Intervention effects of Ganoderma lucidum spores on epileptiform discharge hippocampal neurons and expression of Neurotrophin-4 and N-Cadherin

Epilepsy can cause cerebral transient dysfunctions. Ganoderma lucidum spores (GLS), a traditional Chinese medicinal herb, has shown some antiepileptic effects in our previous studies. This was the first study of the effects of GLS on cultured primary hippocampal neurons, treated with Mg2+ free medium. This in vitro model of epileptiform discharge hippocampal neurons allowed us to investigate the anti-epileptic effects and mechanism of GLS activity. Primary hippocampal neurons from <1 day old rats were cultured and their morphologies observed under fluorescence microscope. Neurons were confirmed by immunofluorescent staining of neuron specific enolase (NSE). Sterile method for GLS generation was investigated and serial dilutions of GLS were used to test the maximum non-toxic concentration of GLS on hippocampal neurons. The optimized concentration of GLS of 0.122 mg/ml was identified and used for subsequent analysis. Using the in vitro model, hippocampal neurons were divided into 4 groups for subsequent treatment i) control, ii) model (incubated with Mg2+ free medium for 3 hours), iii) GLS group I (incubated with Mg2+ free medium containing GLS for 3 hours and replaced with normal medium and incubated for 6 hours) and iv) GLS group II (neurons incubated with Mg2+ free medium for 3 hours then replaced with a normal medium containing GLS for 6 hours). Neurotrophin-4 and N-Cadherin protein expression were detected using Western blot. The results showed that the number of normal hippocampal neurons increased and the morphologies of hippocampal neurons were well preserved after GLS treatment. Furthermore, the expression of neurotrophin-4 was significantly increased while the expression of N-Cadherin was decreased in the GLS treated group compared with the model group. This data indicates that GLS may protect hippocampal neurons by promoting neurotrophin-4 expression and inhibiting N-Cadherin expression

Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50)) ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50) values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50) = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells

Multimodality management and outcomes of brain arterio-venous malformations (AVMs) in children: personal experience and review of the literature, with specific emphasis on age at first AVM bleed.

PURPOSE: The purpose of this paper is to study the presentation and analyse the results of multimodality treatment of brain arterio-venous malformations (AVMs) in children at our centre and review age at first AVM rupture in the literature. METHODS: Of 52 patients aged <18 years, 47 with brain AVMs (27 males and 20 females) aged 4-17 years (mean 12.2) were retrospectively reviewed. PubMed search revealed five additional studies including 267 patients where the prevalence of age-related AVMs rupture was analysed. RESULTS: In our study, 37 patients had bled, 9 were symptomatic without haemorrhage and 1 was incidental. Spetzler-Martin score distribution was 5 cases grade I, 18 grade II, 21 grade III and 3 grade IV. Appropriate imaging was performed, either CT/MRI angiogram only (in emergency cases) or catheter angiogram, prior to definitive treatment. There were 40 supratentorial and 7 infratentorial AVMs. Twenty-nine patients had microsurgery alone and 9 patients were treated by radiosurgery only. Three patients were embolised, all followed by radiosurgery, with one requiring surgery too, while 4 patients had combined surgery and radiosurgery. One patient is awaiting radiosurgery while another was not treated. Good outcomes, classified as modified Rankin score (mRS) 0-2 improved significantly after intervention to 89.4% from 38.3% pre-treatment (p value <0.0001). Angiography confirmed 96.6% obliteration after first planned operation. Repeat cerebral angiogram around age 18 was negative in all previously cured patients. Reviewing the literature, 82.0% (95% CI = [77-87]; N = 267) of children diagnosed with brain AVMs (mean age 11.4 ± 0.4) presented with a bleed in the last 22 years. Males significantly outnumbered females (136 vs 84) (p < 0.001). Ninety-five patients underwent surgical intervention alone when compared to other treatment modalities (p < 0.001). CONCLUSIONS: Microsurgical excision of surgically accessible intracranial AVMs remains the primary treatment option with very good outcomes. A significant number of patients' AVMs ruptured around puberty; therefore, understanding the pathophysiology of AVM instability at this age may aid future therapy