NPS-2143 inhibit glioma progression by suppressing autophagy through mediating AKT-mTOR pathway.

Gliomas are the most common tumours in the central nervous system. In the present study, we aimed to find a promising anti-glioma compound and investigate the underlying molecular mechanism. Glioma cells were subjected to the 50 candidate compounds at a final concentration of 10 µM for 72 h, and CCK-8 was used to evaluate their cytotoxicity. NPS-2143, an antagonist of calcium-sensing receptor (CASR), was selected for further study due to its potent cytotoxicity to glioma cells. Our results showed that NPS-2143 could inhibit the proliferation of glioma cells and induce G1 phase cell cycle arrest. Meanwhile, NPS-2143 could induce glioma cell apoptosis by increasing the caspase-3/6/9 activity. NPS-2143 impaired the immigration and invasion ability of glioma cells by regulating the epithelial-mesenchymal transition process. Mechanically, NPS-2143 could inhibit autophagy by mediating the AKT-mTOR pathway. Bioinformatic analysis showed that the prognosis of glioma patients with low expression of CASR mRNA was better than those with high expression of CASR mRNA. Gene set enrichment analysis showed that CASR was associated with cell adhesion molecules and lysosomes in glioma. The nude mice xenograft model showed NPS-2143 could suppress glioma growth in vivo. In conclusion, NPS-2143 can suppress the glioma progression by inhibiting autophagy.

UPLC-MS/MS method for the determination of voriconazole plasma concentration from pediatric patients with hematologic tumors: An application toward personalized therapy.

Untreated invasive fungal infection is one of the important risk factors affecting the prognosis of pediatric patients with hematologic tumors. Voriconazole (VOR) is the first-line antifungal drug for the treatment of Aspergillus infections. In order to reduce the risk of adverse drug reactions while producing an ideal antifungal effect, therapeutic drug monitoring was performed to maintain the VOR plasma concentration in a range of 1,000-5,500 ng/ml. In the present study, a reliable, accurate, sensitive and quick ultra-high performance liquid chromatograph-tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of the VOR level. Protein precipitation was performed using acetonitrile, and then the chromatographic separation was carried out by UPLC using a C18 column with the gradient mobile phases comprising 0.1% methanoic acid in acetonitrile (A) and 0.1% methanoic acid in water (B). In the selective reaction monitor mode, the mass spectrometric detection was carried out using an TSQ Endura triple quadruple mass spectrometer. The performance of this UPLC-MS/MS method was validated as per the National Medical Products Administration for Bioanalytical Method Validation. Additionally, the plasma concentrations of VOR in pediatric patients with hematologic tumors were detected using this method, and the analyzed results were used for personalized therapy.

Daurisoline suppress glioma progression by inhibiting autophagy through PI3K/AKT/mTOR pathway and increases TMZ sensitivity.

Glioma is one of the most common primary malignant tumors of the central nervous system. Temozolomide (TMZ) is the only effective chemotherapeutic agent, but it easily develops resistance and has unsatisfactory efficacy. Consequently, there is an urgent need to develop safe and effective compounds for glioma treatment. The cytotoxicity of 30 candidate compounds to glioma cells was detected by the CCK-8 assay. Daurisoline (DAS) was selected for further investigation due to its potent anti-glioma effects. Our study revealed that DAS induced glioma cell apoptosis through increasing caspase-3/6/9 activity. DAS significantly inhibited the proliferation of glioma cells by inducing G1-phase cell cycle arrest. Meanwhile, DAS remarkably suppressed the migration and invasion of glioma cells by regulating epithelial-mesenchymal transition. Mechanistically, our results revealed that DAS impaired the autophagic flux of glioma cells at a late stage by mediating the PI3K/AKT/mTOR pathway. DAS could inhibit TMZ-induced autophagy and then significantly promote TMZ chemosensitivity. Nude mice xenograft model revealed that DAS could restrain glioma proliferation and promote TMZ chemosensitivity. Thus, DAS is a potential anti-glioma drug that can improve glioma sensitivity to TMZ and provide a new therapeutic strategy for glioma in chemoresistance.

Ganoderma lucidum spore extract improves sleep disturbances in a rat model of sporadic Alzheimer's disease.

Introduction: Ganoderma lucidum (G. lucidum, Lingzhi) has long been listed as a premium tonic that can be used to improve restlessness, insomnia, and forgetfulness. We previously reported that a rat model of sporadic Alzheimer's disease (sAD) that was induced by an intracerebroventricular injection of streptozotocin (ICV-STZ) showed significant learning and cognitive deficits and sleep disturbances. Treatment with a G. lucidum spore extract with the sporoderm removed (RGLS) prevented learning and memory impairments in sAD model rats. Method: The present study was conducted to further elucidate the preventive action of RGLS on sleep disturbances in sAD rats by EEG analysis, immunofluorescence staining, HPLC-MS/MS and Western blot. Results: Treatment with 720 mg/kg RGLS for 14 days significantly improved the reduction of total sleep time, rapid eye movement (REM) sleep time, and non-REM sleep time in sAD rats. The novelty recognition experiment further confirmed that RGLS prevented cognitive impairments in sAD rats. We also found that RGLS inhibited the nuclear factor-κB (NF-κB)/Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammatory pathway in the medial prefrontal cortex (mPFC) in sAD rats and ameliorated the lower activity of γ-aminobutyric acid (GABA)-ergic neurons in the parabrachial nucleus (PBN). Discussion: These results suggest that inhibiting the neuroinflammatory response in the mPFC may be a mechanism by which RGLS improves cognitive impairment. Additionally, improvements in PBN-GABAergic activity and the suppression of neuroinflammation in the mPFC in sAD rats might be a critical pathway to explain the preventive effects of RGLS on sleep disturbances in sAD.

Sporoderm-removed Ganoderma lucidum spores ameliorated early depression-like behavior in a rat model of sporadic Alzheimer's disease.

Introduction: Ganoderma lucidum: (G. lucidum, Lingzhi) is a medicinal and edible homologous traditional Chinese medicine that is used to treat various diseases, including Alzheimer's disease and mood disorders. We previously reported that the sporoderm-removed G. lucidum spore extract (RGLS) prevented learning and memory impairments in a rat model of sporadic Alzheimer's disease (sAD), but the effect of RGLS on depression-like behaviors in this model and its underlying molecular mechanisms of action remain unclear. Method: The present study investigated protective effects of RGLS against intracerebroventricular streptozotocin (ICV-STZ)-induced depression in a rat model of sAD and its underlying mechanism. Effects of RGLS on depression- and anxiety-like behaviors in ICV-STZ rats were assessed in the forced swim test, sucrose preference test, novelty-suppressed feeding test, and open field test. Results: Behavioral tests demonstrated that RGLS (360 and 720 mg/kg) significantly ameliorated ICV-STZ-induced depression- and anxiety-like behaviors. Immunofluorescence, Western blot and enzyme-linked immunosorbent assay results further demonstrated that ICV-STZ rats exhibited microglia activation and neuroinflammatory response in the medial prefrontal cortex (mPFC), and RGLS treatment reversed these changes, reflected by the normalization of morphological changes in microglia and the expression of NF-κB, NLRP3, ASC, caspase-1 and proinflammatory cytokines. Golgi staining revealed that treatment with RGLS increased the density of mushroom spines in neurons. This increase was associated with elevated expression of brain-derived neurotrophic protein in the mPFC. Discussion: In a rat model of ICV-STZ-induced sAD, RGLS exhibits antidepressant-like effects, the mechanism of which may be related to suppression of the inflammatory response modulated by the NF-κB/NLRP3 pathway and enhancement of synaptic plasticity in the mPFC.

[Reduced estimated glomerular filtration rate and proteinuria are associated with increased cardiovascular events rate in octogenarian population].

OBJECTIVE: To determine the relationship between estimated glomerular filtration rate (eGFR) and proteinuria with cardiovascular events in subjects aged 80 years or older. METHODS: Data for this retrospective prognostic study were drawn from the patient database for routine checkup in Beijing hospital between January 2001 to December 2001. Baseline eGFR and proteinuria were evaluated in 340 subjects [mean age: (85.6 ± 4.0) years]. eGFR was calculated using the modified abbreviated MDRD equations based on the Chinese chronic kidney disease patients. The subjects were divided into normal renal function group and reduced renal function group (eGFR <60 ml·min(-1)·1.73 m(-2)). The subjects were divided into subjects without proteinuria and subjects with proteinuria group. Cardiovascular events included cardiovascular death, nonfatal myocardial infarction, nonfatal stroke. RESULTS: The proportion of reduced renal function was 36.8% (125/340). The proportion of proteinuria was 10.3% (35/340). The proportion of reduced renal function or proteinuria was 41.8% (142/340). Follow-up time was 79 months (40-114 months). Cardiovascular events rate was significantly higher in reduced renal function group than in normal renal function group [37.6% (47/125) vs. 26.2% (55/210), P < 0.05 ] and in proteinuria group than in without proteinuria group [50.0% (17/34) vs. 28.2% (85/301), P < 0.01 ]. Cox multivariate analysis revealed that both eGFR (HR = 0.978, 95%CI:0.961-0.994, P < 0.05 ) and proteinuria (HR = 2.049, 95%CI:1.132-3.709, P < 0.05) were independent risk factors for cardiovascular events after adjusting for age, gender, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, uric acid, hypertension, coronary heart disease, diabetes mellitus. CONCLUSIONS: Reduced eGFR and presence of proteinuria are independent risk factors for cardiovascular event in subjects aged 80 years or older. eGFR and proteinuria can thus be used for cardiovascular event risk stratification in subjects aged 80 years or older.

Loss of ATOH1 in Pit Cell Drives Stemness and Progression of Gastric Adenocarcinoma by Activating AKT/mTOR Signaling through GAS1.

Gastric cancer stem cells (GCSCs) are self-renewing tumor cells that govern chemoresistance in gastric adenocarcinoma (GAC), whereas their regulatory mechanisms remain elusive. Here, the study aims to elucidate the role of ATOH1 in the maintenance of GCSCs. The preclinical model and GAC sample analysis indicate that ATOH1 deficiency is correlated with poor GAC prognosis and chemoresistance. ScRNA-seq reveals that ATOH1 is downregulated in the pit cells of GAC compared with those in paracarcinoma samples. Lineage tracing reveals that Atoh1 deletion strongly confers pit cell stemness. ATOH1 depletion significantly accelerates cancer stemness and chemoresistance in Tff1-CreERT2; Rosa26Tdtomato and Tff1-CreERT2; Apcfl/fl ; p53fl/fl (TcPP) mouse models and organoids. ATOH1 deficiency downregulates growth arrest-specific protein 1 (GAS1) by suppressing GAS1 promoter transcription. GAS1 forms a complex with RET, which inhibits Tyr1062 phosphorylation, and consequently activates the RET/AKT/mTOR signaling pathway by ATOH1 deficiency. Combining chemotherapy with drugs targeting AKT/mTOR signaling can overcome ATOH1 deficiency-induced chemoresistance. Moreover, it is confirmed that abnormal DNA hypermethylation induces ATOH1 deficiency. Taken together, the results demonstrate that ATOH1 loss promotes cancer stemness through the ATOH1/GAS1/RET/AKT/mTOR signaling pathway in GAC, thus providing a potential therapeutic strategy for AKT/mTOR inhibitors in GAC patients with ATOH1 deficiency.

Transcatheter Arterial Chemoembolisation Combined with Radiofrequency Ablation on Hepatocellular Carcinoma and Levels of Relevant Markers.

OBJECTIVE: To investigate the ablative effect and safety of trans-catheter arterial chemoembolisation (TACE) combined with radiofrequency ablation (RFA), and TACE alone for the treatment of hepatocellular carcinoma and compare the changes in the level of relevant serum inflammatory and tumor markers. STUDY DESIGN: Descriptive comparative study. PLACE AND DURATION OF STUDY: Department of Hepatobiliary Surgery, Affiliated Hospital of Hebei University, from January 2016 to June 2018. METHODOLOGY: Patients with hepatocellular carcinoma were randomly chosen and classified into combination group and TACE group, according to the treatment method. The 106 patients in the combination group were given RFA combined with TACE for treatment. The 112 patients in TACE group were given only TACE treatment. The objective response rate (ORR) and disease control rate (DCR) of short-term ablative effect, and adverse effect, serum inflammatory, and tumor markers' levels were compared for both groups before and one month after treatment. RESULTS: ORR and DCR of combination group were significantly higher than those of TACE group: 84 vs. 58%, and 99 vs. 80%, respectively (p=0.013). The differences in the frequency of adverse effects were statistically significant (p<0.05). After treatment, vascular endothelial growth factor (VEGF), alpha fetoprotein (AFP), and matrix metalloproteinase (MMP) of both groups declined significantly (p<0.05), that of the combination group significantly lower than those of TACE group (p<0.05). After treatment, tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and hypersensitivity C reactive protein (hsCRP) of both groups declined significantly (p<0.05), that of combination group significantly lower than those of TACE group (p<0.05). CONCLUSION: TACE combined with RFA has better ablative effect than pure TACE in the treatment of hepatocellular carcinoma. It can effectively reduce the level of tumor active factor and improve microinflammed state of the body.

Inflammatory cytokine-induced expression of MASTL is involved in hepatocarcinogenesis by regulating cell cycle progression.

Microtubule associated serine/threonine kinase-like (MASTL) is the functional mammalian ortholog of Greatwall kinase (Gwl), which was originally discovered in Drosophila. Gwl is an essential kinase for accurate chromosome condensation and mitotic progression, and inhibits protein phosphatase 2A (PP2A), which subsequently dephosphorylates the substrates of cyclin B1-cyclin-dependent kinase 1, leading to mitotic exit. Previous studies have indicated that MASTL has a critical function in the regulation of mitosis in HeLa and U2OS cell lines, though there is currently limited evidence for the involvement of MASTL in hepatocarcinogenesis. The results of the present study revealed that MASTL was inducible by the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), which promoted the proliferation and mitotic entry of human liver cancer cells. It was also determined that MASTL was significantly overexpressed in cancerous liver tissues compared with non-tumor liver tissues. Mechanistically, stimulation by IL-6 and TNF-α induced the trimethylation of histone H3 lysine 4 (H3K4Me3) at the MASTL promoter to facilitate chromatin accessibility. Additionally, H3K4Me3 was associated with the activation of nuclear factor-κB, which subsequently upregulated MASTL expression. These findings suggested that MASTL may have pivotal functions in the development of hepatocarcinoma, and that it may be a potential target for treatment.

Antibiotic resistance profiles and multidrug resistance patterns of Streptococcus pneumoniae in pediatrics: A multicenter retrospective study in mainland China.

Emergent resistance to antibiotics among Streptococcus pneumoniae isolates is a severe problem worldwide. Antibiotic resistance profiles for S pneumoniae isolates identified from pediatric patients in mainland China remains to be established.The clinical features, antimicrobial resistance, and multidrug resistance patterns of S pneumoniae were retrospectively analyzed at 10 children's hospitals in mainland China in 2016.Among the collected 6132 S pneumoniae isolates, pneumococcal diseases mainly occurred in children younger than 5 years old (85.1%). The resistance rate of S pneumoniae to clindamycin, erythromycin, tetracycline, and trimethoprim/sulfamethoxazole was 95.8%, 95.2%, 93.6%, and 66.7%, respectively. The resistance rates of S pneumoniae to penicillin were 86.9% and 1.4% in non-meningitis and meningitis isolates, while the proportions of ceftriaxone resistance were 8.2% and 18.1%, respectively. Pneumococcal conjugate vaccine was administered to only 4.1% of patients. Penicillin and ceftriaxone resistance, underling diseases, antibiotic resistant risk factors, and poor prognosis appeared more frequently in invasive pneumococcal diseases. The incidence of multidrug resistance (MDR) was 46.1% in patients with invasive pneumococcal disease which was more than in patients with non-invasive pneumococcal disease (18.3%). Patients with invasive pneumococcal disease usually have several MDR coexistence.S pneumoniae isolates showed high resistance to common antibiotics in mainland China. Penicillin and ceftriaxone resistance rate of invasive streptococcal pneumonia patients were significantly higher than that of non-invasive S pneumoniae patients. Alarmingly, 46.1% of invasive clinical isolates were multidrug resistant, so it is important to continued monitor the resistance of S pneumoniae when protein conjugate vaccine (PCV13) is coming in mainland China.

Acute myocardial ischemia up-regulates substance P in the retina of rats.

Evidence indicates that sympathetic nerves and substance P (SP) are involved in some physiological and pathophysiological changes and activities in retina. The aim of this study was to investigate whether SP participates in the stress reaction and possible involvement of adrenergic mechanisms in modulation of the changes of SP in the retina of the rats suffering from acute stress evoked by coronary artery occlusion (CAO). The changes of SP in retina were examined and analyzed within 6h of CAO using immunohistochemistry, in situ hybridization and EIA approaches. The effects of phentolamine, an antagonist of alpha1-adrenergic receptor, and esmolol, an antagonist of beta1-adrenergic receptor, on the changes of SP were investigated to examine the role of adrenergic mechanisms in modulation of expression of SP in the retina under the stressful condition. It was observed that SP was markedly up-regulated in the layers of ganglion cells, inner plexiform, inner nuclear and pigment epithelium within 6h of the CAO. Intravenous administration of the adrenergic antagonists attenuated the up-regulation of SP. The results may indicate that SP in retina was involved in the stress response induced by acute myocardial ischemia. Adrenergic mechanisms may modulate the process in the retina.

Volume overload in patients treated with continuous ambulatory peritoneal dialysis associated with reduced circadian blood pressure variation.

BACKGROUND: While a high blood pressure (BP) in patients receiving maintenance ambulatory peritoneal dialysis (CAPD) is associated with congestive heart failure and an increased mortality, the relevance of diurnal variations in BP is unknown. METHODS: In a prospective study, we enrolled 76 prevalent patients receiving stable CAPD (age 60.4 +/- 13.8 years; 37 males). BP was measured over 24 h using an automated device. We also performed routine clinical and biochemical measurements, as well as the Karnofsky index to evaluate physical activity. Volume status was assessed using bioimpedance analysis. RESULTS: 69 patients (with an average time on dialysis of 24.4 +/- 22.5 months) completed the study. Of these, 16 patients experienced a drop in BP >10% at night (dippers), while 53 patients did not (non-dippers). Comparing these two groups, dippers had significantly lower extracellular water (ECW) (13.8 +/- 2.1 vs. 15.9 +/- 3.3 l; p < 0.05), normalized extracellular water (nECW) (0.22 +/- 0.05 vs. 0.26 +/- 0.04 l/m; p < 0.05), and serum albumin (38.2 +/- 2.9 vs. 35.9 +/- 3.7 g/l; p < 0.05). Age, Karnofsky index, vintage, residual renal Kt/V and peritoneal Kt/V, total Kt/V, dose of antihypertensive drugs, mean systolic and diastolic BP did not significantly differ between these groups. Correlation analysis showed the coefficient of variation (CV) of BP positively correlated with E/T (r(2) = 0.292; p < 0.05), diabetic (r(2) = 0.267; p < 0.05), male (r(2) = 0.257; p < 0.05), nECW (r(2) = 0.278; p < 0.05) and ECW (r(2) = 0.249; p < 0.05) negatively correlated with albumin (r(2) = -0.280; p < 0.05). Furthermore, in a multivariate linear regression model, E/T, albumin and sex were independently associated with CV for BP. CONCLUSIONS: We show that reduced BP variation is common in CAPD patients and associated with volume overload and hypoalbuminemia. Furthermore, the relationship between nutritional, inflammatory status and dipping needs further studies.

Autoimmune responses induced by Helicobacter pylori improve the prognosis of gastric carcinoma.

Gastric carcinoma (GC) remains one of most serious malignant tumors worldwide, with Helicobacter pylori being the definite carcinogen. The H. pylori components, cytotoxin-associated gene A (CagA), vacuolating toxin A (VacA) and blood-group antigen-binding adhesin gene (BabA), can mimic and bind to specific receptors or surface molecules both on gastric epithelial cells and platelets, in which CagA and VacA may also be directly involved in loosening of tight junctions in monolayers of polarized gastric epithelial cells. It has been shown that a history of H. pylori infection is found in the majority of patients with GC, and that anti-CagA, anti-VacA and anti-BabA antibodies targeting both H. pylori components and host mimic molecules can be detected in them with increased levels. Patients with GC who are positive for H. pylori prospectively have a better outlook than those negative. The stimulation of mentioned autoantibodies in antigen processing and presentation and subsequent T-cell activation and proliferation improves host immune status. On the other hand, in an autoimmune response, autoantibodies can induce the cross-reaction against those localized or circulating GC cells, which are characterized by mimic or absorbed H. pylori antigens, and lead to the killing and even suppressing of metastasis of cancer cells. Therefore, we here hypothesize that autoimmune responses induced by H. pylori components may play a key role in improving the prognosis of patients with gastric carcinoma.

A non-peptide substance P antagonist down-regulates SP mRNA expression in human mononuclear phagocytes.

Substance P (SP), a potent modulator of neuroimmunoregulation, exerts its activity by binding to the neurokinin-1 receptor (NK-1R). The SP-NK-1R interaction is important in inflammation and viral infections, including HIV infection of human immune cells. We recently demonstrated that SP modulates HIV replication and that a non-peptide SP antagonist CP-96,345 inhibits HIV replication in human monocyte-derived macrophages (MDM) by affecting the SP-NK-1R interaction. In order to examine the effect of the SP antagonist on SP mRNA expression, MDM was incubated with or without CP-96,345 in the presence or absence of HIV infection. SP mRNA expression in these cells was then determined by real-time PCR technology. The effect of CP-96,345 on chemokine gene expression was also investigated by using a cDNA array assay. CP-96,345 down-regulated SP mRNA expression and antagonized exogenous SP-enhanced SP expression at the mRNA level, suggesting that SP autocrine regulation was interrupted by CP-96,345. CP-96,345 inhibited HIV replication in MDM, associated with down-regulated SP mRNA expression in comparison to HIV infection controls. In parallel with down-regulated SP and CCR5 mRNA expression, cDNA array assays indicated that CP-96,345 treatment also inhibited IL-8 gene expression, while enhancing expression of fractalkine and monocyte chemotactic protein-3 (MCP-3). Since SP plays an important role in inflammation and viral infections, these studies may have potential applications for therapeutic intervention of inflammation and viral infection of immune cells.

A visual DNA chip for simultaneous detection of hepatitis B virus, hepatitis C virus and human immunodeficiency virus type-1.

For the simultaneously visual detection of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type-1 (HIV-1), a qualitative DNA chip method, combining multiplex and nested polymerase chain reaction (PCR) with arrayed anchored primer PCR and a biotin-avidin alkaline phosphatase (Av-AP) indicator system, was developed. After pretreatment of infected blood samples and reverse transcription of the RNA virus genome, PCR was performed in a single tube by using the outer primer pairs. Second round nested multiplex PCR was performed on the DNA chip, on which the primers array had already been prepared. During the arrayed anchored multiplex PCR, 5[N-(N-biotinylaminocaproyl)-epsilon-3-aminoallyl]-2-deoxy-uridine-5-triphosphate (biotin-11-dUTP) was incorporated into the extended DNA chains in order to bind avidin alkaline phosphatase via avidin and biotin. To produce purple precipitates on the chips, the enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) was used in conjunction with the enhancer, nitro blue tetrazolium (NBT). Blood samples containing the three viruses were tested using this DNA chip and about 1 pg of specific viral DNA fragments were detected on the chip wells after nested PCR.

Real-time RT-PCR for quantitation of hepatitis C virus RNA.

A newly developed real-time RT-polymerase chain reaction assay for quantitation of hepatitis C virus (HCV) RNA in human plasma and serum was applied. A pair of primers and a probe (molecular beacon) were designed that are specific for the recognition of a highly conservative 5'-non-coding region (5'-NCR) in HCV genome. HCV real-time RT-PCR assay had a sensitivity of 1000 RNA copies per reaction, with a dynamic range of detection between 10(3) and 10(7) RNA copies. The coefficient variation of threshold cycle (Ct) values in intra- and inter-runs were less than 1.37 and 4.66%, respectively. The real-time RT-PCR assay on the HCV sero-positive samples yielded reproducible data, with less than 2.09% of the inter-assay variation. In order to determine its potential for clinical diagnosis, real-time RT-PCR was used to examine the HCV RNA levels in plasma from sero-positive and negative subjects, showing that the assay is highly sensitive and has specificity of 100%. It was demonstrated that the real-time RT-PCR was able to amplify HCV RNA in reference sera with seven genotypes (1A, 1B, 2B, 3A, 4, 5A and 6A) that include six major HCV genotypes circulated in the world. Since HCV is a major pathogen of post-transfusion and community-transmitted non-A, non-B hepatitis, this assay has a broad application for basic and clinical investigations.

[Orexin A and neuropeptide Y levels in plasma and hypothalamus of rats with chronic renal failure].

OBJECTIVE: To explore the changes of orexin A and neuropeptide (NPY) in plasma and hypothalamus of rats with chronic renal failure (CRF). METHODS: 41 male Wister rats weighing 200 approximately 250 g were randomly divided into three groups: normal group, sham operation group, and CRF group (with the right kidney and 2/3 of the left kidney resected). A certain number of rats were decapitated 4, 8,and 12 weeks after respectively. Their hypothalami were removed and blood collected. Radioimmunoassay was used to measure the levels of orexin A and NPY in hypothalamus and plasma. Automatic biochemical analyzer was used to measure the serum creatinine. RESULTS: The serum creatinine level of CRF rats was both significantly higher than those of the sham operation rats at week 8 and week 12, respectively. The plasma orexin A level of CRF rats at week 12 was 264 pg/ml +/- 62 pg/ml, significantly higher than that of sham operation group (183 pg/ml +/- 56pg/ml, P = 0.039). The hypothalamus orexin A level of CRF rats were 10.5 fmol/mg +/- 2.7 fmol/mg wet weight at week 12, significantly lower than that of sham operation rats (17.4 fmol/mg +/- 3.9 fmol/mg wet weight, P = 0.023). The plasma NPY levels of CRF rats at week 8 and week 12 were significantly higher than those of the sham operation rats (7.1 pmol/ml +/- 1.7 pmol/ml vs 5.0 pmol/ml +/- 0.5 pmol/ml, P = 0.01; and 7.9 pmol/ml +/- 1.1 pmol/ml vs 4.8 pmol/ml +/- 1.1 pmol/ml, P = 0.0008). The hypothalamus NPY level of CRF rats at week 12 were 70 fmol/mg +/- 23 fmol/mg wet weight, significantly lower than that of the sham operation rats (113 fmol/mg +/- 31 fmol/mg wet weight, P = 0.03). CONCLUSION: Loss of renal function may diminish the excretion of orexin A and neuropeptide. The lowering of hypothalamus orexin A and neuropeptide Y levels may be one of the causes inducing anorexia in CRF.

[Effect of BMP-7 on the transdifferentiation of cultured human tubular epithelial cell induced by TGF-beta1].

OBJECTIVE: To observe the effect of bone morphogenetic protein-7 (BMP-7) on the transdifferentiation of cultured human tubular epithelial cell (HKC) induced by TGF-beta1 and to elucidate its possible mechanism. METHODS: The cultured HKC cells were divided into 5 groups: serum-free group (negative control); single TGF-beta1 treated group (positive control); single BMP-7 treated group; combined TGF-beta1 and BMP-7 treated group; and BMP-7 pre-treated group. Expression of keratin of HKC cells was assessed by indirect enzyme immunohistochemistry (IEI), expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin by immunohistological method, percentage of alpha-SMA positive HKC cells by flow cytometry, and mRNA expression of alpha-SMA, TGF-beta1, and TGF-beta type II receptor by reverse transcription PCR. RESULTS: The expression of alpha-SMA and the percentage of alpha-SMA positive HKC cells markedly increased after having been treated by TGF-beta1 while the expression of E-cadherin and keratin decreased. In the group pre-treated with BMP-7 (50 ng/ml) and then added with TGF-beta1 (8 ng/ml), expression of alpha-SMA was significantly lower than in the positive control group, while expression of E-cadherin and keratin significantly higher than in the positive control group. Measurement of the percentage of alpha-SMA positive HKC found significant deference between the combined TGF-beta1 and BMP-7 treated group and the positive control group (9.7% vs 19.8%; 5.8% vs 19.8%; P < 0.05). Significant difference existed between the BMP-7 (50 ng/ml) pre-treated group and the positive control group (8.7% vs 19.8%, P < 0.05). mRNA expression of alpha-SMA was measured by RT-PCR and the results showed that it significantly decreased in the group treated or pre-treated with BMP-7 (50 ng/ml) (15% and 12% of the results in the positive control group, respectively). The mRNA expression levels of both TGF-beta1 and its type II receptor significantly decreased (28% and 19%; 47% and 36%, compared with the positive control group, respectively). CONCLUSION: Transdifferentiation of cultured renal epithelial cell induced by TGF-beta1 can be inhibittd by certain levels of BMP-7, cultured together with TGF-beta1 or pretreated. BMP-7 can prevent and inhibit the mRNA expression of TGF-beta1 and its type II receptor, which may be an important mechanism by which BMP-7 inhibit the transdifferentiation of renal tubular epithelial cell.

Restoration of endogenous substance P is associated with inhibition of apoptosis of retinal cells in diabetic rats.

This study was designed to investigate the alterations of substance P (SP) and its correlation with apoptosis of the retinal neurons in diabetic rats. The study was carried out with diabetic rats induced by streptozotocin. Changes of SP and its mRNA were examined using enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. The effect of restoration of SP level by capsaicin (20mg/kg, s.c.) on the apoptosis of the retinal cells was studied. The apoptosis was evaluated by change of ratio of the apoptotic cells and caspase-3 activity in the retina. It was found that increase in apoptosis of retinal cells, by 3.5 fold of control, was accompanied by reduction of SP, by 28% in protein and 32% in the mRNA in the retina at 10 weeks of induction of diabetes, compared to the controls. Capsaicin significantly elevated endogenous SP, by 29% in the mRNA and 17% in protein in the retina, with marked inhibition of the apoptosis and the activity of caspase-3 in the diabetic rats. Induction of diabetes leads to the increase of cell apoptosis and the decrease of SP in the retina. The reduction of the endogenous SP and the increase of the cell apoptosis in the retina of the diabetic rats were reversed by pretreatment with capsaicin. Restoration of SP in the retina may be a novel option for prevention of the retinal injury during development of diabetes.