The Effect of Seed Cryopreservation on the Development of Protocorms by the Hybrid Orchid Bratonia

The aim of this work was to study the influence of storage in liquid nitrogen on the viability of seeds of the hybrid orchid Bratonia and further development of its protocorms in vitro. Seeds were frozen in ampoules by direct immersion in liquid nitrogen and stored in the cryobank for a month. The germination rates of cryopreserved and control (nonfrozen) seeds did not differ and remained as high as 100%. The protocorms derived were cultured on the agar-solidified Murashige and Skoog nutrient medium (MS), half-strength MS and Knop media and also in Morel liquid medium. During the first 45 days of culturing, protocorms derived from cryopreserved seeds grew faster than control protocorms on the MS and half-strength MS media but, at longer culturing (496 days), the size of control protocorms was significantly larger. After 639 days of culturing, there was no difference in the amount of perished, budding, and newly formed protocorms obtained from cryopreserved and control seeds, except half-strength MS medium where the number of budding protocorms in the case of cryopreserved seeds was a little greater than in the control treatment. After seed cryopreservation, the frequency of budding and newly formed protocorms was greater on the agarized MS and in liquid Morel media. Cryopreservation had little effect on the subsequent growth of protocorms in vitro. The preferable nutrient media for culturing the protocorms have been suggested.     

Potassium chloride released from contracting skeletal muscle may stimulate development of its hypertrophy

The effects of potassium chloride on the expression of IGF-1 splice forms and myoblast proliferation were investigated. KCl at the concentrations of 7–12 mM stimulated the synthesis of IGF-1 and mechano growth factor (MGF) in murine myoblasts as well as in myotubes both at the mRNA and protein levels. Pan-calcium channel blocker CdCl₂ completely abolished stimulation of growth factor expression, whereas blocker of HCN and Na_v1.4 channels ZD7288 drastically reduced it. In addition, potassium chloride stimulated myoblast proliferation, while IGF-1 autocrine signaling inhibition partially suppressed these mitogenic effects.     

Methylome analysis identifies a Wilms tumor epigenetic biomarker detectable in blood

Background

Wilms tumor is the most common pediatric renal malignancy and there is a clinical need for a molecular biomarker to assess treatment response and predict relapse. The known mutated genes in this tumor type show low mutation frequencies, whereas aberrant methylation at 11p15 is by far the most common aberration. We therefore analyzed the epigenome, rather than the genome, to identify ubiquitous tumor-specific biomarkers.

Results

Methylome analysis of matched normal kidney and Wilms tumor identifies 309 preliminary methylation variable positions which we translate into three differentially methylated regions (DMRs) for use as tumor-specific biomarkers. Using two novel algorithms we show that these three DMRs are not confounded by cell type composition. We further show that these DMRs are not methylated in embryonic blastema but are intermediately methylated in Wilms tumor precursor lesions. We validate the biomarker DMRs using two independent sample sets of normal kidney and Wilms tumor and seven Wilms tumor histological subtypes, achieving 100% and 98% correct classification, respectively. As proof-of-principle for clinical utility, we successfully use biomarker DMR-2 in a pilot analysis of cell-free circulating DNA to monitor tumor response during treatment in ten patients.

Conclusions

These findings define the most common methylated regions in Wilms tumor known to date which are not associated with their embryonic origin or precursor stage. We show that this tumor-specific methylated DNA is released into the blood circulation where it can be detected non-invasively showing potential for clinical utility.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0434-y) contains supplementary material, which is available to authorized users.     

Wavelet analysis of m. vastus lateralis surface EMG activity in incremental tests till exhaustion using bicycle and knee extension exercises

Continuous wavelet analyses was applied to investigate the spectral characteristics of m. vastus lateralis EMG activity in two incremental tests till exhaustion: rhythmic knee-joint extensions and cycling. Wavelet analysis of surface EMG revealed differences in the dynamics of time-frequency characteristics of the signal during single cycle of two types of movements with different loads, as well as differences in the slow variations of spectral characteristics associated with the development of muscle fatigue during the tests. It was shown that during cycling with low loads (beginning of the test) maximum of EMG activity was confined within the second half of muscle contraction (the angle in the knee joint approximately 140 degrees), increase of load at the end of the test led to a shift of the peak to the beginning of the active phase of movement, while the median frequency of the "instant" wavelet spectra during muscle contraction remained almost unchanged. During knee-joint extensions the maximum of EMG activity was observed at the end of the active phase of movement for all loads, median frequency increased significantly with increasing the angle at the knee joint. Long-term dynamics of EMG intensity growth during these tests differed as well, whereas dynamics of wavelet-spectra median frequencies were practically the same--during both tests their growths were observed.     

Isolation of a rickettsial pathogen from a non-hematophagous arthropod

Rickettsial diversity is intriguing in that some species are transmissible to vertebrates, while others appear exclusive to invertebrate hosts. Of particular interest is Rickettsia felis, identifiable in both stored product insect pests and hematophagous disease vectors. To understand rickettsial survival tactics in, and probable movement between, both insect systems will explicate the determinants of rickettsial pathogenicity. Towards this objective, a population of Liposcelis bostrychophila, common booklice, was successfully used for rickettsial isolation in ISE6 (tick-derived cells). Rickettsiae were also observed in L. bostrychophila by electron microscopy and in paraffin sections of booklice by immunofluorescence assay using anti-R. felis polyclonal antibody. The isolate, designated R. felis strain LSU-Lb, resembles typical rickettsiae when examined by microscopy. Sequence analysis of portions of the Rickettsia specific 17-kDa antigen gene, citrate synthase (gltA) gene, rickettsial outer membrane protein A (ompA) gene, and the presence of the R. felis plasmid in the cell culture isolate confirmed the isolate as R. felis. Variable nucleotide sequences from the isolate were obtained for R. felis-specific pRF-associated putative tldD/pmbA. Expression of rickettsial outer membrane protein B (OmpB) was verified in R. felis (LSU-Lb) using a monoclonal antibody. Additionally, a quantitative real-time PCR assay was used to identify a significantly greater median rickettsial load in the booklice, compared to cat flea hosts. With the potential to manipulate arthropod host biology and infect vertebrate hosts, the dual nature of R. felis provides an excellent model for the study of rickettsial pathogenesis and transmission. In addition, this study is the first isolation of a rickettsial pathogen from a non-hematophagous arthropod.     

Chemical composition of Cystoseira crinita Bory from the Eastern Mediterranean

The chemical composition of the brown alga Cystoseira crinita Bory from the Eastern Mediterranean was investigated. Fourteen sterols have been identified, five of them for the first time in algae. The structure of one new sterol was established. The origin of seven sterols with short side chains was discussed. In the volatile fraction 19 compounds and in the polar fraction 15 compounds were identified. The main lipid classes were isolated and their fatty acid composition was established.     

Octaheme nitrite reductases: Structure and properties

Octaheme oxidoreductases are widespread among various bacterial taxa involved in the biogeochemical nitrogen cycle. The evolution of octaheme oxidoreductases of the nitrogen cycle from the evolutionarily more ancient pentaheme nitrite reductases was accompanied by changes in function from reduction of nitrogen oxides to their oxidation under changing environmental conditions. Octaheme nitrite reductases, which are the subject of the present review, are of a transitional form that combines structural and functional characteristics of pentaheme reductases and octaheme oxidases and possesses a number of unique features typical of only this family of enzymes. The review summarizes data on structure-function investigations of the family of octaheme nitrite reductases. Emphasis is given to comparison of the structures and functions of octaheme nitrite reductases and other multiheme oxidoreductases of the nitrogen cycle.     

Allosteric coupling in pyruvate dehydrogenase kinase 2

Mitochondrial pyruvate dehydrogenase kinase 2 (PDHK2) phosphorylates the pyruvate dehydrogenase multienzyme complex (PDC) and thereby controls the rate of oxidative decarboxylation of pyruvate. The activity of PDHK2 is regulated by a variety of metabolites such as pyruvate, NAD (+), NADH, CoA, and acetyl-CoA. The inhibitory effect of pyruvate occurs through the unique binding site, which is specific for pyruvate and its synthetic analogue dichloroacetate (DCA). The effects of NAD (+), NADH, CoA, and acetyl-CoA are mediated by the binding site that recognizes the inner lipoyl-bearing domain (L2) of the dihydrolipoyl transacetylase (E2). Both allosteric sites are separated from the active site of PDHK2 by more than 20 A. Here we show that mutations of three amino acid residues located in the vicinity of the active site of PDHK2 (R250, T302, and Y320) make the kinase resistant to the inhibitory effect of DCA, thereby uncoupling the active site from the allosteric site. In addition, we provide evidence that substitutions of R250 and T302 can partially or completely uncouple the L2-binding site. Based on the available structural data, R250, T302, and Y320 stabilize the "open" and "closed" conformations of the built-in lid that controls the access of a nucleotide into the nucleotide-binding cavity. This strongly suggests that the mobility of ATP lid is central to the allosteric regulation of PDHK2 activity serving as a conformational switch required for communication between the active site and allosteric sites in the kinase molecule.     

Protein engineering of formate dehydrogenase

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is one of the best enzymes for the purpose of NADH regeneration in dehydrogenase-based synthesis of optically active compounds. Low operational stability and high production cost of native FDHs limit their application in commercial production of chiral compounds. The review summarizes the results on engineering of bacterial and yeast FDHs aimed at improving their chemical and thermal stability, catalytic activity, switch in coenzyme specificity from NAD+ to NADP+ and overexpression in Escherichia coli cells.