Morphogenetic potential and in vitro micropropagation of endangered plant species Leucojum aestivum L. and Lilium rhodopaeum Delip.

Summary The present work deals with the in vitro methods for rapid propagation, and morphogenetic potential of the rare and endangered bulb species Leucojum aestivum L., Amaryllidaceae, and Lilium rhodopaeum Delip., Liliaceae. The morphogenetic potential of different plant organs (bulb, stem, leaves and ovaries) was studied. Leaves of Leucojum aestivum L. and basal parts of the bulb in Lilium rhodopaeum Delip. possess the highest regeneration activity. Murashige and Skoog (MS) medium + 1 mg/l 6-benzylaminopurine (BAP) + 1 mg/l kinetin and Linsmaier and Skoog (LS) medium + 0.5 mg/l 1-naphthaleneacetic acid (NAA) + 0.1 mg/l kinetin were favourable for direct organogenisis from these explants. A stimulating effect of alow gamma-irradiation dose (5 Gy) upon the quantity and growth intensity of the bulbs formed by the explants in in vitro conditions is observed.     

Temperature dependence of the transition enthalpy of core particle DNA and of long DNA

The denaturation of short (145 base pairs) and long (about 8000 base pairs) DNA moelucules has been studied by adiabitic differential microcalorimetry in solutions with different NaCl content. It is found that the enthalpy of denaturation of short DNA is more sensitive to changes in T_m than that of long DNA. A comparison with other data is also given.     

Eligibility and Safety of Triple Therapy for Hepatitis C: Lessons Learned from the First Experience in a Real World Setting

Background

HCV protease inhibitors (PIs) boceprevir and telaprevir in combination with PEG-Interferon alfa and Ribavirin (P/R) is the new standard of care in the treatment of chronic HCV genotype 1 (GT1) infection. However, not every HCV GT1 infected patient is eligible for P/R/PI therapy. Furthermore phase III studies did not necessarily reflect real world as patients with advanced liver disease or comorbidities were underrepresented. The aim of our study was to analyze the eligibility and safety of P/R/PI treatment in a real world setting of a tertiary referral center.

Methods

All consecutive HCV GT1 infected patients who were referred to our hepatitis treatment unit between June and November 2011 were included. Patients were evaluated for P/R/PI according to their individual risk/benefit ratio based on 4 factors: Treatment-associated safety concerns, chance for SVR, treatment urgency and nonmedical patient related reasons. On treatment data were analyzed until week 12.

Results

208 patients were included (F3/F4 64%, mean platelet count 169/nl, 40% treatment-naïve). Treatment was not initiated in 103 patients most frequently due to safety concerns. 19 patients were treated in phase II/III trials or by local centers and a triple therapy concept was initiated at our unit in 86 patients. Hospitalization was required in 16 patients; one patient died due to a gastrointestinal infection possibly related to treatment. A platelet count of <110/nl was associated with hospitalization as well as treatment failure. Overall, 128 patients were either not eligible for therapy or experienced a treatment failure at week 12.

Conclusions

P/R/PI therapies are complex, time-consuming and sometimes dangerous in a real world setting, especially in patients with advanced liver disease. A careful patient selection plays a crucial role to improve safety of PI based therapies. A significant number of patients are not eligible for P/R/PI, emphasizing the need for alternative therapeutic options.     

Tumor necrosis factor-alpha binding in porcine primary stromal-vascular cell cultures

Summary The binding characteristics of tumor necrosis factor-α receptors (TNFRs) in primary stromal-vascular cultures from fat tissue of 7-d-old pigs were analyzed. Cells were plated and maintained in 10% fetal bovine serum from day 0 to day 3 and then switched to serum-free medium from day 3 to day 6 to induce lipid filling. On days 3 and 6 of culture, some of the cells were lysed for ligand and immunoblotting and the remainder subjected to competitive and inhibitory-binding assays. Media from day 6 of culture were subjected to ligand and immunoblotting. Competitive binding analysis showed one-site bindings, with IC₅₀s in the nanomolar andK _ds in the picomolar ranges, that were not signficantly different at both time-points of measurement. However, the B_max decreased significantly with differentiation. Preincubation with antibody against TNF receptor type 1 (TNFR1) or TNF receptor type 2 reduced the specific binding by 95 and 15%, respectively, suggesting a dominating role of TNFR1 in¹²⁵I-labeled TNFα (¹²⁵I-TNFα) binding. This was further supported by ligand blotting of cell lysates. Ligand and immunoblotting of cell lysates indicated that TNFα utilizes both types of surface receptors and their isoforms which were not modified during differentiation. Ligand blotting of media revealed soluble receptors with high M_r implying the formation of multimers. Immunoblotting suggested the presence of both types of TNFRs, but a greater abundance of soluble TNFR1. Also, it indicated the additional formation of smaller oligomers from both types of soluble receptors suggesting higher affinity of larger multimers for¹²⁵I-TNFα.     

Influence of antigen stimulation on the oxidative stress parameters in outbred and inbred rabbits

The influence of antigen stimulation on the oxidative stress parameters in two groups of rabbits-inbred and outbred were explored by evaluation of the level of lipid peroxidation products (MDA) in the plasma membrane, and the activity of erythrocyte antioxidant defense enzymes superoxide dismutase (SOD) and catalase (CAT). There was not a significant difference between levels of MDA in inbred and outbred rabbits before immunization. However, SOD activity in inbred rabbits was significantly increased in comparison with that of outbred (p = 0.006). Significantly higher plasma levels of lipid peroxidation products were detected in both inbred and outbred rabbits during immune response in comparison to the corresponding groups before immunization (p = 0.008 and p = 0.002). SOD and CAT activities in erythrocytes of rabbits during immune response were also significantly increased compared to that before immunization. In addition, during immune response SOD and CAT activities were found to be positively correlated to each other in both inbred and outbred rabbits (r = 0.727 and r = 0.916). In conclusion, our results suggest the presence of an increased oxidative stress during the antigen stimulation accompanied by an adaptive increase of SOD and CAT activities. 30 days after immunization, the plasma levels of MDA and the activities of SOD and CAT in erythrocytes decreased and reached values close to the controls.     

ELF fields and photooxidation yielding lethal effects on cancer cells

The lethal effect on human cancer cells was studied under three types of treatment: A) an ELF pulsed sinusoidal of 50 Hz electromagnetic field (PEMF) with amplitudes between 10 and 55 mT; B) the field and a cytostatic agent (actinomycin-C); and C) the field, the cytostatic agent, which has a photodynamic effect, and exposure to a halogen lamp. The results show a decreasing vitality of human K-562 and U-937 cancer cells in suspension with each additional treatment. Combination with other parameters as hyperthermia and/or hyperacidity could yield high killing rates by this noninvasive method.     

SiRNA-Induced Mutation in HIV-1 Polypurine Tract Region and Its Influence on Viral Fitness

Converting single-stranded viral RNA into double stranded DNA for integration is an essential step in HIV-1 replication. Initial polymerization of minus-strand DNA is primed from a host derived tRNA, whereas subsequent plus-strand synthesis requires viral primers derived from the 3′ and central polypurine tracts (3′ and cPPTs). The 5′ and 3′ termini of these conserved RNA sequence elements are precisely cleaved by RT-associated RNase H to generate specific primers that are used to initiate plus-strand DNA synthesis. In this study, siRNA wad used to produce a replicative HIV-1 variant contained G(-1)A and T(-16)A substitutions within/adjacent to the 3′PPT sequence. Introducing either or both mutations into the 3′PPT region or only the G(-1)A substitution in the cPPT region of NL4-3 produced infectious virus with decreased fitness relative to the wild-type virus. In contrast, introducing the T(-16)A or both mutations into the cPPT rendered the virus(es) incapable of replication, most likely due to the F185L integrase mutation produced by this nucleotide substitution. Finally, the effects of G(-1)A and T(-16)A mutations on cleavage of the 3′PPT were examined using an in vitro RNase H cleavage assay. Substrate containing both mutations was mis-cleaved to a greater extent than either wild-type substrate or substrate containing the T(-16)A mutation alone, which is consistent with the observed effects of the equivalent nucleotide substitutions on the replication fitness of NL4-3 virus. In conclusion, siRNA targeting of the HIV-1 3′PPT region can substantially suppress virus replication, and this selective pressure can be used to generate infectious virus containing mutations within or near the HIV-1 PPT. Moreover, in-depth analysis of the resistance mutations demonstrates that although virus containing a G(-1)A mutation within the 3′PPT is capable of replication, this nucleotide substitution shifts the 3′-terminal cleavage site in the 3′PPT by one nucleotide (nt) and significantly reduces viral fitness.     

Investigation of iron-containing products from natural and laboratory cultivated Sphaerotilus-Leptothrix bacteria

Bacterial biomass collected from sheath-forming bacteria of the genera Sphaerotilus and Leptothrix was collected from a high-mountain natural stream water source. The elemental constitution and oxide phases of the products after selective cultivation of the bacteria on two different elective media using neutron activation analysis (NAA), electron microscopy (SEM, TEM), and X-ray diffraction (XRD) were studied. A high enrichment level of iron was revealed by the NAA technique in cultivated isolates as compared to the reference sample from nature. Three types of iron oxide compounds were established after cultivation in Adler’s medium: lepidocrocite (γ-FeOOH), magnetite (Fe₃O₄), and goethite (α-FeOOH). The cultivation in the Isolation medium yielded a single phase, that of goethite, excluding one sample with a distinguishable amount of lepidocrocite. XRD and EM investigations show that the biogenic oxides are nanosized. Our study exemplifies the possibilities of the biotechnology approach for obtaining, under artificial conditions, large quantities of iron-containing by-products that could be of further used in appropriate nano- and biotechnologies.