Endopeptidase-24.11, a Cell-Surface Peptidace of Central Nervous System Neurons, Is Expressed by Schwann Cells in the Pig Peripheral Nervous System

Endopeptidase-24.11 is a 90-kDa surface glycoprotein with the ability to hydrolyze a variety of biologically active peptides. Interest in this enzyme is based on the consensus that it may play a role in the termination of peptide signals in the central nervous system. In the present study, we have investigated the distribution of endopeptidase-24.11 in two nerves of the peripheral nervous system of newborn pigs: the sciatic, composed of a mixture of myelinated and nonmyelinated axons, and cervical sympathetic trunk in which greater than 99% of the axons are nonmyelinated. The endopeptidase was monitored enzymatically, as well as by immunoblotting and immunocytochemistry using mono- and polyclonal anti-endopeptidase antibodies. Endopeptidase-24.11 was detected in both the sciatic nerve and the cervical sympathetic trunk. Membrane preparations from sciatic nerve hydrolyzed 125I-insulin B-chain, and more than 50% of the activity was inhibited by phosphoramidon with an IC50 concentration of 3.2 nM. Moreover, a 90-kDa polypeptide was detected by immunoblotting of sciatic nerve membranes. The type of cells expressing the endopeptidase was determined by immunohistochemistry. In teased nerve preparations, these cells were identified morphologically as myelin- and non-myelin-forming Schwann cells. Endopeptidase-24.11 was also expressed by cultured Schwann cells from sciatic nerve and cervical sympathetic trunk maintained for 3 h in vitro. The presence of endopeptidase-24.11 on the Schwann cell surface raises the possibility of a potential role for the enzyme in nerve development and/or regeneration.     

Economic Recession and Emergence of an HIV-1 Outbreak among Drug Injectors in Athens Metropolitan Area: A Longitudinal Study

Background

During 2011, a dramatic increase (1600%) of reported HIV-1 infections among injecting drug users (IDUs) was noted in Athens, Greece. We herein assess the potential causal pathways associated with this outbreak.

Methods

Our study employed high resolution HIV-1 phylogenetic and phylogeographic analyses. We examined also longitudinal data of ecological variables such as the annual growth of gross domestic product (GDP) of Greece in association with HIV-1 and HCV sentinel prevalence in IDUs, unemployment and homelessness rates and HIV transmission networks in Athens IDUs before and during economic recession (2008–2012).

Results

IDU isolates sampled in 2011 and 2012 suggested transmission networks in 94.6% and 92.7% of the cases in striking contrast with the sporadic networking (5%) during 1998–2009. The geographic origin of most HIV-1 isolates was consistent with the recently documented migratory waves in Greece. The decline in GDP was inversely correlated with annual prevalence rates of HIV and HCV and with unemployment and homelessness rates in IDUs (all p<0.001). The slope of anti-HCV prevalence in the sentinel populations of IDUs and in “new” drug injectors was found 120 and 1.9-fold (p = 0.007, p = 0.08 respectively) higher in 2008–2012 (economic recession) compared with 2002–2006. The median (25th, 75th) size of transmission networks were 34 (12, 58) and 2 (2, 2) (p = 0.057) in 2008–2012 and 1998–2007, respectively. The coverage of harm reduction services was low throughout the study period.

Conclusions

Scaling-up harm reduction services and addressing social and structural factors related to the current economic crisis should be urgently considered in environments where HIV-1 outbreaks may occur.     

Proteomics studies of childhood pilocytic astrocytoma

Childhood pilocytic astrocytoma is the most frequent brain tumor affecting children. Proteomics analysis is currently considered a powerful tool for global evaluation of protein expression and has been widely applied in the field of cancer research. In the present study, a series of proteomics, genomics, and bioinformatics approaches were employed to identify, classify and characterize the proteome content of low-grade brain tumors as it appears in early childhood. Through bioinformatics database construction, protein profiles generated from pathological tissue samples were compared against profiles of normal brain tissues. Additionally, experiments of comparative genomic hybridization arrays were employed to monitor for genetic aberrations and sustain the interpretation and evaluation of the proteomic data. The current study confirms the dominance of MAPK pathway for the childhood pilocytic astrocytoma occurrence and novel findings regarding the ERK-2 expression are reported.     

Microbiological quality of grey-mullet roe

The Greek grey-mullet roe is produced from the fully developed gonads of the female mullet (Mugil cephalus) couth in lagoons during their reproductive migration. The traditional processing method of the roe includes, air drying, salting, shape formation and covering with multiple layers of natural beeswax for preservation and distribution. Fish Roe brands have been a staple in local diet and is increasingly becoming popular in the international market. As a ready-to-eat food it’s microbial quality should be of concern for the protection of consumers health. In this study, 48 samples of fish roe, just before waxing, were collected from various local processors for microbiological examination by using selective media and incubated under aerobic and anaerobic conditions. The identification of the bacteria was carried out according to the Bergey’s manual. Microscopic examination of Gram stained cells, catalase, oxidase and biochemical tests were performed when necessary to further identify.V. parahaemolyticus, Vibrio spp., Salmonella spp., and Aeromonas hydrophila were detected in one sample (2%). Shigella spp., and Flavobacterium spp. in two samples (4%), Clotriduim perfringens (vegetative forms), E. coli, and spores of Bacillus spp., were detected in three samples (6%), Staphylococcus aureus in four samples (8%). Various Micrococcus spp., and spores of C. perfringens in 16% and 35% of the samples respectively. From the Listeria genus, only the species Listeria innocua, Listeria welshimeri, Listeria seeligeri Listeria ivanovii and Listeria grayi were recovered from 2 to 10% of the samples.Microbiological analyses revealed the presence of a small number of pathogens in grey-mullet roe samples which are in accordance with the findings of similar studies. Traditional processing of the fish roe, seems inadequate to ensure the food safety and even waxing isn’t expected to fully protect them against facultative anaerobes with salt tolerance. Therefore, additional measures should be taken during processing and marketing of fish roe to minimize potential health risks for the consumers.     

Antibacterial activity of different honeys against pathogenic bacteria

To study the antimicrobial activity of honey, 60 samples of various botanical origin were evaluated for their antimicrobial activities against 16 clinical pathogens and their respective reference strains. The microbiological quality of honeys and the antibiotic susceptibility of the various isolates were also examined. The bioassay applied for determining the antimicrobial effect employs the well-agar diffusion method and the estimation of minimum active dilution which produces a 1 mm diameter inhibition zone. All honey samples, despite their origin (coniferous, citrus, thyme or polyfloral), showed antibacterial activity against the pathogenic and their respective reference strains at variable levels. Coniferous and thyme honeys showed the highest activity with an average minimum dilution of 17.4 and 19.2% (w/v) followed by citrus and polyfloral honeys with 20.8 and 23.8% respectively. Clinical isolates of Staphylococcus aureus subsp. aureus, Escherichia coli, Salmonella enterica subsp. Enterica, Streptococcus pyogenes, Bacillus cereus and Bacillus subtilis were proven to be up to 60% more resistant than their equal reference strains thus emphasizing the variability in the antibacterial effect of honey and the need for further research.     

Isolation of microbial pathogens of subclinical mastitis from raw sheep's milk of Epirus (Greece) and their role in its hygiene

The natural raw milk microflora is a factor that expresses its sensorial characteristics. The microbial charge into the mammary gland of healthy animal is low and the application of right and healthy conditions during milking and cheese making procedure, prevents from contaminating as well as maintains the natural microflora in order to lend the particular characteristics of milk.The purpose of the present project was the study of the Total Viable Count (T.V.C.) and the count of total psychrotropic bacteria of raw sheep milk from Boutsiko and Karamaniko breeds, collected from healthy animals, as well as the isolation, identification and enumeration of pathogenic bacteria related with the hygiene and the quality of raw sheep milk (with a particular interest in bacteria that may cause human infection).During the experiment we examined two hundred forty (240) samples of raw sheep milk. In these samples a) Staphylococcus aureus, Salmonella sp., Escherichia coli, Clostridium perfringens (vegetative cells and spores) and Bacillus sp. were isolated and identified b) the Total Viable Count and the total number of psychrotropic bacteria were also specified. The sampling, the preparation of samples and decimal dilutions were based on international methods. The Total viable count was determined using the standard methods of the American Public Health Association, 2002. The total number of psychrotropic bacteria was determined using APHA 1976, 1978 rules. The identification of the bacteria was carried out according to the Bergey’s manual. Microscopic examination of Gram stained cells, catalase, oxidase and biochemical tests were performed when necessary to further identify.From the 240 milk samples tested, only 5% were E. coli positive, with mean counts ranged from 2 × 10³ to 2.4 × 10⁴ cfu/ml. S. aureus was isolated from 24% of the samples and the mean count per ml was ranged from <10 to 3.4 × 10². Meanwhile, Bacillus spp. was also detected in 29% samples. Vegetative forms and spores of C. perfringens were detected in 13% and 63% of the samples respectively. However, microbiological analyses revealed the presence of a small number of selected pathogens in milk samples such as Salmonella, which was only detected in 5% of the samples. Listeria sp., Pseudomonas sp. and Vibrio cholerae were never found.From the experimental results, the Total Viable Count from raw sheep milk samples, fulfils the microbiological criteria of EU Legislation in a percentage of approximately 97%.     

Specific interference of urokinase-type plasminogen activator receptor and matrix metalloproteinase-9 gene expression induced by double-stranded RNA results in decreased invasion, tumor growth, and angiogenesis in gliomas

We have previously demonstrated the effectiveness of adenovirus-mediated expression of antisense urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9) in inhibiting tumor invasion in vitro and ex vivo. However, the therapeutic effect of the adenovirus-mediated antisense approach was shown to be transient and required potentially toxic, high viral doses. In contrast, RNA interference (RNAi)-mediated gene targeting may be superior to the traditional antisense approach, because the target mRNA is completely degraded and the molar ratio of siRNA required to degrade the target mRNA is very low. Here, we have examined the siRNA-mediated target RNA degradation of uPAR and MMP-9 in human glioma cell lines. Using RNAi directed toward uPAR and MMP-9, we achieved specific inhibition of uPAR and MMP-9. This bicistronic construct (pUM) inhibited the formation of capillary-like structures in both in vitro and in vivo models of angiogenesis. We demonstrated that blocking the expression of these genes results in significant inhibition of glioma tumor invasion in Matrigel and spheroid invasion assay models. RNAi for uPAR and MMP-9 inhibited cell proliferation, and significantly reduced the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules when compared with parental and empty vector/scrambled vector-transfected SNB19 cells. Furthermore, using RNAi to simultaneously target two proteases resulted in total regression of pre-established intracerebral tumor growth. Our results provide evidence that the use of hairpin siRNA expression vectors for uPAR and MMP-9 may provide an effective tool for cancer therapy.