Inflammatory bowel diseases as an intermediate stage between normal and cancer: a FTIR-microspectroscopy approach

Elucidation of the evolution of inflammatory bowel disease (IBD) to cancer by clinical symptoms and histopathology of biopsies is important. Fourier transform infrared microspectroscopy (FTIR-MSP) has shown promise as a diagnostic tool for distinction of normal and cancer cells and tissues. In the present work, FTIR-MSP is used to evaluate IBD cases and to study the IR spectral characteristic with respect to cancer and normal tissues from formalin-fixed colonic biopsies from patients. Specific regions of the spectra were analyzed by statistical tools to study variations in metabolites that signified changes between the two pathological conditions: IBD and cancer. IBD tissues can be grouped with cancer or normal tissue using certain parameters such as phosphate content and RNA/DNA ratio as calculated from the spectra and show intermediate levels with regard to these metabolites. Further classification of the spectra by cluster analysis indicated which cases of Crohn's disease (3 of 10 cases) or ulcerative colitis (7 of 10 cases) were more likely to progress to cancer. The study exhibits that FTIR-MSP can detect gross biochemical changes in morphologically identical IBD and cancer tissues and suggest which cases of IBD may require further evaluation for carcinogenesis.     

Detection of nitric oxide from pig trachea by a fluorescence method

A nitronyl nitroxide radical covalently linked to an organic fluorophore, pyrene, was used to detect nitric oxide (NO) from freshly excited tissues. This approach is based on the phenomenon of the intramolecular fluorescence quenching of the fluorophore fragment by the nitroxide. The pyrene-nitronyl (PN) reacts with NO to yield a pyrene-imino nitroxide radical (PI) and NO(2). Conversion of PN to PI is accompanied by changes in the electron paramagnetic resonance (EPR) spectrum from a five-line pattern (two equivalent N nuclei) into a seven-line pattern (two nonequivalent N nuclei). The transformation of the EPR signal is accompanied by an increase in the fluorescence intensity since the imino nitroxide radical is a weaker quencher than the nitronyl one. The results indicate that the fluorescence measurements enable detection of nanomolar concentrations of NO compared to a sensitivity threshold of only several micromolar for the EPR technique. The method was applied to the determination of NO and S-nitroso compounds in tissue from pig trachea epithelia. The measured basal flux of S-nitroso compounds obtained from the tissues was about 1.2 nmol/g x min, and NO-synthase stimulated by extracellular adenosine 5'-triphosphate produced NO flux of 0.9 nmol/g x min.     

Monolithic peptidyl sorbents for comparison of affinity properties of plasminogen activators

Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein's denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA-EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.     

Carbon sequestration and turnover in soil under the energy crop Miscanthus: repeated 13C natural abundance approach and literature synthesis

The stability and turnover of soil organic matter (SOM) are a very important but poorly understood part of carbon (C) cycling. Conversion of C₃ grassland to the C₄ energy crop Miscanthus provides an ideal opportunity to quantify medium‐term SOM dynamics without disturbance (e.g., plowing), due to the natural shift in the δ¹³C signature of soil C. For the first time, we used a repeated ¹³C natural abundance approach to measure C turnover in a loamy Gleyic Cambisol after 9 and 21 years of Miscanthus cultivation. This is the longest C₃–C₄ vegetation change study on C turnover in soil under energy crops. SOM stocks under Miscanthus and reference grassland were similar down to 1 m depth. However, both increased between 9 and 21 years from 105 to 140 mg C ha^?1 (P < 0.05), indicating nonsteady state of SOM. This calls for caution when estimating SOM turnover based on a single sampling. The mean residence time (MRT) of old C (>9 years) increased with depth from 19 years (0–10 cm) to 30–152 years (10–50 cm), and remained stable below 50 cm. From 41 literature observations, the average SOM increase after conversion from cropland or grassland to Miscanthus was 6.4 and 0.4 mg C ha^?1, respectively. The MRT of total C in topsoil under Miscanthus remained stable at ~60 years, independent of plantation age, corroborating the idea that C dynamics are dominated by recycling processes rather than by C stabilization. In conclusion, growing Miscanthus on C‐poor arable soils caused immediate C sequestration because of higher C input and decreased SOM decomposition. However, after replacing grasslands with Miscanthus, SOM stocks remained stable and the MRT of old C₃‐C increased strongly with depth.     

Variation of sperm morphology in Pacific oyster precludes its use as a species marker but enables intraspecific geo-authentification and aquatic monitoring

According to recent reports, shell morphology is unreliable for the identification of oysters because of the high phenotypic plasticity of these bivalves. Using COI DNA barcoding and sperm morphology, we reinvestigated the species validity of wild Pacific oyster Crassostrea gigas habituating the Peter the Great Bay (Sea of Japan). DNA barcoding confirmed the species validity of samples collected. Application of the single sperm pattern was not possible for species identification due to pronounced sperm plasticity being found. Six sperm morphs were discovered in the testes of each oyster collected. The amount of abundant sperm morphs and the type of the most dominant sperm pattern are particular to geographical localities that are individual depending on the environmental factors. Ecological monitoring of marine areas and commercially assigned intraspecific geo-authentification of the Pacific oyster seems possible based on the analysis of this species’ heterogenic sperm. Further work will be needed to test if sperm heterogeneity exists in other Ostreidae species and if heterogenic sperms could be used for interspecific analysis.     

Exploring phylogeography and species limits in the Altai vole (Rodentia: Cricetidae)

Natural hybridization between species is not a rare event. In arvicoline rodents, hybridization is known to occur in the wild and/or in captivity. In the Microtus arvalis group, cytogenetic studies revealed that there were two distinct chromosomal forms (2n = 46 but a different fundamental number of autosomes). These forms have been attributed to two cryptic species: the common (arvalis) and Altai (obscurus) voles. Recently, individuals with intermediate karyotypes (F₁ and backcrosses) were discovered in central European Russia, and, for this reason, other studies have regarded obscurus and arvalis as conspecific. In the present study, to address the question of the species limits in the Altai vole and to infer its evolutionary history, a phylogeographical analysis combined with multivariate morphometric methods and original chromosome data was performed. Two obscurus lineages were identified: the Sino‐Russian and South Caucasian lineages. Both lineages are characterized by low genetic diversity, resulting, in the former, from a past bottleneck event caused by encroaching periglacial areas and, in the latter, from recent rapid population divergence. Introgressive hybridization between the Altai and common voles appears to be the result of a secondary contact following the Last Glacial Maximum in central European Russia. Despite the fact that speciation is an ongoing process in most arvicoline species, the common and Altai voles are genetically divergent, morphologically and karyologically distinct, and exhibit contrasting evolutionary histories. For all these reasons, they should be ranked as species: M. arvalis and M. obscurus. © 2013 The Linnean Society of London     

A strategy of tumor treatment in mice with doxorubicin-cyclophosphamide combination based on dendritic cell activation by human double-stranded DNA preparation

Background

Immunization of mice with tumor homogenate after combined treatment with cyclophosphamide (CP) and double-stranded DNA (dsDNA) preparation is effective at inhibition of growth of tumor challenged after the treatment. It was assumed that this inhibition might be due to activation of the antigen-presenting cells. The purpose was to develop improved antitumor strategy using mice. We studied the combined action of cytostatics doxorubicin (Dox) plus CP with subsequent dsDNA preparation on tumor growth.

Methods

Three-month old CBA/Lac mice were used in the experiments. Mice were injected with CP and human dsDNA preparation. The percentage of mature dendritic cells (DCs) was estimated by staining of mononuclear cells isolated from spleen and bone marrow 3, 6, and 9 days later with monoclonal antibodies CD34, CD80, and CD86. In the next set of experiments, mice were given intramuscularly injections of 1-3 × 10⁵ tumor cells. Four days later, they were injected intravenously with 6-6.7 mg/kg Dox and intraperitoneally with 100-200 mg/kg CP; 200 mkg human DNA was injected intraperitoneally after CP administration. Differences in tumor size between groups were analyzed for statistical significance by Student's t-test. The MTT-test was done to determine the cytotoxic index of mouse leucocytes from treated groups.

Results

The conducted experiments showed that combined treatment with CP and dsDNA preparation produce an increase in the total amount of mature DCs in vivo. Treatment of tumor bearers with preparation of fragmented dsDNA on the background of pretreatment with Dox plus CP demonstrated a strong suppression of tumor growth in two models. RLS, a weakly immunogenic, resistant to alkalyting cytostatics tumor, grew 3.4-fold slower when compared with the control (p < 0.001). In experiment with Krebs-2 tumor, only 2 of the 10 mice in the Dox+CP+DNA group had a palpable tumor on day 16. The cytotoxic index of leucocytes was 86.5% in the Dox+CP+DNA group, but it was 0% in the Dox+CP group.

Conclusions

Thus, the set of experiments we performed showed that exogenous dsDNA, when administered on the background of pretreatment with Dox plus CP, has an antitumor effect possibly due to DC activation.     

The C. elegans developmental fusogen EFF-1 mediates homotypic fusion in heterologous cells and in vivo

During cell-cell fusion, two cells' plasma membranes merge, allowing the cytoplasms to mix and form a syncytium. Little is known about the mechanisms of cell fusion. Here, we asked whether eff-1, shown previously to be essential for fusion in Caenorhabditis elegans, acts directly in the fusion machinery. We show that expression of EFF-1 transmembrane protein drives fusion of heterologous cells into multinucleate syncytia. We obtained evidence that EFF-1-mediated fusion involves a hemifusion intermediate characterized by membrane mixing without cytoplasm mixing. Furthermore, syncytiogenesis requires EFF-1 in both fusing cells. To test whether this mechanism also applies in vivo, we conducted genetic mosaic analysis of C. elegans and found that homotypic epidermal fusion requires EFF-1 in both cells. Thus, although EFF-1-mediated fusion shares characteristics with viral and intracellular fusion, including an apparent hemifusion step, it differs from these reactions in the homotypic organization of the fusion machinery.