Evaluation of large scale quantitative proteomic assay development using peptide affinity-based mass spectrometry

Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays.     

Involvement of the sun and the magnetic compass of domestic fowl in its spatial orientation

Domestic chicks are able to find a food goal at different times of day, with the sun as the only consistent visual cue. This suggests that domestic chickens may use the sun as a time-compensated compass, rather than as a beacon. An alternative explanation is that the birds might use the earth's magnetic field. In this study, we investigated the role of the sun compass in a spatial orientation task using a clock-shift procedure. Furthermore, we investigated whether domestic chickens use magnetic compass information when tested under sunny conditions.Ten ISA Brown chicks were housed in outdoor pens. A separate test arena comprised an open-topped, opaque-sided, wooden octagonal maze. Eight goal boxes with food pots were attached one to each of the arena sides. A barrier inside each goal box prevented the birds from seeing the food pot before entering. After habituation, we tested in five daily 5-min trials whether chicks were able to find food in an systematically allocated goal direction. We controlled for the use of olfactory cues and intra-maze cues. No external landmarks were visible. All tests were done under sunny conditions. Circular statistics showed that nine chicks significantly oriented goalwards using the sun as the only consistent visual cue during directional testing. Next, these nine chicks were subjected to a clock-shift procedure to test for the role of sun-compass information. The chicks were housed indoors for 6 days on a light-schedule that was 6 h ahead of the natural light–dark schedule. After clock-shifting, the birds were tested again and all birds except one were disrupted in their goalward orientation. For the second experiment, six birds were re-trained and fitted with a tiny, powerful magnet on the head to disrupt their magnetic sense. The magnets did not affect the chicks’ goalward orientation.In conclusion, although the strongest prediction of the sun-compass hypothesis (significant re-orientation after clock-shifting) was neither confirmed nor refuted, our results suggest that domestic chicks use the sun as a compass rather than as a beacon. These findings suggest that hens housed indoors in large non-cage systems may experience difficulties in orientation if adequate alternative cues are unavailable. Further research should elucidate how hens kept in non-cage systems orient in space in relation to available resources.     

Temperature impact on localization of "free" phenolic compounds in the root tissues and deformation of root hairs in pea seedlings inoculated by Rhizobium

The study was focused on localization of "free" phenolic compounds in pea Pisum sativum L. seedling roots grown at 22 and 8 degrees C 24 h after their inoculation with Rhizobium leguminosarum bv. viceae bacteria. A comparison of phenolic compound distribution along the root in root tissues, and results of observation of root hair development on the root surface, response of root hairs to inoculation, manifesting itself in various deformation degree (bends, twists, ect.) enabled us to reveal differences between roots grown at different temperatures. These differences are basically referred to a sector localized 0-5 mm away from the root tip containing meristematic and extending cells. A distribution of phenolic compounds in sectors with root hairs responding to inoculation by various degrees of contortion practically did not depend on the temperature of plant growth. The evidence provided in the course of this work enabled us to suggest that inhibition of pea root infection at low temperature is caused by decelerated growth processes characteristic of both the root itself and root hairs, as well by a slow increase in the root hair zone.     

Crosslinking of mRNA analog, pUUAGUAUUUAUU derivative with a photoactivated group at the guanosine residue, with human 80S ribosomes

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg2+, or 4 mM Mg2+ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840-1849 of 18S rRNA, but in the complexes formed with participation of Phe-TPHKPhe (where the G residue carrying the arylazido group occupied position-3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position-3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg2+ concentration and the presence of polyamines.     

Encapsulation of recombinant <Emphasis Type="Italic">E. coli</Emphasis> expressing cyclopentanone monooxygenase in polyelectrolyte complex capsules for Baeyer–Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one

Recombinant Escherichia coli cells, over-expressing cyclopentanone monooxygenase activity, were immobilized in polyelectrolyte complex capsules, made of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine), CaCl₂ and NaCl. More than 90% of the cell viability was preserved during the encapsulation process. Moreover, the initial enzyme activity was fully maintained within encapsulated cells while it halved in free cells. Both encapsulated and free cells reached the end point of the Baeyer–Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one to 4,9-dioxabicyclo[4.2.1]non-7-en-3-one at the same time (48 h). Similarly, the enantiomeric excess above 94% was identical for encapsulated and free cells.     

The enzyme of carbon metabolism in the thermotolerant sulfobacillus strain K1

To determine enzymatic activities in the thermotolerant strain K1 (formerly "Sulfobacillus thermosulfidooxidans subsp. thermotolerans"), it was grown in a mineral medium with (1) thiosulfate and Fe2+ or pyrite (autotrophic conditions), (2) Fe2+, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. An increased content of carbon dioxide (up to 5 vol%) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide was fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO2 in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase decreased with the increasing content of CO2 in the medium.     

Specific features of the structure of the Z-pinch emitting region formed during the implosion of a foam-wire load at the ANGARA-5-1 facility

Results are presented from experimental studies of the structure of the compressed plasma of a Z-pinch produced during the implosion of a foam-wire load at the current of up to 3 MA. The foam-wire load consisted of two nested cylindrical cascades, one of which was a solid or hollow cylinder made of low-density agar-agar foam, while the other was a wire array. The wall thickness of a hollow foam cylinder was 100–200 μm. The images of the pinch and its spectrum obtained with the help of multiframe X-ray cameras and a grazing incidence spectrograph with a spatial resolution were analyzed. Data on the spatial structure of the emitting regions and the soft X-ray (SXR) spectrum of the Z-pinch in the final stage of compression of a foam-wire load were obtained. The implosion modes characterized by the formation of hot regions during implosion of such loads were revealed. The characteristic scale lengths of the hot regions were determined. It is shown that the energy distribution of SXR photons in the energy range from 80 eV to 1 keV forms the spatial structure of Z-pinch images recorded during the implosion of foam-wire loads. It is revealed that the spectral density of SXR emission in the photon energy range of 300–600 eV from hot Z-pinch regions exceeds the spectral density of radiation from the neighboring Z-pinch regions by more than one order of magnitude. Groups of lines related to the absorption and emission of radiation by atoms and multicharged ions of carbon and oxygen in the outer foam cascade of a foam-wire load were recorded for the first time by analyzing the spatial distribution of the SXR spectra of multicharged ions of the Z-pinch. The groups of absorption lines of ions (C III, O III, O IV, and O VI) corresponding to absorption of SXR photons in the Z-pinch of a tungsten wire array, which served as the inner cascade of a foam-wire load, were identified. The plasma electron temperature measured from the charge composition of carbon and oxygen ions in the outer agar-agar foam cascade was 10–40 eV. During the implosion of foam-wire loads at currents of up to 3 MA, SXR pulses (hν > 100 eV) with a duration of 10 ns and peak power of 3 TW were detected. It is shown that the temporal profile of single-peak and double-peak SXR pulses can be controlled by varying the parameters of the outer and inner cascades of the foam-wire load.