Impact of plasmid architecture on stability and yEGFP3 reporter gene expression in a set of isomeric multicopy vectors in yeast

Multicopy episomal plasmids in yeast, used whenever elevated levels of foreign or homologous gene expression are necessary, are known to be less stable compared to the endogenous 2-μm plasmid they are based on, at least without selective pressure. Considering that rich medium favors growth rate and, simultaneously, is less expensive than selective medium, enhancing stability in non-selective medium is extremely desirable. In this study, we changed the architecture of a multicopy model expression plasmid, creating six isoforms (same size, same DNA content but different positions and orientations of the expression block) and studied mitotic stability, copy number, as well as reporter yEGFP3 expression between isoforms. With one isoform being significantly more stable than the others and another one exhibiting elevated plasmid copy numbers in rich medium, we show that consideration of the arrangement of the plasmid elements might be crucial for productivity employing Saccharomyces cerevisiae as a host. We strongly believe that the ideal architecture has to be assessed for each case and assembly strategy has to begin by evaluating the stability of the vector backbone before insertion of the desired gene. For the plasmid set studied, yEGFP3 reporter production depends more on mitotic stability than on elevated plasmid copy numbers in a small number of cells retaining the plasmid under non-selective conditions.

     

Enlargement of Cerebral Ventricles as an Early Indicator of Encephalomyelitis

Inflammatory disorders of the central nervous system such as multiple sclerosis and acute disseminated encephalomyelitis involve an invasion of immune cells that ultimately leads to white matter demyelination, neurodegeneration and development of neurological symptoms. A clinical diagnosis is often made when neurodegenerative processes are already ongoing. In an attempt to seek early indicators of disease, we studied the temporal and spatial distribution of brain modifications in experimental autoimmune encephalomyelitis (EAE). In a thorough magnetic resonance imaging study performed with EAE mice, we observed significant enlargement of the ventricles prior to disease clinical manifestation and an increase in free water content within the cerebrospinal fluid as demonstrated by changes in T₂ relaxation times. The increase in ventricle size was seen in the lateral, third and fourth ventricles. In some EAE mice the ventricle size started returning to normal values during disease remission. In parallel to this macroscopic phenomenon, we studied the temporal evolution of microscopic lesions commonly observed in the cerebellum also starting prior to disease onset. Our data suggest that changes in ventricle size during the early stages of brain inflammation could be an early indicator of the events preceding neurological disease and warrant further exploration in preclinical and clinical studies.     

Development of Novel Chitosan Microcapsules for Pulmonary Delivery of Dapsone: Characterization,Aerosol Performance,and In Vivo Toxicity Evaluation

Pneumocystis carinii pneumonia (PCP) is a major opportunistic infection that affects patients with human immunodeficiency virus. Although orally administered dapsone leads to high hepatic metabolism, decreasing the therapeutic index and causing severe side effects, this drug is an effective alternative for the treatment of PCP. In this context, microencapsulation for pulmonary administration can offer an alternative to increase the bioavailability of dapsone, reducing its adverse effects. The aim of this work was to develop novel dapsone-loaded chitosan microcapsules intended for deep-lung aerosolized drug delivery. The geometric particle size (D_4,3) was approximately 7 μm, the calculated aerodynamic diameter (d_aero) was approximately 4.5 μm, and the mass median aerodynamic diameter from an Andersen cascade impactor was 4.7 μm. The in vitro dissolution profile showed an efficient dapsone encapsulation, demonstrating the sustained release of the drug. The in vitro deposition (measured by the Andersen cascade impactor) showed an adequate distribution and a high fine particles fraction (FPF = 50%). Scanning electron microscopy of the pulmonary tissues demonstrated an adequate deposition of these particles in the deepest part of the lung. An in vivo toxicity experiment showed the low toxicity of the drug-loaded microcapsules, indicating a protective effect of the microencapsulation process when the particles are microencapsulated. In conclusion, the pulmonary administration of the novel dapsone-loaded microcapsules could be a promising alternative for PCP treatment.KEY WORDS: dapsone, dry powders inhalers, in vivo toxicity, microparticles, pulmonary drug delivery     

A proteomic view of the facultatively chemolithoautotrophic lifestyle of Ralstonia eutropha H16

Ralstonia eutropha H16 is an H₂‐oxidizing, facultative chemolithoautotroph. Using 2‐DE in conjunction with peptide mass spectrometry we have cataloged the soluble proteins of this bacterium during growth on different substrates: (i) H₂ and CO₂, (ii) succinate and (iii) glycerol. The first and second conditions represent purely lithoautotrophic and purely organoheterotrophic nutrition, respectively. The third growth regime permits formation of the H₂‐oxidizing and CO₂‐fixing systems concomitant to utilization of an organic substrate, thus enabling mixotrophic growth. The latter type of nutrition is probably the relevant one with respect to the situation faced by the organism in its natural habitats, i.e. soil and mud. Aside from the hydrogenase and Calvin‐cycle enzymes, the protein inventories of the H₂‐CO₂‐ and succinate‐grown cells did not reveal major qualitative differences. The protein complement of the glycerol‐grown cells resembled that of the lithoautotrophic cells. Phosphoenolpyruvate (PEP) carboxykinase was present under all three growth conditions, whereas PEP carboxylase was not detectable, supporting earlier findings that PEP carboxykinase is alone responsible for the anaplerotic production of oxaloacetate from PEP. The elevated levels of oxidative stress proteins in the glycerol‐grown cells point to a significant challenge by ROS under these conditions. The results reported here are in agreement with earlier physiological and enzymological studies indicating that R. eutropha H16 has a heterotrophic core metabolism onto which the functions of lithoautotrophy have been grafted.     

Diversity and abundance of freshwater Actinobacteria along environmental gradients in the brackish northern Baltic Sea

Actinobacteria are highly abundant in pelagic freshwater habitats and also occur in estuarine environments such as the Baltic Sea. Because of gradients in salinity and other environmental variables estuaries offer natural systems for examining factors that determine Actinobacteria distribution. We studied abundance and community structure of Bacteria and Actinobacteria along two transects in the northern Baltic Sea. Quantitative (CARD-FISH) and qualitative (DGGE and clone libraries) analyses of community composition were compared with environmental parameters. Actinobacteria accounted for 22–27% of all bacteria and the abundance changed with temperature. Analysis of 549 actinobacterial 16S rRNA sequences from four clone libraries revealed a dominance of the freshwater clusters ac I and ac IV, and two new subclusters ( ac I-B scB-5 and ac IV-E) were assigned. Whereas ac I was present at all stations, occurrence of ac II and ac IV differed between stations and was related to dissolved organic carbon (DOC) and chlorophyll a (Chl a ) respectively. The prevalence of the ac I-A and ac I-B subclusters changed in relation to total phosphorus (Tot-P) and Chl a respectively. Community structure of Bacteria and Actinobacteria differed between the river station and all other stations, responding to differences in DOC, Chl a and bacterial production. In contrast, the composition of active Actinobacteria (analysis based on reversely transcribed RNA) changed in relation to salinity and Tot-P. Our study suggests an important ecological role of Actinobacteria in the brackish northern Baltic Sea. It highlights the need to address dynamics at the cluster or subcluster phylogenetic levels to gain insights into the factors regulating distribution and composition of Actinobacteria in aquatic environments.     

ClassyFlu: Classification of Influenza A Viruses with Discriminatively Trained Profile-HMMs

Accurate and rapid characterization of influenza A virus (IAV) hemagglutinin (HA) and neuraminidase (NA) sequences with respect to subtype and clade is at the basis of extended diagnostic services and implicit to molecular epidemiologic studies. ClassyFlu is a new tool and web service for the classification of IAV sequences of the HA and NA gene into subtypes and phylogenetic clades using discriminatively trained profile hidden Markov models (HMMs), one for each subtype or clade. ClassyFlu merely requires as input unaligned, full-length or partial HA or NA DNA sequences. It enables rapid and highly accurate assignment of HA sequences to subtypes H1–H17 but particularly focusses on the finer grained assignment of sequences of highly pathogenic avian influenza viruses of subtype H5N1 according to the cladistics proposed by the H5N1 Evolution Working Group. NA sequences are classified into subtypes N1–N10. ClassyFlu was compared to semiautomatic classification approaches using BLAST and phylogenetics and additionally for H5 sequences to the new “Highly Pathogenic H5N1 Clade Classification Tool” (IRD-CT) proposed by the Influenza Research Database. Our results show that both web tools (ClassyFlu and IRD-CT), although based on different methods, are nearly equivalent in performance and both are more accurate and faster than semiautomatic classification. A retraining of ClassyFlu to altered cladistics as well as an extension of ClassyFlu to other IAV genome segments or fragments thereof is undemanding. This is exemplified by unambiguous assignment to a distinct cluster within subtype H7 of sequences of H7N9 viruses which emerged in China early in 2013 and caused more than 130 human infections. http://bioinf.uni-greifswald.de/ClassyFlu is a free web service. For local execution, the ClassyFlu source code in PERL is freely available.     

Die cucurbitacine in Bryonia alba und Bryonia dioica

The cucurbitacins in roots of Bryonia dioica and B. alba have been investigated. Both species contain the cucurbitacins E, B, I, D, J, K and L, the dihydrocucurbitacins E and B, and tetrahydrocucurbitacin I. The detection of certain cucurbitacin aglycones depends upon the date of harvest, the duration of storage and the methods used for extraction.     

Hepatic Rac1 GTPase contributes to liver-mediated basal immune homeostasis and LPS-induced endotoxemia

Background

The Ras-homologous GTPase Rac1 plays a key role in the regulation of gene expression, cytoskeleton-associated processes and cell death as well as carcinogenesis and inflammation. Here, we investigated the impact of Rac1 signaling on liver-mediated immune homeostasis.

Nanoencapsulation of Rose-Hip Oil Prevents Oil Oxidation and Allows Obtainment of Gel and Film Topical Formulations

The rose-hip oil holds skin regenerating properties with applications in the dermatological and cosmetic area. Its nanoencapsulation might favor the oil stability and its incorporation into hydrophilic formulations, besides increasing the contact with the skin and prolonging its effect. The aim of the present investigation was to develop suitable rose-hip-oil-loaded nanocapsules, to verify the nanocapsule effect on the UV-induced oxidation of the oil and to obtain topical formulations by the incorporation of the nanocapsules into chitosan gel and film. The rose-hip oil (500 or 600 μL), polymer (Eudragit RS100®, 100 or 200 mg), and acetone (50 or 100 mL) contents were separately varied aiming to obtain an adequate size distribution. The results led to a combination of the factors acetone and oil. The developed formulation showed average diameter of 158?±?6 nm with low polydispersity, pH of 5.8?±?0.9, zeta potential of +9.8?±?1.5 mV, rose-hip oil content of 54?±?1 μL/mL and tendency to reversible creaming. No differences were observed in the nanocapsules properties after storage. The nanoencapsulation of rose-hip oil decreased the UVA and UVC oxidation of the oil. The chitosan gel and film containing rose-hip-oil-loaded nanocapsules showed suitable properties for cutaneous use. In conclusion, it was possible to successfully obtain rose-hip-oil-loaded nanocapsules and to confirm the nanocapsules effect in protecting the oil from the UV rays. The chitosan gel and film were considered interesting alternatives for incorporating the nanoencapsulated rose-hip oil, combining the advantages of the nanoparticles to the advantages of chitosan.