Synergism of rMV-Hu191 with cisplatin to treat gastric cancer by acid sphingomyelinase-mediated apoptosis requiring integrity of lipid raft microdomains.

BACKGROUND: DDP-based chemotherapy is one of the first-line treatment in GC. However, the therapeutic efficacy of DDP is limited due to side effects. Therefore, it is of great significance to develop novel adjuvants to synergize with DDP. We had demonstrated previously that rMV-Hu191 had antitumor activity in GC. Here we examined the synergism of rMV-Hu191 with DDP in vitro and in vivo. METHODS: Cellular proliferation, the synergistic effect and cell apoptosis were evaluated by CCK-8 assay, ZIP analysis and flow cytometry, respectively. The protein levels and location of ASMase were monitored by western blot and immunofluorescence assay. shRNA and imipramine were used to regulate the expression and activity of ASMase. MßCD was administrated to disrupt lipid rafts. Mice bearing GC xenografts were used to confirm the synergism in vivo. RESULTS: From our data, combinational therapy demonstrated synergistic cytotoxicity both in resistant GC cell lines from a Chinese patient and drug-nonresistant GC cell lines, and increased cell apoptosis, instead of viral replication. Integrity of lipid rafts and ASMase were required for rMV-Hu191- and combination-induced apoptosis. The ASMase was delivered to the lipid raft microdomains at the initial stage of rMV-Hu191 treatment. In vivo GC mice xenografts confirmed the synergism of combinational treatment, together with increased apoptosis and trivial side-effects. CONCLUSIONS: This is the first study to demonstrate that rMV-Hu191 combined with DDP could be used as a potential therapeutic strategy in GC treatment and the ASMase and the integrity of lipid rafts are required for the synergistic effects.

[Clinical effect of "head-nine-needle" therapy on tension headache].

OBJECTIVE: To observe the effect of the "head-nine-needle" therapy on tension headache. METHODS: A total of 150 patients with tension headache were divided into a head-nine-needle therapy group, a western medicine control group and an acupuncture control group according to the random number table, 50 cases in each one. In the head-nine-needle therapy group, the head-nine-needle therapy was adopted. In the western medicine control group, epiperisone hydrochloride tablets were prescribed for oral administration, 50 mg, three times a day as well as fluoguili hydrochloride capsules, 5 mg, once a day, taking orally before sleep. In the acupuncture control group, the routine acupuncture technique was used at Baihui (GV20), Taiyang (EX-HN5), Fengchi (GB20) and Ashi (extra), etc. The clinical effect was observed in each group. The scores of visual analogy scale (VAS) for headache severity and the scores of headache duration were assessed before and after treatment in the patients of each group. RESULTS: In comparison of the total effective rate among the groups, there was no significant difference between the head-nine-needle therapy group (48/50，96.0%) and the acupuncture control group (47/50，94.0%). The total effective rate of either of the two groups was higher than that of the western medicine control group (40/50，80.0%，P<0.05). The VAS score and the score of headache duration were reduced obviously as compared with those before treatment in each group (P<0.05), and the score was not different significantly between the head-nine-needle therapy group and the acupuncture control group (P>0.05). After 3 weeks and 4 weeks of treatment, the scores in the two acupuncture groups were all better than those in the western medicine control group (P<0.05). CONCLUSION: The "head-nine-needle" therapy achieves the obviously advantages in the alleviation of headache degree as compared with the simple western medicine and its effect is similar to the common acupuncture therapy in the patients with tension headache.

Construction of Multiple-Substituted Chiral Cyclohexanes through Hydrogenative Desymmetrization of 2,2,5-Trisubstituted 1,3-Cyclohexanediones.

The construction of chiral multiple-substituted cyclohexanes motifs is a challenging topic in organic synthesis. By the combination of desymmetrization and remote stereocontrol, a ruthenium-catalyzed transfer hydrogenative desymmetrization of 2,2,5-trisubstituted 1,3-cyclohexanediones has been successfully developed for the construction of chiral multiple-substituted cyclohexanes with high enantioselectivity and diastereoselectivity. When an ester group was introduced to the two-position, a hydrogenative desymmetrization/transesterification cascade occurred, affording the bicyclic lactones bearing three stereocenters, including two discrete stereocenters and one quaternary stereogenic center, with high enantioselectivity. The products are the multiple-substituted chiral cyclohexanes bearing the hydroxyl and carbonyl functional groups, which provide a new opportunity for further precise elaboration.

A recombinant measles virus vaccine strain rMV-Hu191 has oncolytic effect against human gastric cancer by inducing apoptotic cell death requiring integrity of lipid raft microdomains.

Live-attenuated strain of measles virus (MV) has oncolytic effect. In this study, the antitumor effect of rMV-Hu191, a recombinant Chinese Hu191 MV generated in our laboratory by efficient reverse genetics system, was evaluated in gastric cancer (GC). From our data, rMV-Hu191 induced cytopathic effects and inhibited tumor proliferation both in vitro and in vivo by inducing caspase-dependent apoptosis. In mice bearing GC xenografts, tumor size was reduced and survival was prolonged significantly after intratumoral injections of rMV-Hu191. Furthermore, lipid rafts, a type of membrane microdomain with specific lipid compositions, played an important role in facilitating entry of rMV-Hu191. Integrity of lipid rafts was required for successful viral infection as well as subsequent cell apoptosis, but was not required for viral binding and replication. CD46, a MV membrane receptor, was found to be partially localized in lipid rafts microdomains. This is the first study to demonstrate that Chinese Hu191 MV vaccine strain could be used as a potentially effective therapeutic agent in GC treatment. As part of the underlying cellular mechanism, the integrity of lipid rafts is required for viral entry and to exercise the oncolytic effect.

Preparation of Axially Chiral 2,2'-Biimidazole Ligands through Remote Chirality Delivery and Their Application in Asymmetric Carbene Insertion into N-H of Carbazoles.

Axially chiral biimidazole ligands have been rarely synthesized and studied, in contrast to the significant achievements in the synthesis and application of central chiral imidazole ligands. Herein, a series of novel axially chiral 2,2'-biimidazole ligands were synthesized from the reaction of 2,2'-bis(bromomethyl)-1,1'-binaphthalene and 2,2'-biimidazole in one step through the strategy of remote chirality delivery. These ligands have been proven to be efficient for Cu- or Fe-catalyzed asymmetric insertion of α-aryl-α-diazoacetates into the N-H bond of carbazoles with up to 96% ee.

Ruthenium-Catalyzed Hydrogenation of Carbocyclic Aromatic Amines: Access to Chiral Exocyclic Amines.

The first highly enantioselective hydrogenation of carbocyclic aromatic amines has been successfully realized using in situ-generated chiral ruthenium complex as catalyst, affording facile access to chiral exocyclic amines with up to 98% ee.

Evaluation of a real-time method of simultaneous amplification and testing in diagnosis of Mycoplasma pneumoniae infection in children with pneumonia.

Mycoplasma pneumoniae (M. pneumoniae) infection can cause community acquired pneumonia in children. A real-time method of simultaneous amplification and testing of M. pneumoniae (SAT-MP) was developed to diagnose M. pneumoniae targeting a region of the ribosomal RNA. The SAT-MP assay can accurately identify M. pneumoniae with a detection range from 101 to 107 CFU/ml. In this study, the specimens from 315 children with pneumonia were collected and analyzed by SAT-MP in parallel with real-time PCR method and IgM ELISA assay. The positive rates of these specimens examined by SAT-MP assay, real-time PCR method and IgM ELISA assay were 16.51%, 15.56% and 12.70% respectively. While there was statistical significance (p = 0.04) between SAT-MP assay and IgM ELISA assay, no statistical significance (p = 0.25) was found between SAT-MP assay and real-time PCR method and these two methods had high consistency (kappa value = 0.97). These findings indicate that the newly developed SAT-MP assay is a rapid, sensitive and specific method for identifying M. pneumoniae with potential clinical application in the early diagnosis of M. pneumoniae infection.

A Hydrogenation/Oxidative Fragmentation Cascade for Synthesis of Chiral 4,5-Dihydro-1H-benzo[d]azepin-1-ones.

An iridium-catalyzed asymmetric hydrogenation/oxidative fragmentation of 6-substituted 5H-benzo[d] benzofuro[3,2-b]azepines has been developed, providing an efficient access to optically active 4-substituted 4,5-dihydro-1H-benzo[d]azepin-1-ones with up to 91% ee. A possible reaction pathway includes the asymmetric hydrogenation to furnish chiral cyclic amines and oxidative fragmentation under an air atmosphere.

Haze is an important medium for the spread of rotavirus.

This study investigated whether the rotavirus infection rate in children is associated with temperature and air pollutants in Hangzhou, China. This study applied a distributed lag non-linear model (DLNM) to assess the effects of daily meteorological data and air pollutants on the rotavirus positive rate among outpatient children. There was a negative correlation between temperature and the rotavirus infection rate. The impact of temperature on the detection rate of rotavirus presented an evident lag effect, the temperature change shows the greatest impact on the detection rate of rotavirus approximate at lag one day, and the maximum relative risk (RR) was approximately 1.3. In 2015, the maximum cumulative RR due to the cumulative effect caused by the temperature drop was 2.5. Particulate matter (PM) 2.5 and PM10 were the primary air pollutants in Hangzhou. The highest RR of rotavirus infection occurred at lag 1-1.5 days after the increase in the concentration of these pollutants, and the RR increased gradually with the increase in concentration. Based on the average concentrations of PM2.5 of 53.9 µg/m(3) and PM10 of 80.6 µg/m(3) in Hangzhou in 2015, the cumulative RR caused by the cumulative effect was 2.5 and 2.2, respectively. The current study suggests that temperature is an important factor impacting the rotavirus infection rate of children in Hangzhou. Air pollutants significantly increased the risk of rotavirus infection, and dosage, lag and cumulative effects were observed.

Clinical Value of Assessing Cytokine Levels for the Differential Diagnosis of Bacterial Meningitis in a Pediatric Population.

We performed a prospective observational study to evaluate the utility of measuring inflammatory cytokine levels to discriminate bacterial meningitis from similar common pediatric diseases. Inflammatory cytokine levels and other cerebrospinal fluid (CSF) physicochemical indicators were evaluated in 140 patients who were diagnosed with bacterial meningitis via microbiological culture or PCR assay. The CSF concentrations of interleukin (IL)-6 and IL-10, CSF/blood IL-6 and IL-10 ratios, CSF white blood cell count, and CSF micro total protein were significantly elevated in bacterial meningitis patients compared with healthy children or patients with viral encephalitis, epilepsy, or febrile convulsions (P < 0.001). The area under the curve values for CSF concentrations of IL-6 and IL-10, CSF/blood IL-6 and IL-10 ratios, CSF white blood cell count, and CSF micro total protein to identify bacterial meningitis episodes by receiver-operating characteristic analysis were 0.988, 0.949, 0.995, 0.924, 0.945, and 0.928, respectively. The area under the curve for the combination of CSF IL-6 and CSF/blood IL-6 ratio was larger than that for either parameter alone, and the combination exhibited enhanced specificity and positive predictive value. After effective meningitis treatment, CSF IL-6 levels dropped significantly. These results suggest that CSF IL-6 and CSF/blood IL-6 ratio are good biomarkers in discriminating bacterial meningitis. Evaluating CSF IL-6 and CSF/blood IL-6 ratio in combination can improve diagnostic efficiency. Additionally, CSF IL-6 levels can be used to monitor the effects of bacterial meningitis treatment.

Low concentrations of chloroquine and 3-methyladenine suppress the viability of retinoblastoma cells synergistically with vincristine independent of autophagy inhibition.

BACKGROUND: To study the inhibition of retinoblastoma cell viability by two commonly used autophagy inhibitors, chloroquine (CQ) and 3-methyladenine (3-MA), alone or in combination with the conventional chemotherapeutic drug vincristine (VCR), and to investigate whether the mechanisms of these drugs are related to inhibition of autophagy. METHODS: On retinoblastoma cell line HXO-Rb44, VCR, CQ and 3-MA were used individually or combined. The cell viability was determined by CCK8 method, and the cellular autophagic activity was determined by Western blotting of LC3 and p62. Caspase 3 fragmentation and Akt activation was also determined by Western blotting. RESULTS: VCR induced cell cycle arrest and apoptosis in HXO-Rb44 cells, but only inhibited autophagy at relatively high doses. Both CQ and 3-MA were synergistic with VCR to inhibit the growth of retinoblastoma cells and the combinational use significantly reduced the dosage of each drug. The lowest effective dose of CQ and 3-MA was most efficient to add on VCR; however, such dose was not sufficient to suppress autophagy in these cells. CQ could directly induce caspase activation, while 3-MA significantly inhibited Akt phosphorylation. CONCLUSIONS: CQ and 3-MA were synergistic with VCR to inhibit retinoblastoma cells. Our result suggested a novel strategy to combine CQ or 3-MA with VCR to reduce the side effects of each drug. However, lack of change in the autophagic activity when using the two drugs at lower doses suggests multiple mechanisms of action of the same drug at different doses. At higher doses, the drugs could inhibit autophagy, while at lower doses, they suppress tumor growth via autophagy-independent mechanisms.

24h Urinary Protein Levels and Urine Protein/Creatinine Ratios Could Probably Forecast the Pathological Classification of HSPN.

This study aimed to assess the relevance of laboratory tests in Henoch-Schönlein purpura nephritis (HSPN) classification, and determine accurate classification factors. This prospective study included 694 HSPN patients who underwent ultrasound-guided percutaneous renal biopsy (PRB). Renal specimens were scored according to International Study of Kidney Disease in Children (ISKDC) classification. Meanwhile, blood samples were immediately collected for laboratory examination. The associations between laboratory parameters and HSPN classification were assessed. Significant differences in levels of serum Th1/Th2 cytokines, immunoglobulins, T-lymphocyte subsets, complement, and coagulation markers were obtained between HSPN patients and healthy children. Interestingly, 24h urinary protein (24h-UPRO) levels and urine protein/urine creatinine ratios could determine HPSN grade IIb, IIIa, and IIIb incidences, with areas under ROC curve of 0.767 and 0.731, respectively. At 24h-UPRO >580.35mg/L, prediction sensitivity and specificity were 75.2% and 70.0%, respectively. These values became 53.0% and 82.3%, respectively, with 24h-UPRO exceeding 1006.25mg/L. At urine protein/urine creatinine > 0.97, prediction sensitivity and specificity were 65.5% and 67.2%, respectively, values that became 57.4% and 80.0%, respectively, at ratios exceeding 1.2. Cell and humoral immunity, coagulation and fibrinolytic systems are all involved in the pathogenesis of HSPN, and type I hypersensitivity may be the disease trigger of HSPN. 24h-UPRO levels and urine protein/creatinine ratios could probably forecast the pathological classification of HSPN.

Dual Inhibition of Cdc7 and Cdk9 by PHA-767491 Suppresses Hepatocarcinoma Synergistically with 5-Fluorouracil.

Activation of checkpoint kinase 1 (Chk1) is essential in chemoresistance of hepatocarcinoma (HCC) to 5-fluorouracil (5-FU) and other antimetabolite family of drugs. In this study, we demonstrated that PHA-767491, a dual inhibitor of two cell cycle checkpoint kinases, cell division cycle kinase 7 (Cdc7) and cyclin-dependent kinase 9 (Cdk9), has synergistic antitumor effect with 5-FU to suppress human HCC cells both in vitro and in vivo. Compared with the sole use of each agent, PHA-767491 in combination with 5-FU exhibited much stronger cytotoxicity and induced significant apoptosis manifested by remarkably increased caspase 3 activation and poly(ADP-Ribose) polymerase fragmentation in HCC cells. PHA-767491 directly counteracted the 5-FU-induced phosphorylation of Chk1, a substrate of Cdc7; and decreased the expression of the anti-apoptotic protein myeloid leukemia cell 1, a downstream target of Cdk9. In tumor tissues sectioned from nude mice HCC xenografts, administration of PHA-767491 also decreased Chk1 phosphorylation and increased in situ cell apoptosis. Our study suggests that PHA- 767491 could enhance the efficacy of 5-FU by inhibiting Chk1 phosphorylation and down-regulating Mcl1 expression through inhibition of Cdc7 and Cdk9, thus combinational administration of PHA-767491 with 5-FU could be potentially beneficial to patients with advanced and resistant HCC.

Isolation of autophagosome subpopulations after induction of autophagy by calcium.

Autophagy is a dynamic process accomplished by the generation and maturation of autophagosomes. Isolation of autophagosomes and subsequent compositional analysis can provide information about their biogenesis mechanism. In this article, HEK293 cells expressing GFP-LC3 were treated by calcium phosphate precipitates (CPP) to induce autophagy. The autophaogomes induced by CPP were tubular and vesicular structures, extensively formed in the cytosol. After all membranes in the cell lysate were fractionated by differential centrifugation, autophagosomes from light and heavy membranes were isolated by immuno-precipitation, using antibodies against GFP-LC3 and Atg5. We found that GFP-LC3 and Atg5 positive autophagosomes represented distinctive subpopulations. Judged from the molecular markers associated, including organelle markers and Atg proteins, GFP-LC3 positive autophagosomes were overall at the later biogenetic stage. Furthermore, both GFP-LC3 and Atg5 positive autophagosomes from light membranes were less mature than those from heavy membranes. We have established a method to isolate subpopulations of autophagsomes for further characterization.

Epidemiological characteristics and immune status of children with Respiratory Syncytial Virus.

Respiratory Syncytial Virus (RSV) infections are the dominant cause of pneumonia in children. In order to determine the epidemiological characteristics and immune status of children with Respiratory Syncytial Virus, a prospective study was performed among patients with RSV infection. Comparisons between RSV pneumonia group and normal control group, RSV pneumonia group had lower IL-2 (median levels, pg/ml: 3.8 vs. 5.1, P < 0.01), and higher IL-4 (median levels, pg/ml: 3.2 vs. 2.4, P < 0.01), IL-10 (median levels, pg/ml: 12.2 vs. 2.3, P < 0.01), and IFN-γ (median levels, pg/ml: 13.4 vs. 4.6, P < 0.01). The level of IgE among pneumonia patients caused by RSV increased sharply (median levels, mg/L: 48.1 vs. 8.8, P < 0.01). Another amazing finding is that after birth, the degree of IgE of the children infected by RSV increases gradually with age. This effect is at its peak in 0.6 years old. The IgE and eosinophil levels were higher when patients suffered from RSV pneumonia with wheeze (IgE median levels, IU/ml: with wheeze: 72.74 vs. without wheeze: 11.5, P < 0.05; eosinophil median levels, ×10(9) /l: with wheeze: 0.21 vs. without wheeze: 0.05, P < 0.05). The main morbidity crowd is the children under the age of 1 year old. The downregulation of IL2 and the upregulation of IL-4, IL-10, IFN-γ, and IgE happen after RSV infection.

Relationship between immune parameters and organ involvement in children with Henoch-Schonlein purpura.

Henoch-Schonlein purpura (HSP) is the most common type of connective tissue diseases which increasingly occurs in children in recent years and its pathogenesis remains unclear. In order to explore the immune parameters and underlying pathogenesis mechanism of children with HSP, the study involved 1232 patients with HSP having different clinical symptoms and their laboratory indicators were evaluated. Th1/Th2 imbalance and overactivity of Th2 cells can cause increase in the synthesis and release of immunoglobulins in children with HSP. The number of red blood cells and white blood cells in urine was directly proportional to the level of IgA and inversely proportional to the level of serum complements (C3 and C4). Activation of these complements caused by immunoglobulin in patients with HSP plays an important role in renal injury. The urinary protein content in children with HSP along with proteinuria was positively correlated with IgE level, and IgE mediated type 1 hypersensitivity can cause increase in capillary permeability and weakened the charge barrier; hence, it could be considered as one of the causes of proteinuria in HSP. Additionally, the NK cells percentage was reduced and impaired immune function of NK cells were related to the immune injury of the digestive tract and kidney.

Absolute lymphocyte count is associated with minimal residual disease level in childhood B-cell precursor acute lymphoblastic leukemia.

The prognostic value of absolute lymphocyte count (ALC) has been a recent matter of debate in childhood acute lymphoblastic leukemia (ALL). In the current study, ALCs at the time of diagnosis (ALC-0), after 7 days of initial therapy (ALC-8) and at interim of the induction therapy (ALC-22) were examined in Chinese children with B-cell precursor (BCP) ALL and correlated with the level of minimal residual disease (MRD) at day 22 of induction therapy. Medical and laboratory records of 140 patients diagnosed with childhood BCP ALL were retrieved and analyzed. ALC-22 is significantly correlated with MRD level at day 22 of therapy and can be a good prognostic factor for childhood BCP-ALL. Furthermore, lymphocyte count at initial diagnosis is correlated with MRD level at day 22 in childhood BCP-ALL with the immnunophenotype of CD19(pos)/CD10(pos)/CD34(pos)/CD45(neg) and role as a new prognostic factor was determined.

Prognostic significance of flow cytometric minimal residual disease assessment after the first induction course in Chinese childhood acute myeloid leukemia.

Flow cytometry based minimal residual disease (MRD) was evaluated for outcome prediction in childhood acute myeloid leukemia (AML). The median levels of MRD in relapsed and nonrelapsed patients were different after the first induction (0.64% vs. 0.18%, P=0.030). A cutoff level of ≥ 0.25% after the first course of induction was correlated with a high risk of relapse in both univariate analysis (5-year cumulative incidence of relapse: 66.8% vs. 21.2%, P=0.002) and multivariate analyses (hazard ratio: 3.70, 95% CI, 1.23-11.08, P=0.020). Our results showed that MRD level after the first induction therapy provides important information for risk assessment in childhood AML.