The clinical application value of 3.0T magnetic resonance T2 mapping imaging in evaluating the degree of acetabular cartilage degeneration in joint replacement surgery running title: MRI and acetabular cartilage degeneration.

BACKGROUND: To explore and compare the values of 3.0T magnetic resonance imaging (MRI) T2 mapping in evaluating the degree of acetabular cartilage degeneration in hip replacement surgery. METHODS: A total of 26 elderly patients with femoral neck fractures who were scanned in 3.0T MRI T2 mapping quantification technique were included. Basing on MRI images, the degree of acetabular cartilage degeneration was classified into Grade 0, 1, 2, 3 and 4, according to the International Cartilage Repair Society (ICRS) scores. In addition, 8 healthy volunteers were included for control group. RESULTS: By comparison with health population, T2 relaxation values in the anterior, superior, and posterior regions of acetabular cartilage in patients with femoral neck fracture were obviously increased (P < 0.001). Among the patients with femoral neck fractures, there were 16 hip joint with Grade 1-2 (mild degeneration subgroup) and 10 hip joints with Grade 3-4 (severe degeneration subgroup), accounting for 61.54% and 38.46%, respectively. Additionally, T2 relaxation values in the anterior and superior bands of articular cartilage were positively related to the MRI-based grading (P < 0.05); while there was no significant difference of T2 relaxation values in the posterior areas of articular cartilage between severe degeneration subgroup and mild degeneration subgroup (P > 0.05). Importantly, acetabular cartilage degeneration can be detected through signal changes of T2 mapping pseudo-color images. CONCLUSION: 3.0T MRI T2 mapping technology can be used to determine the degree of acetabular cartilage degeneration, which can effectively monitor the disease course.

Association of serum interleukin-6 with negative symptoms in stable early-onset schizophrenia.

BACKGROUND: Accumulating evidence suggests that the inflammatory cytokine interleukin-6 (IL-6) contributes to the pathophysiology of psychiatric disorders. However, there was no study concerning the relationship between IL-6 concentrations and clinical features in the chronic phase of early-onset schizophrenia (EOS). AIM: To investigate the relationship between serum IL-6 concentration and the clinical features of EOS. METHODS: We measured serum IL-6 Levels from 74 patients with chronic schizophrenia, including 33 with age at onset < 21 years (EOS group) and 41 with onset ≥ 21 years in [adult-onset schizophrenia (AOS) group], and from 41 healthy controls. Symptom severities were evaluated using the Positive and Negative Syndrome Scale (PANSS). RESULTS: Serum IL-6 concentrations were higher in both EOS and AOS groups than healthy controls (F = 22.32, P < 0.01), but did not differ significantly between EOS and AOS groups (P > 0.05) after controlling for age, body mass index, and other covariates. Negative symptom scores were higher in the EOS group than the AOS group (F = 6.199, P = 0.015). Serum IL-6 concentrations in the EOS group were negatively correlated with both total PANSS-negative symptom score (r = -0.389, P = 0.032) and avolition/asociality subscore (r = -0.387, P = 0.026). CONCLUSION: Patients with EOS may have more severe negative symptoms than those with adult-onset schizophrenia during the chronic phase of the illness. IL-6 signaling may regulate negative symptoms and its avolition/asociality subsymptoms among the early-onset chronic schizophrenic patients.

NAC transcription factor SlNOR-like1 plays a dual regulatory role in tomato fruit cuticle formation.

The plant cuticle is an important protective barrier on the plant surface, constructed mainly by polymerized cutin matrix and a complex wax mixture. Although the pathway of plant cuticle biosynthesis has been clarified, knowledge of the transcriptional regulation network underlying fruit cuticle formation remains limited. In the present work, we discovered that tomato fruits of the NAC transcription factor SlNOR-like1 knockout mutants (nor-like1) produced by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] displayed reduced cutin deposition and cuticle thickness, with a microcracking phenotype, while wax accumulation was promoted. Further research revealed that SlNOR-like1 promotes cutin deposition by binding to the promoters of glycerol-3-phosphate acyltransferase6 (SlGPAT6; a key gene for cutin monomer formation) and CUTIN DEFICIENT2 (SlCD2; a positive regulator of cutin production) to activate their expression. Meanwhile, SlNOR-like1 inhibits wax accumulation, acting as a transcriptional repressor by targeting wax biosynthesis, and transport-related genes 3-ketoacyl-CoA synthase1 (SlKCS1), ECERIFERUM 1-2 (SlCER1-2), SlWAX2, and glycosylphosphatidylinositol-anchored lipid transfer protein 1-like (SlLTPG1-like). In conclusion, SlNOR-like1 executes a dual regulatory effect on tomato fruit cuticle development. Our results provide a new model for the transcriptional regulation of fruit cuticle formation.

Aberrant degree centrality profiles during rumination in major depressive disorder.

Rumination is closely linked to the onset and maintenance of major depressive disorder (MDD). Prior neuroimaging studies have identified the association between self-reported rumination trait and the functional coupling among a network of brain regions using resting-state functional magnetic resonance imaging (MRI). However, little is known about the underlying neural circuitry mechanism during active rumination in MDD. Degree centrality (DC) is a simple metric to denote network integration, which is critical for higher-order psychological processes such as rumination. During an MRI scan, individuals with MDD (N = 45) and healthy controls (HC, N = 46) completed a rumination state task. We examined the interaction effect between the group (MDD vs. HC) and condition (rumination vs. distraction) on vertex-wise DC. We further characterized the identified brain region's functional involvement with Neurosynth and BrainMap. Network-wise seed-based functional connectivity (FC) analysis was also conducted for the identified region of interest. Finally, exploratory correlation analysis was conducted between the identified region of interest's network FCs and self-reported in-scanner affect levels. We found that a left superior frontal gyrus (SFG) region, generally overlapped with the frontal eye field, showed a significant interaction effect. Further analysis revealed its involvement with executive functions. FCs between this region, the frontoparietal, and the dorsal attention network (DAN) also showed significant interaction effects. Furthermore, its FC to DAN during distraction showed a marginally significant negative association with in-scanner affect level at the baseline. Our results implicated an essential role of the left SFG in the rumination's underlying neural circuitry mechanism in MDD and provided novel evidence for the conceptualization of rumination in terms of impaired executive control.

Phylogenomic analyses provide insights into primate evolution.

Comparative analysis of primate genomes within a phylogenetic context is essential for understanding the evolution of human genetic architecture and primate diversity. We present such a study of 50 primate species spanning 38 genera and 14 families, including 27 genomes first reported here, with many from previously less well represented groups, the New World monkeys and the Strepsirrhini. Our analyses reveal heterogeneous rates of genomic rearrangement and gene evolution across primate lineages. Thousands of genes under positive selection in different lineages play roles in the nervous, skeletal, and digestive systems and may have contributed to primate innovations and adaptations. Our study reveals that many key genomic innovations occurred in the Simiiformes ancestral node and may have had an impact on the adaptive radiation of the Simiiformes and human evolution.

Association between increased serum interleukin-8 levels and improved cognition in major depressive patients with SSRIs.

BACKGROUND: The effect of neuroinflammatory cytokines on cognitive deficits in patients with major depressive disorder (MDD) can be altered by selective serotonin reuptake inhibitors (SSRIs). This study aimed to examine serum interleukin-8 (IL-8) levels, cognitive function, and their associations in MDD patients with SSRIs. METHODS: Thirty SSRI-treated MDD patients and 101 healthy controls were recruited for this study. We examined cognitive performance using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and serum IL-8 levels using the Human Inflammatory Cytokine Cytometric Bead Array in both cases and controls. RESULTS: The RBANS test scores were significantly lower in MDD patients with SSRIs than in healthy controls after controlling for covariates (all p < 0.001). Serum levels of IL-8 were higher in MDD patients with SSRIs than in healthy controls after adjusting for covariates (F = 3.82, p = 0.05). Serum IL-8 levels were positively correlated with sub-scores of delayed memory (r = 0.37, p = 0.04) and visuospatial/constructional (r = 0.43, p = 0.02) in MDD patients with SSRIs but not in in healthy controls (delayed memory score: r = -0.12, p = 0.24; visuospatial/constructional score: r = 0.02, p = 0.81). CONCLUSIONS: Our findings suggested that increased serum IL-8 level might not only be involved in the MDD psychopathology or the use of SSRIs but also correspond to improving MDD delayed memory and visuospatial/constructional function.

Transcription factor SlBEL2 interferes with GOLDEN2-LIKE and influences green shoulder formation in tomato fruits.

Chloroplasts play a crucial role in plant growth and fruit quality. However, the molecular mechanisms of chloroplast development are still poorly understood in fruits. In this study, we investigated the role of the transcription factor SlBEL2 (BEL1-LIKE HOMEODOMAIN 2) in fruit of Solanum lycopersicum (tomato). Phenotypic analysis of SlBEL2 overexpression (OE-SlBEL2) and SlBEL2 knockout (KO-SlBEL2) plants revealed that SlBEL2 has the function of inhibiting green shoulder formation in tomato fruits by affecting the development of fruit chloroplasts. Transcriptome profiling revealed that the expression of chloroplast-related genes such as SlGLK2 and SlLHCB1 changed significantly in the fruit of OE-SlBEL2 and KO-SlBEL2 plants. Further analysis showed that SlBEL2 could not only bind to the promoter of SlGLK2 to inhibit its transcription, but also interacted with the SlGLK2 protein to inhibit the transcriptional activity of SlGLK2 and its downstream target genes. SlGLK2 knockout (KO-SlGLK2) plants exhibited a complete absence of the green shoulder, which was consistent with the fruit phenotype of OE-SlBEL2 plants. SlBEL2 showed an expression gradient in fruits, in contrast with that reported for SlGLK2. In conclusion, our study reveals that SlBEL2 affects the formation of green shoulder in tomato fruits by negatively regulating the gradient expression of SlGLK2, thus providing new insights into the molecular mechanism of fruit green shoulder formation.

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.

Association between decreased HDL levels and cognitive deficits in patients with bipolar disorder: a pilot study.

BACKGROUND: Cognitive deficits are common in patients with bipolar disorder (BD). Abnormal high density lipoprotein (HDL) levels have been implicated in cognitive deficits associated with ageing and neurodegenerative disorders. The present study aimed to investigate serum HDL levels, cognitive deficits and their association in patients with BD. METHODS: Thirty-seven patients with BD and 37 gender- and age-matched healthy controls (HCs) were recruited in a case-control study. Cognition was assessed using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), and serum HDL levels were measured using enzymatic colourimetry. RESULTS: There was no difference in serum HDL levels between patients with BD and HCs after adjusting for gender, age, education and body mass index (BMI). Cognitive test scores in patients with BD were significantly lower than those in HCs except for the visuospatial/constructional index after adjusting for confounding variables. Serum HDL levels were positively correlated with RBANS total score and language score in patients with BD. Stepwise multiple regression analysis showed that serum HDL levels were significantly correlated with RBANS total score and subscale scores on immediate memory and language in patients with BD after adjusting for confounding factors. CONCLUSIONS: Our findings suggest that patients with BD had poorer cognitive performance than HCs except for the visuospatial/constructional domain, and decreased serum HDL levels were correlated with cognitive deficits, especially in immediate memory and language domains in patients with BD.

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.

Association between PLA2G12A polymorphism and patients with schizophrenia in a southern Chinese Han population.

BACKGROUND: Elevated phospholipase A2 (PLA2) activity is reported to be involved in the development of schizophrenia. Further study revealed an association between PLA2 groups XIIA (PLA2G12A) polymorphism and patients with schizophrenia in a northeast Chinese Han population. OBJECTIVE: This study will further examine whether PLA2G12A rs3087494 polymorphism is associated with patients with schizophrenia in a southern Chinese Han population. METHODS: This polymorphism was genotyped in 438 patients with schizophrenia (diagnosed according to Diagnostic and Statistical Manual of Mental Disorders-IV) and 876 healthy controls using a case-control design. Demographic and clinical data were collected in all subjects. RESULTS: The allele and genotype frequencies of PLA2G12A rs3087494 polymorphism significantly differed between groups (both, p < .001). These differences still were significant by adjusting for sex and age. However, there was no difference in age at onset among 3 genotype groups in patients with schizophrenia by adjusting for the variables (F = 0.22, p = .80). Stepwise multivariate regression analysis showed that this polymorphism was not associated with age at onset in patients with schizophrenia (ß = .008, t = .07, p = .94). CONCLUSIONS: Our results indicated that even though PLA2G12A rs3087494 polymorphism did not influence age at onset in patients with schizophrenia, it may play an important role in the susceptibility to schizophrenia in a southern Chinese Han population.

Elevated triglyceride levels are associated with cognitive impairments among patients with major depressive disorder.

BACKGROUND: Cognitive deficits have been identified as one of core clinical symptoms of major depressive disorder (MDD). Accumulating evidence indicated that triglycerides (TG) might be associated with MDD and cognitive decline. OBJECTIVE: This study examined whether patients with MDD had poorer cognitive functions than healthy controls, and further investigate whether TG levels were involved in MDD, and its cognitive impairments in a Han Chinese population. METHOD: 115 patients with MDD and 119 healthy controls were enrolled. Cognitive functions were assessed by the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), and serum TG levels were examined using enzymatic colorimetry. RESULTS: TG levels were higher in patients with MDD than healthy controls after controlling for the variables. Cognitive test scores were lower in patients with MDD than healthy controls except for visuospatial/constructional index after controlling for the variables. TG levels were negatively correlated with visuospatial/constructional score, delayed memory score and RBANS total score of MDD. Further multivariate regression analysis showed that TG levels were negatively associated with visuospatial/constructional score, attention score, delayed memory score and RBANS total score of MDD. CONCLUSIONS: Our findings supported that serum TG levels might be involved in MDD, and play an important role in cognitive impairments of MDD, especially in delayed memory. Moreover, patients with MDD experienced greater cognitive impairments than healthy controls except for visuospatial/constructional index.

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.

Silencing of the SlNAP7 gene influences plastid development and lycopene accumulation in tomato.

Ripening is an important stage of fruit development. To screen the genes associated with pigment formation in tomato fruit, a suppression subtractive hybridization (SSH) cDNA library was constructed by using tomato fruit in the green ripe and break ripe stages, and 129 differential genes were obtained. Using redness as a screening marker, virus-induced gene silencing (VIGS) of the differential genes was performed with a sprout vacuum-infiltration system (SVI). The results showed that silencing the SlNAP7 gene affected the chloroplast development of tomato leaves, manifesting as a photo-bleaching phenotype, and silenced fruit significantly affected the accumulation of lycopene, manifested as a yellow phenotype. In our study, we found that silencing the SlNAP7 gene downregulates the expression of the POR and PORA genes and destroys the normal development of the chloroplast. The expression of related genes included in the lycopene biosynthesis pathway was not significantly changed, but lycopene accumulation was significantly reduced in tomato fruit. Perhaps it was caused by the destruction of the chromoplast, which leads to the oxidation of lycopene. The results show that the SlNAP7 gene influences chloroplast development and lycopene accumulation in tomato.

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.