Recently Evolved Tumor Suppressor Transcript TP73-AS1 Functions as Sponge of Human-Specific miR-941.

MicroRNA (miRNA) sponges are vital components of posttranscriptional gene regulation. Yet, only a limited number of miRNA sponges have been identified. Here, we show that the recently evolved noncoding tumor suppressor transcript, antisense RNA to TP73 gene (TP73-AS1), functions as a natural sponge of human-specific miRNA miR-941. We find unusually nine high-affinity miR-941 binding sites clustering within 1 kb region on TP73-AS1, which forms miR-941 sponge region. This sponge region displays increased sequence constraint only in humans, and its formation can be traced to the tandem expansion of a 71-nt-long sequence containing a single miR-941 binding site in old world monkeys. We further confirm TP73-AS1 functions as an efficient miR-941 sponge based on massive transcriptome data analyses, wound-healing assay, and Argonaute protein immunoprecipitation experiments conducted in cell lines. The expression of miR-941 and its sponge correlate inversely across multiple healthy and cancerous tissues, with miR-941 being highly expressed in tumors and preferentially repressing tumor suppressors. Thus, the TP73-AS1 and miR-941 duo represents an unusual case of the extremely rapid evolution of noncoding regulators controlling cell migration, proliferation, and tumorigenesis.

[Effects of sublingual immunotherapy on serum IL-17 and IL-35 levels in children with allergic rhinitis or asthma].

OBJECTIVE: To study the clinical effect and mechanisms of specific sublingual immunotherapy (SLIT) for the treatment of allergic rhinitis or asthma in children. METHODS: Thirty children suffering from Dermatophagoides farinae-allergic rhinitis or asthma (case group) and 30 healthy children (control group) were enrolled in this study. The case group accepted SLIT between January and December 2011. The ratio of forced expiratory volume in one second (FEV1) and its expected value, the ratio of airway resistance and its expected value, peripheral blood eosinophil (Eos) count and serum levels of IL-17 and IL-35 were measured before treatment and one and two years after treatment. The rhinitis or asthma symptom scores were rated and the level of asthma control was monitored. RESULTS: Serum IL-17 level in the case group was significantly higher than in the control group before treatment and one year after treatment (P<0.01). Furthermore, serum IL-17 level in the case group gradually decreased from before treatment to 1 year to 2 years after treatment (P<0.01). By two years of treatment, there was no significant difference in serum IL-17 level between the case and control groups (P>0.05). The changes of serum IL-35 level after treatment were opposite to serum IL-17 in the case group. The ratio of FEV1 and its expected value gradually increased from before treatment to 1 year to 2 years after treatment (P<0.01) in the case group. In contrast, the change of the ratio of airway resistance and its expected value and Eos count gradually decreased from before treatment to 1 year to 2 years after treatment (P<0.01) in the case group. More patients achieved improved rhinitis or asthma symptom scores two years after treatment than one year after treatment in the case group (P<0.01). SLIT was effective in 85% of children with allergic rhinitis one after treatment vs 100% two years after treatment. Asthma control was observed in 76% of the asthmatic patients one after treatment vs 92% two years after treatment. CONCLUSIONS: SLIT is effective for allergic rhinitis and asthma in children, and the treatment period of two years seems to be superior to one year. The mechanism of action of SLIT for the treatment of allergic rhinitis and asthma may be associated with inhibition of IL-17 expression and promotion of IL-35 expression.

[Characteristics of recidivism in patients with mental disorders: 156 cases analysis].

OBJECTIVE: To analyze the characteristics of recidivism in patients with mental disorders, including criminology, clinic and guardianship, in order to provide references for preventing recidivism. METHODS: Using the self-designed questionnaire, 156 psychotic patients who had repeated crimes were appraised by West China Forensic Science Center of Sichuan University from 2007 to 2011 and the data were collected and analyzed. RESULTS: In the majority of these cases, patients were male, 26-45 years old, junior high school or below diploma, unmarried, and farmers or jobless. Each patient broke law 3.26 times on average. The main crimes were intentional injury (34.6%) and murder (15.7%). Within 5 years after diagnosis with mental disorder, 56.4% of the patients committed first crime. Within 1 year after the first time breaking the law, 55.8% of them repeated crimes. The diagnoses of schizophrenia (63.5%) were in the majority. The assessment results were mostly irresponsibility (61.5%). Among the patients, 44.9% of them didn't receive treatment while 34.6% of them were out of supervision. After the first crime, 66.1% of them didn't receive criminal prosecution while only 7.1% of them went through the appraisement of forensic psychiatry. CONCLUSION: Most of the patients had low education and low income. Meanwhile, low outpatient rate and pool supervision occurred in this special crowd. A good system for care and treatment of these mental patients should be built to prevent them from recidivism.

[Clinical analysis of immune function changes in children with bronchial pneumonia].

OBJECTIVE: To investigate changes in serum complement, immunoglobulins and lymphocyte subsets in children with common and severe bronchial pneumonia, and the role of immune function testing in bronchial pneumonia. METHODS: Twenty children with common bronchial pneumonia, 20 with severe bronchial pneumonia and 20 healthy children (as controls) were enrolled in this study. Immunization rate scattering turbidimetry and six-color flow cytometry were used to detect changes in serum levels of IgA, IgG and IgM, complement C3 and C4 and CD3(+), CD4(+), CD8(+), CD16(+), CD56(+) and CD19(+) cells. RESULTS: The IgA levels of children with common and severe pneumonia were significantly lower than in the control group (P<0.05). The IgG level of children with severe pneumonia was significantly lower than in the control group (P<0.05). There were no significant differences in the levels of IgM and complement C3 and C4 between the two pneumonia groups and the control group (P>0.05). Compared with the controls, the children with severe pneumonia showed significantly lower CD4(+) and CD3(+) counts (P<0.05) and a significantly higher CD19(+) count (P<0.05), and the CD16(+) and CD56(+) counts of children with severe pneumonia were significantly lower than in the controls and in children with common pneumonia (P<0.05). There were no differences in CD8(+) count and CD4(+)/CD8(+) ratio between the two pneumonia groups and the control group (P>0.05). CONCLUSIONS: Immune dysfunction exists in children with bronchial pneumonia, especially those with severe pneumonia. Changes in immune function are correlated with the severity of pneumonia. Immune function testing in children with pneumonia has important clinical significance.

[Application of bacterial cultures of bronchoalveolar lavage fluids in children with pulmonary infection].

OBJECTIVE: To study the value of bacterial cultures of bronchoalveolar lavage fluids (BALF) in children with pulmonary infection. METHODS: Bacterial cultures sampled from both sputum and BALF were performed on 80 hospitalized children with pulmonary infection between June 2008 and February 2011.Culture results between the two samples were compared. RESULTS: In the 80 children with pulmonary infection, bacterial cultures of BALF showed that Viridans Streptococci were found in 72 cases (90%), Neisseria in 41 cases (51%), Streptococcus pneumoniae in 11 cases (14%), Staphylococcus Aureus in 3 cases (4%) and Escherichia coli in 3 cases (4%). The positive rates of Viridans Streptococci in the bacterial cultures of BALF was not significantly different from the bacterial cultures of sputum, but the positive rate of Streptococcus pneumoniae in the bacterial cultures of BALF was significantly higher than in the bacterial cultures of sputum (4%). Moreover, Escherichia coli were found only by bacterial cultures of BALF. CONCLUSIONS: Bacterial cultures of BALF are useful in the identification of pathogenic bacteria for pulmonary infection in children. Due to the samples taken from the lesion regions in bacterial cultures of BALF, the results of may be more reliable.

[Criminological characteristics of female violent criminal suspects].

OBJECTIVE: To study criminological characteristics of female violent criminal suspects who accepted forensic psychiatry assessment. METHODS: Information of the suspects involved in judicial appraisal between 2000 and 2009 were collected and analyzed according to our self-made scale. RESULTS: The age of 259 suspects were between 16 and 81 years old. There were 205 (79.2%) suspects who were younger than 45 years old. There were 225 (86.9%) suspects who were married. There were 14 different appraisal results: schizophrenia 47.1%, without psychosis 15.4%, depression 10.4% and others 27.1%. Irresponsibility involved with 59.5%, partial responsibility 18.5% and full responsibility 22.0%. Murder were 85.7%, arson 10.4%, inflicted injury 1.9% and robbery 1.9%. A total of 191 cases resulted in death, accounting for 82.3% of all cases. In 34.9% of all cases, the victims were male spouse of the suspects. Main weapons used in the crime were cutters and other working related tools (36.3%). There were 66.8% of all cases were with pathological motivation, 29.7% with reality motivation and 3.5% cases were with unknown motivation. CONCLUSION: Female violent suspects in our cases often suffered from various kinds of mental diseases. Their motivations were pathological primarily. Most cases were intentional killing with family members as victim mainly. Major weapons used were daily working related tools.

[Study on physicochemical characteristics and denitrogenation of sludge during granulation].

The physicochemical characteristics and denitrogenation of sludge during granulation were investigated in SBR. It was revealed that the process of granulation can be divided into three stages, including formation, growth and mature stage. In the first stage, the settling ability of sludge was improved obviously, SVI decreased from 110 mL/g to 23 mL/g during the first 30 days; In the second phase, aerobic granule grew rapidly, the mean diameter increased to 0.82 mm from 0.25 mm in the following 15 days, the value of SOUR(h) decreased from 80 mg/(g x h) to 35 mg/(g x h), denitrification rate was highly improved during granulation, the removal efficiency of TN enhanced to 80% from 55%; In the last stage, the physicochemical characteristics of sludge and denitrogenation became stable, and the removal efficiency of TN could achieve 85%, that means simultaneous nitrification and denitrification was realized.

High-level expression of functional recombinant human butyrylcholinesterase in silkworm larvae by Bac-to-Bac system.

Butyrylcholinesterase (BChE: EC 3.1.1.8) serves as a natural scavenger for a variety of drugs, poisons, and organophosphorous compounds by hydrolyzing their ester bonds. Large scale production of recombinant human BChE (rhBChE) has been reported in transgenic goat. Here we demonstrate high-level expression of rhBChE with biological activity comparable to that of natural and recombinant enzymes, through the Bac-to-Bac baculovirus expression system in silkworm Bombyx mori larvae. We constructed the full-length hBChE cDNA into the plasmid pFastBac. To monitor the level of expression, the cDNA coding for an orange fluorescent protein (OFP) was cloned downstream to the polyhedron (pH) promoter. Transfection was carried out by subcutaneous injection of 4-5th instar silkworm larvae. Approximately 4-7 days after infection, high-level expression of recombinant proteins was observed as indicated by the orange fluorescence of the larvae under blue light illumination. The hemolymph of the infected larvae was harvested, purified and assayed for BChE activity. The total units of BChE activity after purification were around 6.4 units per larvae. The K(m) and V(max) values of rhBChE were determined to be 17.7 microM and 2194 U/l hemolymph, respectively. By SDS-PAGE and Western analysis, the size of silkworm rhBChE was estimated to be 85 kDa. The results indicate that the silkworm larva is a good alternative system to produce bioactive rhBChE. Further optimization and modifications will be necessary for large-scale production of rhBChE. This should provide a rapid, low-cost, and high yield rhBChE for therapeutic applications.

Quantification of silkworm coactivator of MBF1 mRNA by SYBR Green I real-time RT-PCR reveals tissue- and stage-specific transcription levels.

Transcriptional coactivators play a crucial role in gene transcription and expression. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA-binding activators, such as FTZ-F1 and GCN4. Until now, very few studies have been reported in the silkworm. We selected the Bombyx mori because it is a model insect and acts as an economic animal for silk industry. In this study, we conducted the quantitative analysis of MBF1 mRNA in silkworm B. mori L. with actin (A3) as internal standard by means of SYBR Green I real-time RT-PCR method. The total RNA was extracted from the silk gland, epidermis, fat body, and midguts of the fifth instar B. mori larvae. The mRNA was reverse transcripted, and the cDNA fragments of MBF1 mRNA and actin gene were amplified by RT-PCR using specific primers. MBF1 mRNA expression in different tissues of silkworm B. mori L. was quantified using standardized SYBR Green I RT-PCR. The results suggested MBF1 gene was expressed in all investigated organs but highly expressed in the silk gland, showing its relation to biosynthesis of silk proteins.

[Urinary leukotrience E(4) level in children with asthma].

OBJECTIVE: Cysteinyl leukotriene (CysLTs) plays an important role in airway inflammation and remodeling in asthma. Measurement of urinary leukotriene E(4) (LTE(4)) is a sensitive and noninvasive method of assaying total body CysLTs level. This study aimed to evaluate the clinical significance of urinary leukotriene E(4) (LTE(4)) in childhood asthma. METHODS: Sixty children with acute asthma were randomly divided into montelukast (leukotriene receptor antagonist) treatment and conventional treatment groups (n = 30 each). Urinary LTE(4) levels were measured using ELISA and the airway resistance Rint was assessed by the lung function instrument at the acute and the convalescence phases. Twenty healthy children were used as the control group. RESULTS: Urinary LTE(4) levels in asthmatic children at the acute and the convalescence phases were significantly higher than those in the control group (p<0.01). The urinary LTE(4) levels at the convalescence phase were significantly reduced compared with those at the acute phase in asthmatic children (p<0.01). More significantly decreased urinary LTE(4) levels were noted in the montelukast treatment group than the conventional treatment group at the convalescence phase (p<0.01). In the acute phase, there was no correlation between urinary LTE4 level and Rint in asthmatic children. CONCLUSIONS: Urinary LTE(4) level is significantly increased in children with acute asthma. Urinary LTE(4) is a useful marker for the diagnosis of asthma and can be as a predictor of asthma control and marker of susceptibility to treatment with leukotriene receptor antagonists.

Manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L enhances the NK activity and splenocyte proliferation against Sarcoma 180 tumor cells in vivo.

Natural killer cell (NK) is known as a major immune system in body through mediating cell death via several possible pathways, and one of three subpopulations of lymphocytes functioning as scavenger of tumor, virus infected cells etc. Our present results found that the SOD-contained silkworm larvae powder caused an enhancement of the effect on NK cell cytotoxicity, which implied this material modulated the immune system in mice in vivo. The NK cell activities of S180 tumor modeled mice treated with silkworm powder including SOD were enhanced significantly ranging from 30% to 48%, respectively, compare to a distilled water feeding control and silkworm powder without SOD. Meanwhile, the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control both in T cells or B cells. The average tumor weight of S180 modeled mice treated with doses of SOD-contained silkworm powder was lighter than that of water control showing the tumor inhibition rates (IR) reached to 22.51% to 37%, respectively. In conclusion, these findings demonstrate that administration of silkworm larvae powder containing SOD results in activation of NK cells and immune T-cell and B-cell, suggesting the silkworm larvae powder containing SOD play a positive role in tumor inhibition.

High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system.

This novel orange fluorescent protein (OFP) emits brilliant orange fluorescent light. OFP has high fluorescence quantum yield, fast maturation rate, and stability, which imply this protein should be the most favorable biotechnological tools used to investigate the function of target gene by visualizing, monitoring, and quantifying in living cells. B. mori, silkworm has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). In this paper, we used infection technique which introduced the baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus, and demonstrated a high-level expression of the orange fluorescent protein (OFP) gene in the Bombyx mori, silkworm larvae. When recombinant rBacmid/BmNPV/OFP DNA ranging from 50-100 ng/larval was injected, a sufficient OFP expression in hemolymph was harvested. The recombinant viruses could be obtained from the hemolymph of infected larvae and stored as seed which could be used for the large-scale expression. This procedure omitted the costly and labor-consumed insect cell culture. Further investigation of OFP should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.

Silkworm powder containing manganese superoxide dismutase regulated the immunity and inhibited the growth of Hepatoma 22 cell in mice.

The effects of SOD contained silkworm powder on immune regulation and inhibition against Hepatoma 22 tumor cells in vivo were investigated. The activity of natural killer cell (NK) and the ConA-stimulated spleen proliferation were measured. The results found that the SOD-contained silkworm powder caused an enhancement on NK cell activity, which implied this material modulated the immune system in mice in vivo. The NK cell activities of Hepatoma 22 tumor modeled mice treated with silkworm powder including SOD were increased significantly compared to a modeled control and silkworm powder without SOD, reaching 36.18%. In addition, the ConA-stimulated spleen proliferation of SOD treated mice was higher than that of the controls. The treatment of SOD contained silkworm powder presented 40.3% of average inhibition rate to Hepatoma 22 tumor, showing stronger inhibition against tumor. There were no significant difference in body weight between modeled control and SOD silkworm powder feeding in Hepatoma 22 tumor modeled mice, suggesting the SOD silkworm powder is safety as an inhabitant to tumor. In conclusion, these findings demonstrate that administration of silkworm powder containing SOD results in activation of NK cells and immunity, suggesting the silkworm powder containing SOD plays a positive role in tumor inhibition.

Proteomic identification of the silkworm (Bombyx mori L) prothoracic glands during the fifth instar stage.

Although the ecdysteroid of the silkworm had been studied for decades, the proteome of the prothoracic gland, the primary source of ecdysteroid hormones, has not been studied previously. In the present paper, we utilized a proteomic approach to investigate the fifth instar prothoracic gland during the growth and development of the silkworm, Bombyx mori L. The two-dimensional electrophoresis results showed that the majority of proteins were acidic proteins, especially concentrated in the area of 25-65 kDa, with pI values of between 4 and 7, and the difference was not distinct. When compared with Qiufeng (Japanese strain), the interspecific distinction was larger than the intraspecific distinction, and 19 particular spots, excized from the third, fifth and ninth days of p50 (Chinese strain) and Qiufeng were subjected to MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight MS) analysis. We sorted them into seven catagories: energetics and/or metabolism, storage proteins, protection, lipid metabolism, signal transduction, cell function and unknown function proteins. Of these proteins, arginine methyltransferase is discussed as playing an important role in regulating the activation of ecdysteroidogenesis via transcription or translation.

Improvement of recombinant baculovirus infection efficiency to express manganese superoxide dismutase in silkworm larvae through dual promoters of Pph and Pp10.

The silkworm, Bombyx mori, has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). There are several problems which will probably be the bottleneck for practical and industrial utilization of silkworm bioreactor. Traditionally, the recombinant virus should infect the larvae through individual dorsal injection by a syringe. This is a time- and labor-consuming procedure. This drawback has become a bottleneck for practical and industrial utilization of baculovirus expression system in the silkworm bioreactor. In this paper, we constructed a dual expression baculovirus to express the renovated polyhedron and target manganese superoxide dismutase (SOD) gene under P10 and polyhedron promoters, respectively, through oral infection. The results showed that the direct injection of recombinant rBacmid/BmNPV/SOD DNA with cellfectin reagent infected the silkworm larvae partially. When next batches of larvae were fed orally with hemolymph, which was collected from first batch of injected and infected larvae, the obvious symptom of infection was found and high target SOD was expressed. These results imply it is feasible to express target genes through combination of recombinant bacmid DNA injection and oral feeding by a dual expression bacmid baculovirus.

Effects of silkworm larvae powder containing manganese superoxide dismutase on immune activity of mice.

To study the function of silkworm larvae powder containing superoxide dismutase and potential practical development, we investigated the safety assessment and effects on immune activity of mice such as the growth of immunity-related organs, delayed-type hypersensitivity (DTH) and charcoal particle clearance ability. The mean body weights in treated mice were significantly heavier than that of control, meanwhile, the ratio of splenocytes/body weight and the thoracic gland/body weight in treated mice was significantly enhanced after 30 days treated with silkworm larvae powder containing manganese superoxide dismutase. The treated mice resulted in a profound activation of the DTH and charcoal particle clearance, and indicated the treated mice have stronger phagocytic activity to exogenous materials. Our data also indicated the feeding treatment was safe with 360 folds of recommended human dosage in acute toxic test. In long-term test, there were no effects of silkworm larvae powder containing SOD on treated mice's growth and inside organs as long as 90 days. Further the electronic microscope investigation showed the intestine, liver, splenocyte and stomach in mice were no obvious changes both in organs and sub-organs such as nucleus, endoplasmic reticulum, mitochondrion, Golgi and peroxisomes after treated for as long as 90 days.

Anti-oxidation and immune responses in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L.

With manganese superoxide dismutase expressed in silkworm larvae, Bomby mori L, we investigate the effects of silkworm larvae powder containing SOD on the antioxidation and the immune system of mouse. The contents of MDA both in mice plasma or liver organ treated with silkworm larvae powder containing manganese superoxide dismutase were reduced compare to control. The superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities both in plasma or liver organ of the treated mice were significantly higher than that of both control and bromobenzene treated mice (group-BM), suggesting the silkworm larvae powder containing SOD play a positive role in anti-oxidation in mice. This experiment was also designed to investigate the effects of silkworm larvae powder containing SOD on the immune system of mouse, focused on hemolysin response, hemagglutination against SRBC and the activity of natural killer (NK) cells. All treated mice showed significant increase in hemolysin response to SRBC and demonstrated an activation of NK cell function by the SOD-contained silkworm larvae powder, which suggest a promotion in humoral immunity. The results suggested the SOD expressed in silkworm maybe have potential application in medicine.

Immunity promotion and proteomic identification in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae.

With manganese superoxide dismutase (SOD) expressed in silkworm larvae, Bomby mori L, we investigated the effects of silkworm larvae powder containing SOD on the immune system of mouse and employed a proteomics approach to examine this phenomenon. Our data on the effects of continuous treatment with SOD-containing silkworm larvae powder showed that the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control. The results of PFC assay also revealed that antibody production was higher in all three treated groups than controlled mice. We investigated the phagocytosis of mouse macrophages. The SOD treatment led to a dose-dependent increase of phagocytic activity. We identified six proteins that related to immunity of mice. The data showed all these six matched proteins related immunity presented the increase of expression level in plasma of mouse administrated with silkworm powder including SOD compared to that of control. These findings demonstrate that administration of silkworm larvae powder containing SOD results in enhancement of immunity activities in the mouse. The results also suggested that the SOD expressed in silkworm maybe have potential application in medicine.