Cloning and characterization of the trout perforin

The pore-forming protein, perforin is one of the effectors of cell-mediated killing. A perforin cDNA clone was isolated from rainbow trout (Oncorhynchus mykiss) after screening of a spleen cDNA library. The full-length cDNA is 2070 bp in size, encoding for a polypeptide of 589 amino acids. The predicted amino acid sequence of the trout perforin is 64, 58 and 40% identical to those of Japanese flounder, zebrafish and human perforins, respectively. Although its membrane attack complex/perforin (MACPF) domain is conserved, trout perforin shows low homology to human and trout terminal complement components (C6, C7, C8 and C9), ranging from 19 to 26% identity. Expression analysis reveals that the trout perforin gene is expressed in the blood, brain, heart, kidney, intestine and spleen. Phylogenetic analysis of proteins which belong to the MACPF superfamily clusters the trout perforin in the same group with other known perforins.     

Solar energy conversion efficiencies in photosynthesis: Minimizing the chlorophyll antennae to maximize efficiency

The theoretical maxima of solar energy conversion efficiencies and productivities in oxygenic photosynthesis are evaluated. These are contrasted with actual measurements in a variety of photosynthetic organisms, including green microalgae, cyanobacteria, C4 and C3 plants. Minimizing, or truncating, the chlorophyll antenna size of the photosystems can improve photosynthetic solar energy conversion efficiency and productivity up to 3-fold. Generation of truncated light-harvesting chlorophyll antenna size (tla) strains, in all classes of photosynthetic organisms would help to alleviate excess absorption of sunlight and the ensuing wasteful dissipation of excitation energy, and to maximize solar-to-product energy conversion efficiency and photosynthetic productivity in high-density mass cultivations. The tla concept may find application in the commercial exploitation of microalgae and plants for the generation of biomass, biofuels, chemical feedstocks, as well as nutraceuticals and pharmaceuticals.     

Embryoid bodies from mouse stem cells express oxytocin receptor, Oct-4 and DAZL

Oxytocin is a nine amino acid peptide involved in a wide spectrum of physiological functions; predominantly those concerning reproduction and differentiation are of interest. Oxytocin receptors are expressed at early developmental stages of mammals, suggesting that oxytocin might be involved in the determination of the germ stem cell line, at the very early stages of mammalian development. In this respect, the proximate aim of the present study was to confirm and further analyze the existence of oxytocin receptors at a very early level of cell commitment, that is, the determination of germ cells derived from embryoid bodies. To achieve our purpose we have cultured mouse embryonic stem cells under conditions inducing formation of embryoid bodies. In this work, ES cells were allowed to aggregate in a novel medium, “Stefanidis medium” from day 0 to day 14 until formed EBs. RNA was isolated from EBs and using RT-PCR we showed that EBs expressed Oct-4, OTR, OT, and DAZL. To demonstrate simultaneous expression immunocytochemistry was preformed, in which EBs showed strong immunoreactivity for both, OTR and DAZL molecular markers. We found that 35% of the cells displayed OTR, using flow cytometry. In addition, this novel medium showed to increase OTR mRNA. We propose, that at least in murine induced embryoid bodies there is simultaneous expression of oxytocin receptors and germ cell markers (DAZL) in many cells (expressing Oct-4). We thus conclude that, the oxytocin might indeed be a molecule playing a leading role in germ cell determination.     

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Monoclonal anti‐HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L ‐Phe/β‐Ala bi‐substituted 1,3,5‐triazine (Trz) scaffold (β‐Ala‐Trz‐L ‐Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10‐binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K_D) of 0.41 ± 0.05 µM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60–80%). Analysis of the antibody preparation by SDS‐PAGE, enzyme‐linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids. Copyright © 2009 John Wiley & Sons, Ltd.     

“Density equilibrium” method for the quantitative and rapid in situ determination of lipid,hydrocarbon, or biopolymer content in microorganisms

The work provides a simple method, based on a direct density equilibrium measurement, for the rapid in situ estimation of total lipid, hydrocarbon or biopolymer content in a variety of prokaryotic and eukaryotic samples. The method can be readily applied to live microalgae and photosynthetic bacteria, single‐celled or colonial microorganisms, as well as cellular fractions and isolated subcellular compartments or components. In this approach, the absolute lipid, hydrocarbon, or biopolymer content of the cells can be readily calculated. This method is especially useful for tracking the oil or polymer content of strains of microalgae and other microorganisms, whose lipid, hydrocarbon or biopolymer content may change with cultivation conditions and/or time, as the case would be in microorganism lipid‐induction industrial processes. The method is also useful for the direct in situ measurement of storage polymer accumulation in live cells, such as starch in microalgae and polyhydroxybutyrate, or other polyhydroxyalkanoates, in photosynthetic and non‐photosynthetic bacteria. Biotechnol. Bioeng. 2009;102: 1406–1415. © 2008 Wiley Periodicals, Inc.     

Adiposity,blood pressure,and carotid intima‐media thickness in greek adolescents

Objective:

In children and adolescents with cardiovascular risk factors, the assessment of subclinical target‐organ damage is of paramount importance. This study investigated factors associated with carotid intima‐media thickness (cIMT) in adolescents.

Design and Methods:

A cross‐sectional study was performed in 448 apparently healthy adolescents recruited from schools (mean age 14 ± 2.2 years, 211 boys), which involved cIMT measurements (common carotid artery) and assessment of lipid profile, glucose, and blood pressure (BP).

Results:

The prevalence of overweight/obesity was 28.1%/12.7% and of BP ≥95th percentile 19.6%. Left cIMT was correlated with age (r = 0.10), waist circumference (WC) (0.15), and BP (0.21/0.13, systolic/diastolic) (all P < 0.05). Right cIMT was correlated with waist to hip ratio (WHR) (0.10), whereas the mean of left and right cIMT was correlated with WC (0.12), WHR (0.12), and systolic BP (0.14) (all P < 0.05). After the age of 13 years, boys tended to have higher cIMT than girls, which was significant in the 13‐15 years subgroup (P < 0.05). In stepwise multivariate analysis (independent variables: age, gender, WC, WHR, body mass index z‐score, lipid parameters, glucose, BP), left cIMT was independently associated with systolic BP; right cIMT with WHR; mean left and right cIMT with WC. Adolescents with BP ≥90th percentile had higher left cIMT than those <90th percentile (0.63 ± 0.09 vs. 0.61 ± 0.09 mm respectively, P < 0.05).

Conclusion:

Central adiposity and systolic BP appear to be independently associated with increased cIMT values in apparently healthy adolescents. Left side cIMT appears to be superior to right side measurements in terms of association with cardiovascular risk factors.     

Trends of Mortality due to Septicemia in Greece: An 8-Year Analysis

Background

Infectious diseases are among the major causes of death worldwide. We evaluated the trends of mortality due to septicemia in Greece and compared it with mortality due to other infections.

Methods

Data on mortality stratified by cause of death during 2003–2010 was obtained from the Hellenic Statistical Authority. Deaths caused by infectious diseases were grouped by site of infection and analyzed using SPSS 17.0 software.

Results

45,451 deaths due to infections were recorded in Greece during the 8-year period of time, among which 12.2% were due to septicemia, 69.7% pneumonia, 1.5% pulmonary tuberculosis, 0.2% influenza, 0.5% other infections of the respiratory tract, 7.9% intra-abdominal infections (IAIs), 2.5% urinary tract infections (UTIs), 2.2% endocarditis or pericarditis or myocarditis, 1.6% hepatitis, 1% infections of the central nervous system, and 0.7% other infections. A percentage of 99.4% of deaths due to septicemia were caused by bacteria that were not reported on the death certificate (noted as indeterminate septicemia). More deaths due to indeterminate septicemia were observed during 2007–2010 compared to 2003–2006 (3,558 versus 1,966; p<0.05).

Conclusion

Despite the limitations related to the quality of death certificates, this study shows that the mortality rate due to septicemia has almost doubled after 2007 in Greece. Proportionally, septicemia accounted for a greater increase in the mortality rate within the infectious causes of death for the same period of time. The emergence of resistance could partially explain this alarming phenomenon. Therefore, stricter infection control measures should be urgently applied in all Greek healthcare facilities.     

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.