynthesis and the onset of tomato fruit ripening.

Fruit ripening in tomato (Solanum lycopersicum) is the result of selective expression of ripening-related genes, which are regulated by transcription factors (TFs). The NAC (NAM, ATAF1/2, and CUC2) TF family is one of the largest families of plant-specific TFs and members are involved in a variety of plant physiological activities, including fruit ripening. Fruit ripening-associated NAC TFs studied in tomato to date include NAC-NOR (non-ripening), SlNOR-like1 (non-ripening like1), SlNAC1, and SlNAC4. Considering the large number of NAC genes in the tomato genome, there is little information about the possible roles of other NAC members in fruit ripening, and research on their target genes is lacking. In this study, we characterize SlNAM1, a NAC TF, which positively regulates the initiation of tomato fruit ripening via its regulation of ethylene biosynthesis. The onset of fruit ripening in slnam1-deficient mutants created by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) technology was delayed, whereas fruit ripening in OE-SlNAM1 lines was accelerated compared with the wild type. The results of RNA-sequencing (RNA-seq) and promoter analysis suggested that SlNAM1 directly binds to the promoters of two key ethylene biosynthesis genes (1-aminocyclopropane-1-carboxylate synthase: SlACS2 and SlACS4) and activates their expression. This hypothesis was confirmed by electrophoretic mobility shift assays and dual-luciferase reporter assay. Our findings provide insights into the mechanisms of ethylene production and enrich understanding of the tomato fruit ripening regulatory network.

A Y(III)-based Metal-Organic Framework as a Carrier in Chemodynamic Therapy.

A biocompatible Y(III)-based metal-organic framework [Y4(TATB)2]·(DMF)3.5·(H2O) (ZJU-16, H3TATB= 4,4',4''-(1,3,5-triazine-2,4,6-triyl) tribenzoic acid) was synthesized, and it was adopted to load Mn2+ for chemodynamic therapy. Meanwhile, ibuprofen sodium (IBUNa), an anti-inflammatory drug, was introduced to increase the amount of Mn2+ (about 5.66 wt %) due to the low loading capacity of Mn2+. Mn&IBUNa@ZJU-16 which was loaded by Mn2+ and IBUNa exhibited significant effects of chemodynamic therapy and excellent inhibition of the 4T1 tumor cell growth, implying its long-term prospects in chemodynamic therapy and its possibility in bimodal cancer therapy.

Cognitive Impairments in First-Episode Drug-Naïve Versus Medicated Depressive Patients: RBANS in a Chinese Population.

Cognitive deficits are a core feature of major depressive disorder (MDD). However, there are no previous studies that directly compare cognitive performance between first-episode drug-naive depressive patients (FDDP) and medicated depressive patients (MDP). Therefore, the aim of this study was to investigate whether there were the differences in cognitive functions between FDDP and MDP. Sixty-two FDDP, 111 MDP and 90 healthy controls were enrolled in a Chinese population. Cognitive functions were assessed using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). There were the differences in the RBANS total score (F = 26.55, p < 0.001), subscales of immediate memory (F = 3.95, p = 0.02), language (F = 54.11, p < 0.001) and delayed memory (F = 11.19, p = 0.001) among the three groups after controlling for gender, education, smoking and body mass index (BMI). These differences in the RBANS total score, subscales of language and delayed memory passed the Bonferroni corrections (all, p < 0.05). Compared to healthy controls, FDDP and MDP had poorer cognitive performance including the RBANS total score, and subscales of language and delayed memory (all, p < 0.05) after controlling for the variables. FDDP experienced greater language deficits than MDP (p < 0.05) after controlling for the variables. Education was correlated with the language score in FDDP (r = 0.61, p < 0.001). Multivariate regression analysis indicated that education was an independent contributor to the language score in FDDP (ß = 3.11, t = 5.48, p < 0.001). Our findings indicated that FDDP had poorer language performance than MDP. Moreover, education could influence the language performance in FDDP.

Transcriptome Analysis Provides a Preliminary Regulation Route of the Ethylene Signal Transduction Component, SlEIN2, during Tomato Ripening.

Ethylene is crucial in climacteric fruit ripening. The ethylene signal pathway regulates several physiological alterations such as softening, carotenoid accumulation and sugar level reduction, and production of volatile compounds. All these physiological processes are controlled by numerous genes and their expression simultaneously changes at the onset of ripening. Ethylene insensitive 2 (EIN2) is a key component for ethylene signal transduction, and its mutation causes ethylene insensitivity. In tomato, silencing SlEIN2 resulted in a non-ripening phenotype and low ethylene production. RNA sequencing of SlEIN2-silenced and wild type tomato, and differential gene expression analyses, indicated that silencing SlEIN2 caused changes in more than 4,000 genes, including those related to photosynthesis, defense, and secondary metabolism. The relative expression level of 28 genes covering ripening-associated transcription factors, ethylene biosynthesis, ethylene signal pathway, chlorophyll binding proteins, lycopene and aroma biosynthesis, and defense pathway, showed that SlEIN2 influences ripening inhibitor (RIN) in a feedback loop, thus controlling the expression of several other genes. SlEIN2 regulates many aspects of fruit ripening, and is a key factor in the ethylene signal transduction pathway. Silencing SlEIN2 ultimately results in lycopene biosynthesis inhibition, which is the reason why tomato does not turn red, and this gene also affects the expression of several defense-associated genes. Although SlEIN2-silenced and green wild type fruits are similar in appearance, their metabolism is significantly different at the molecular level.

Efficient Virus-Induced Gene Silencing in Solanum rostratum.

Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum.

Attenuation of EGFL7 inhibits human laryngocarcinoma cells growth and invasion.

AIM: To investigate its effect on the proliferation and invasion of laryngeal carcinoma and understand the potential underlying mechanisms to provide new targets for the diagnosis and treatment of recurrent laryngeal cancer metastasis. METHODS: We constructed a lentiviral vector expressing EGFL7 specific shRNA, and introduced it in EGFL7 functions were attenuated by a lentiviral vector harboring shRNA targeting at EGFL7 in laryngeal carcinoma cell line Hep-2. Prolifereation and invasion assays were carried out in vitro. And in vivo tumor burden assay was done in nude mice. RESULTS: The expression of EGFL7 was knocked-down by 80% in hep-2 cells transfected by the lentiviral EGFL7 shRNA vector and EGFL7 gene expression was detected by realtime PCR and Western blotting analysis respectively. The flow cytometric analysis showed that arrested the cell cycle in G1 phase, In tumor burden assay, to parental And vector control cells, the survival rates Of nude mice in EGFL7 shRNA group dropped down from the first day after implantation as indicated by MTT assay (P < 0.05). The formation and growth rate of xenograft tumor in mice transfected with siRNA against Bmi-1 slowed down significantly. CONCLUSION: Attenuation of EGFL7 function significantly suppresses tumor growth and induces apoptosis, both in vitro and in vivo. EGFL7 may be play a key role in invasion and metastasis of Laryngeal squamous cell carcinoma (LSCC), thus would to be a new target for gene therapy in LSCC.

Silencing Bmi-1 expression by RNA interference suppresses the growth of laryngeal carcinoma cells.

The aim of this study was to investigate the effect of a B-cell-specific MLV integration site-1 (Bmi-1) RNA interference (RNAi) expression vector on the proliferation and invasiveness of laryngeal carcinoma. We constructed a lentiviral vector expressing Bmi-1-specific short hairpin RNA (shRNA), and transfected it into HEp-2 cells. Bmi-1 gene expression was detected by real-time RT-PCR and western blot analysis. We used flow cytometry and TUNEL assay to analyze the apoptosis of transfected cells, and examined cellular growth in vitro by MTT assay. We established an animal model and evaluated the therapeutic effects of small interfering RNA (siRNA) against Bmi-1. siRNA against Bmi-1 significantly knocked down Bmi-1 expression in HEp-2 cells, induced cell cycle arrest at the G1 phase, inhibited cell proliferation and promoted cell apoptosis. Lentiviral Bmi-1-shRNA vector transfection also significantly reduced cell migration. The formation and growth rate of xenograft tumors in mice transfected with siRNA against Bmi-1 was significantly reduced. The loss of mitochondrial membrane potential, the release of cytochrome c from the mitochondria into the cytosol, and the increased activity of caspase-3, -8 and -9 occurred concomitantly with the inhibition of Bmi-1. Our data indicate that siRNA against Bmi-1 significantly suppresses tumor growth and induces apoptosis in vitro and in vivo.

[Toll-like receptor 2 and Toll-like receptor 4 participates in mediation of acute otitis media and mortality in pneumococcal infections in mice].

OBJECTIVE: To investigate the roles of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in host defense against Streptococcus pneumoniae infection in the middle ear. METHODS: Wild-type (WT) C57BL/6J, TLR2-deficient (TLR2(-/-)) and TLR4-deficient (TLR4(-/-)) mice were inoculated with Streptococcus pneumoniae (1 × 10(6)CFU) through the tympanic membrane. All animals were tested the mouse ABR thresholds and tympanometry measurement before, and 1 day, 3 days and 7 days following pneumococcal challenge. Blood bacterial titer were determined by plating 50 µl volumes of 10-fold diluted blood. Histological analysis of middle ear and inner ear were performed by fixation, decalcification, embedded section, and counterstained with hematoxylin/eosin and toluidine blue staining. Semi-quantitative RT-PCR was applied to determine mRNA accumulation of TLR2 and TLR4 related genes. RESULTS: Forty of 68 TLR2(-/-) mice and twenty-one of 59 TLR4(-/-) mice showed bacteremia and died within 3 days after the pneumococcal challenge, however, only 9 of 52 WT mice died. The survive mice were shown have more severe hearing loss in the TLR2(-/-) and TLR4(-/-) mice than in the WT mice, indicated by ABR thresholds, at 3 or 7 days postinoculation. The histological pathology was characterized by effusion and tissue damage in the middle ear, and in the TLR2(-/-) and TLR4(-/-) mice, the outcome of infection became more severe at 7 days. At both 3 and 7 days after challenge, the TLR2(-/-) mice had higher blood bacterial titers than WT mice (P < 0.05). Temporal bone histopathologic change indicated that 3 days after the pneumococcal challenge, the TLR2(-/-) and TLR4(-/-) mice showed effusion and tissue damage in the middle ear, and the infection became more severe at 7 days postinoculation. TLR2(-/-) mice showed severe inflammatory cell infiltration in the cochlear, the organ of Corti showed the outer hair cells damage, the tectorial membrane swelling, degeneration of the stria vascularis, and severe loss of spiral ganglion cells; However, the WT mice was not found the cell infiltration and tissue damage in the cochlear, the organ of Corti shown normal of outer hair cells. Mast cells were not found in the middle ear mucosa of TLR2(-/-) mice, but in the TLR4(-/-) and WT mice, more mast cells were found in the middle ear mucosa of effusion ear by 3 and 7 days postchallenge. Moreover, by 3 days postchallenge, the mRNA accumulation levels of NF-κB, tumor necrosis factor alpha (TNFα), interleukin1ß, MIP-1α, MUC5AC and MUC5B were significantly lower in the ears of TLR2(-/-) mice than that in WT and TLR4(-/-) mice. CONCLUSIONS: TLR2(-/-) mice may produce relatively low levels of proinflammatory cytokines following pneumococcal challenge, thus hindering the clearance of bacteria from the middle ear and leading to sepsis and high mortality rate. This study indicated that TLR2 and TLR4 are important in the molecular pathogenesis and host response to otitis media.

[Inhibitory effect of lentiviral vectors expressing small interfering RNA targeting survivin gene on Hep-2 cell growth in vitro and in nude mice].

OBJECTIVE: To observe the effect of the lentiviral vectors expressing small interfering RNA (siRNA) for survivin gene knockdown in inhibiting Hep-2 cell growth in vitro and its tumorigenicity in nude mice. METHODS: The tumorigenicity of Hep-2 cells transfected with the siRNA mediated by the lentiviral vectors was tested in nude mice. The expression of survivin gene of the transfected cells at the mRNA and protein levels were detected by RT-PCR and Western blotting, respectively, and the cell cycle changes were analyzed by flow cytometry. RESULTS: Transfection of the siRNA targeting survivin significantly decreased the expression of survivin mRNA and protein in Hep-2 cells in vitro by 60%-85% and 70%, respectively, resulting also in increased cell apoptosis as shown by flow cytometry (P<0.01). The transfection significantly lowered the tumorigenicity of the cells in nude mice. CONCLUSION: The lentiviral vectors expressing survivin siRNA can significantly inhibit survivin gene expression in Hep-2 cells and induce the cell apoptosis in vitro, and suppress the tumorigenicity of the cells in nude mice.

[Effect of recombinant caspase-3 gene transfection on CNE2 cell apoptosis].

OBJECTIVE: To investigate the apoptosis-inducing effect of caspase-3 on human nasopharyngeal carcinoma cells (CNE2). METHODS: Recombinant caspase-3 was subcloned into the eukaryotic expression vector PEGFP-C1 containing the reporter gene using DNA recombinant technique. CNE2 cells were transfected with the recombinant caspase-3 gene via lipofectamine 2000 and the expression of caspase-3 mRNA was detected by reverse transcription-polymerase chain reaction. The cell morphological changes were observed under fluorescence microscope and electron microscope and the cell survival rate after the transfection was assessed by MTT assay. RESULTS: Transfection with the recombinant caspase-3 gene induced significant apoptosis in CNE2 cells, which exhibited obvious morphological changes typical of apoptotic cells. CONCLUSION: The recombinant caspase-3 gene can inhibit the growth and effectively induce apoptosis of CNE2 cells.

[Adenovirus-mediated antisense HSP70 cDNA transfection inhibits the growth of laryngeal carcinoma Hep-2 cells].

OBJECTIVE: To construct a recombinant adenovirus vector carrying antisense heat shock protein 70 (HSP70) cDNA and observe its effect on inhibiting the growth of laryngeal carcinoma Hep-2 cells. METHODS: The HSP70 gene fragment encoding the 5' region was cloned reversely into the shuttle plasmid PAdTrack-CMV, and the resultant plasmid was recombined with the backbone plasmid PadEasy-1 in the E.coli Bj5183 cells to generate the recombinant adenovirus vector. The adenovirus were then packaged and amplified in 293 cells, and the viral titer was determined using GFP. RESULTS: The recombinant adenovirus vector carrying antisense HSP70 cDNA was constructed successfully with a viral titer of 8 x 10(9). HSP70 expression of Hep-2 cells was obviously blocked by antisense HSP70 RNA, and Western blotting and immuohistochemistry demonstrated that cells transfected with antisense HSP70 did not express or express HSP70 at low levels. Flow cytometry presented apoptotic peak in the antisense HSP70-transfected cells, but not in the control cells. CONCLUSION: The recombinant adenovirus vector containing antisense HSP70 cDNA can effectively deliver antisense HSP70 gene into Hep-2 cells, suggesting the great potential of this gene therapy strategy in management of human laryngeal carcinoma.

[Protective effect of adeno-associated virus-mediated neurotrophin-3 on the cochlea of guinea pigs with gentamicin-induced hearing loss].

OBJECTIVE: To study the protective effect of local gene therapy with adeno-associated virus (AAV)-mediated neurotrophin-3 (NT-3) on the function and morphology of the cochlea of guinea pigs with gentamicin-induced hearing loss. METHODS: Hearing loss was induced with gentamicin (80 mg.kg(-1).day(-1) injected intramuscularly) in 18 pigmented guinea pigs 4 days prior to gene transfer. The guinea pigs were then divided into groups A, B, and C for AAV-mediated NT-3 gene transfer (n=7), AAV infection (n=7) or no particular intervention (n=4), respectively. Mini-Osmotic pump were implanted in either side of the ears in groups A and B, and the guinea pigs were injected with gentamicin (80 mg.kg(-1).day(-1)) intramuscularly since the operation day for 10 consecutive days. In group C, only gentamicin was administrated. Before and 14 days after gentamicin administration, auditory brainstem response audiometry (ABR) and distort-product otoacoustic emissions (DPOAE) were recorded, and the animals sacrificed to observe the morphological changes of the cochlear microscopically. RESULTS: Compared with groups B and C, the animals in group A showed better auditory ability (ABR and DPOAE) and significantly higher surviving rate of the outer hair cells (P<0.05). CONCLUSION: AAV-mediated NT-3 gene transfer may protect and repair the cochlear hair cells and auditory function damaged by aminoglycoside ototoxicity in guinea pigs, and aseptic procedure is of vital importance in cochlear local gene therapy.

Molecular cloning and characterization of ETHYLENE OVERPRODUCER 1-LIKE 1 gene, LeEOL1, from tomato (Lycopersicon esculentum Mill.) fruit.

Recently, ETHYLENE OVERPRODUCER 1 (ETO1) had been cloned and identified as a negative post-transcriptional regulator in the ethylene biosynthesis in Arabidopsis. However, little was known about the role of ETO1 in other species, especially in tomato, which was an ideal model for studying the biosynthesis of ethylene during tomato fruit ripening. In this study, a tomato ETHYLENE OVERPRODUCER 1-LIKE 1 (LeEOL1) was cloned. The LeEOL1 cDNA was 3,515 bp long and carried an ORF that putatively encoded a polypeptide of 886 amino acids with a predicted molecular mass of 95 kDa. It shared 74% identity in amino acid sequence with Arabidopsis EOL1 and had one BTB (Broad-complex, Tramtrack, Bric-à-brac) domain and two TPR (tetratricopeptide repeat) domains, which were also conserved domains in AtEOL1. RT-PCR analysis of the temporal expression of LeEOL1 showed that its transcript decreased companied with increase of ethylene production in tomato ripening. The level of LeEOL1 transcripts in wild type tomato fruit at mature green stage did not distinctively change when treated with exogenous ethylene.

Virus-induced gene silencing in tomato fruit.

Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants. Here we report that either by syringe-infiltrating the tobacco rattle virus (TRV)-vector into the surface, stem or carpopodium of a tomato fruit attached to the plant or by vacuum-infiltrating into a tomato fruit detached from the plant, TRV can efficiently spread and replicate in the tomato fruit. Although VIGS can be performed in tomato fruit by all of the means mentioned above, the most effective method is to inject the TRV-vector into the carpopodium of young fruit attached to the plant about 10 days after pollination. Several reporter genes related to ethylene responses and fruit ripening, including LeCTR1 and LeEILs genes, were also successfully silenced by this method during fruit development. In addition, we found that the silencing of the LeEIN2 gene results in the suppression of tomato fruit ripening. The results of our study indicate that the application of VIGS techniques by the described methods can be successfully applied to tomato fruit and is a valuable tool for studying functions of the relevant genes during fruit developing.