Recombinant Components Research Articles

Combinatory effect of BRCA1 and HERC2 expression on outcome in advanced non-small-cell lung cancer.

BackgroundBRCA1 is a main component of homologous recombination and induces resistance to platinum in preclinical models. It has been studied as a potential predictive marker in lung cancer. Several proteins modulate the function of BRCA1. The E3 ubiquitin ligase HERC2 facilitates the assembly of the RNF8-UBC13 complex to recruit BRCA1 to DNA damage sites. The combined analysis of multiple components of the pathway leading to the recruitment of BRCA1 at DNA damage sites has the potentiality to improve the BRCA1 predictive model.MethodsWe retrospectively analyzed 71 paraffin-embedded tumor samples from advanced non-small-cell lung cancer patients treated with first-line platinum based chemotherapy and measured the mRNA expression levels of BRCA1, RNF8, UBC13 and HERC2 using real-time PCR. The mRNA expression was categorized using median value as cut-off point.ResultsThe median progression-free survival of all 71 patients was 7.2 months whereas the median overall survival of the study population was 10.7 months. Among patients with low BRCA1 expression, the median PFS was 7.4 months in the presence of low HERC2 levels and 5.9 months for patients expressing high HERC2 levels (p = 0.01). The median OS was 15.3 months for patients expressing low levels of both genes and 7.4 months for those with low BRCA1 but high HERC2 (p = 0.008). The multivariate analysis showed that among patients with Eastern Cooperative Oncology Group performance status 0–1, the combined low expression of both BRCA1 and HERC2 clearly reduced the risk of progression (p = 0.03) and of death (p = 0.004).ConclusionsThese findings confirm the potentiality of integrated DNA repair components analysis in predicting the sensitivity to platinum in lung cancer. The study indicates a predictive role for HERC2 mRNA expression and paves the way for further refinement of the BRCA1 predictive model.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2339-5) contains supplementary material, which is available to authorized users.

The role of allergen components for the diagnosis of latex-induced occupational asthma.

Recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergen components have been developed to assess the patients' allergen sensitization profile and to improve the diagnosis of NRL allergy. To examine whether the determination of specific IgE (sIgE) reactivity to a panel of recombinant allergen components would be helpful for diagnosing NRL-induced occupational asthma (OA) in predicting the outcome of a specific inhalation test. sIgE levels to NRL extract and 12 recombinant NRL allergen components were assessed in 82 subjects with OA ascertained by a positive specific inhalation challenge (SIC) with NRL gloves and in 25 symptomatic subjects with a negative challenge. The sensitivity, specificity, positive predictive value, and negative predictive value of a NRL-sIgE level ≥0.35 kUA /l as compared to the result of SICs were 94%, 48%, 86%, and 71%, respectively. The positive predictive value increased above 95% when increasing the cutoff value to 5.41 kUA /l. Subjects with a positive SIC showed a significantly higher rate of sIgE reactivity to rHev b 5, 6.01, 6.02, and 11 than those with a negative SIC. A sIgE sum score against rHev b 5 plus 6.01/6.02 ≥ 1.46 kUA /l provided a positive predictive value >95% with a higher sensitivity (79%) and diagnostic efficiency (Youden index: 0.67) as compared with a NRL-sIgE ≥5.41 kUA /l (49% and 0.41, respectively). In suspected OA, high levels of sIgE against rHev b 5 combined with rHev b 6.01 or 6.02 are the most efficient predictors of a bronchial response to NRL.

The effects of gamma irradiation on neutron displacement sensitivity of lateral PNP bipolar transistors

The effects of gamma irradiation on neutron displacement sensitivity of four types of lateral PNP bipolar transistors (LPNPs) with different neutral base widths, emitter widths and the doping concentrations of the epitaxial base region are studied. The physical mechanisms of the effects are explored by defect analysis using deep level transient spectroscopy (DLTS) techniques and numerical simulations of recombination process in the base region of the lateral PNP bipolar transistors, and are verified by the experiments on gate-controlled lateral PNP bipolar transistors (GCLPNPs) manufactured in the identical commercial bipolar process with different gate bias voltage. The results indicate that gamma irradiation increases neutron displacement damage sensitivity of lateral PNP bipolar transistors and the mechanism of this phenomenon is that positive charge induced by gamma irradiation enhances the recombination process in the defects induced by neutrons in the base region, leading to larger recombination component of base current and greater gain degradation.

Quantitative analysis of optical and recombination losses in Cu(In,Ga)Se2 thin-film solar cells

Optical and recombination losses in a Cu(In,Ga)Se2 thin-film solar cell with a band gap of 1.36–1.38 eV are theoretically analyzed. The optical transmittance of the ZnO and CdS layers through which the radiation penetrates into the absorbing layer is determined. Using optical constants, the optical loss caused by reflection at the interfaces (7.5%) and absorption in the ZnO and CdS layers (10.2%) are found. To calculate the recombination loss, the spectral distribution of the quantum efficiency of CdS/CuIn1–xGaxSe2 is investigated. It is demonstrated that, taking the drift and diffusion components of recombination at the front and rear surfaces of the absorber into account, the quantum efficiency spectra of the investigated solar cell can be analytically described in detail. The real parameters of the solar cell are determined by comparing the calculated results and experimental data. In addition, the losses caused by the recombination of photogenerated carriers at the front and rear surfaces of the absorbing layer (1.8% and <0.1%, respectively), at its neutral part (7.6%), and in the space-charge region of the p–n heterojunction (1.0%) are determined. A correction to the parameters of Cu(In,Ga)Se2 is proposed, which enhances the charge-accumulation efficiency.

Nonsense-mediated decay regulates key components of homologous recombination.

Cells frequently experience DNA damage that requires repair by homologous recombination (HR). Proteins involved in HR are carefully coordinated to ensure proper and efficient repair without interfering with normal cellular processes. In Saccharomyces cerevisiae, Rad55 functions in the early steps of HR and is regulated in response to DNA damage through phosphorylation by the Mec1 and Rad53 kinases of the DNA damage response. To further identify regulatory processes that target HR, we performed a high-throughput genetic interaction screen with RAD55 phosphorylation site mutants. Genes involved in the mRNA quality control process, nonsense-mediated decay (NMD), were found to genetically interact with rad55 phospho-site mutants. Further characterization revealed that RAD55 transcript and protein levels are regulated by NMD. Regulation of HR by NMD extends to multiple targets beyond RAD55, including RAD51, RAD54 and RAD57. Finally, we demonstrate that loss of NMD results in an increase in recombination rates and resistance to the DNA damaging agent methyl methanesulfonate, suggesting this pathway negatively regulates HR under normal growth conditions.

Allergen extracts and recombinant proteins: comparison of efficiency of in vitro allergy diagnostics using multiplex assay on a biological microchip.

BackgroundImmunological test systems for diagnostics of type I hypersensitivity involve the following types of antigens: whole allergen extracts, individual highly purified proteins and their recombinant analogues. The goal of this study was to compare the results obtained with whole allergen extracts (birch pollen, cat dander, and timothy grass pollen) and their respective recombinant proteins in biochip-based immunoassay.MethodsMultiplex fluorescent immunoassay of 139 patients’ blood serum samples was carried out using biological microchips (biochips). sIgE concentrations for the chosen allergens and their recombinant components were measured. ROC analysis was used for comparison of the results and determination of diagnostic accuracy.ResultsThe results for the birch pollen extract and its recombinant allergens have shown that the diagnostic accuracy of the methods utilizing the whole allergen extract, its major component Bet v 1 and the combination of major and minor components (Bet v 1 and Bet v 2) was the same. Values for diagnostic accuracy for the cat dander extract and its major recombinant component Fel d 1 were equal. In contrast with birch pollen and cat dander allergens, using of recombinant components of timothy grass pollen (Phl p 1, Phl p 5, Phl p 7 and Phl p 12) did not allow reaching the diagnostic accuracy of using natural extract.ConclusionsMultiplex analysis of samples obtained from patients with allergy to birch pollen and cat dander using biological microchips has shown that comparable accuracy was observed for the assay with natural extracts and recombinant allergens. In the case of timothy grass allergen, using the recombinant components may be insufficient.

Auger and Carrier Trapping Dynamics in Core/Shell Quantum Dots Having Sharp and Alloyed Interfaces.

The role of interface sharpness in controlling the excited state dynamics in CdSe/ZnSe core/shell particles is examined here. Particles composed of CdSe/ZnSe with 2.4-4.0 nm diameter cores and approximately 4 monolayer shells are synthesized at relatively low temperature, ensuring a sharp core-shell interface. Subsequent annealing results in cadmium and zinc interdiffusion, softening the interface. TEM imaging and absorption spectra reveal that annealing results in no change in the particle sizes. Annealing results in a 5-10 nm blue shift in the absorption spectrum, which is compared to calculated spectral shifts to characterize the extent of metal interdiffusion. The one- and two-photon dynamics are measured using time-resolved absorption spectroscopy. We find that biexcitons undergo biexponential decays, with fast and slow decay times differing by about an order of magnitude. The relative magnitudes of the fast and slow components depend on the sharpness of the core-shell interface, with larger fast component amplitudes associated with a sharp core-shell interface. The slow component is assigned to Auger recombination of band edge carriers and the fast decay component to Auger recombination of holes that are trapped in defects produced by lattice strain. Annealing of these particles softens the core-shell interface and thereby reduces the amount of lattice strain and diminishes the magnitude of the fast decay component. The time constant of the slow biexciton Auger recombination component changes only slightly upon softening of the core-shell interface.

Clinical utility of recombinant allergen components in diagnosing buckwheat allergy

Clinical utility of recombinant allergen components in diagnosing buckwheat allergy

Math modeling of electronic processes and deep level ionization kinetic

Mathematical model of kinetics of charge deep levels in the forbidden band of the semiconductor, which takes into account the processes of carriers charge exchange between deep levels and both allowed bands, which adequately describes the nature of the non-exponential relaxation capacity, is proposed. A method for determining the spectrum of deep level transient spectroscopy having greater accuracy and resolution in comparison with traditional methods using a relaxation time approximation. The results of numerical experiments using the kinetics charge deep levels model in the frameworks of proposed approximations are presented. Account of generational and recombination components of charge exchange processes of all deep levels in the forbidden band of the semiconductor leads to the conclusion that the kinetics of ionization of these centers, in general, does not obey the Boltzmann statistics. Account of charge exchange processes between the deep levels a significantly effects on their recharge kinetics. Numerical analysis results show that the processes of deep levels ionization are more complicated than the staged-type kinetics. It is shown that in most cases stagedtype kinetics at deep level transient spectroscopy leads to significant methodological error in the parameters determination. From the results of numerical analysis follows, that the density of surface electronic states has a significant impact on the overall recharged kinetics of deep levels. Donor deep levels recharge analysis revealed not only the features of the deep levels ionization in semiconductors, but also allowed to answer some questions that are typical to all deep-level transient spectroscopy in general.

Formation of reactive oxygen species by human and bacterial pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components

Individual recombinant components of pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes (PDHc, OGDHc) of human and Escherichia coli (E. coli) origin were expressed and purified from E. coli with optimized protocols. The four multienzyme complexes were each reconstituted under optimal conditions at different stoichiometric ratios. Binding stoichiometries for the highest catalytic efficiency were determined from the rate of NADH generation by the complexes at physiological pH. Since some of these complexes were shown to possess ‘moonlighting’ activities under pathological conditions often accompanied by acidosis, activities were also determined at pH 6.3. As reactive oxygen species (ROS) generation by the E3 component of hOGDHc is a pathologically relevant feature, superoxide generation by the complexes with optimal stoichiometry was measured by the acetylated cytochrome c reduction method in both the forward and the reverse catalytic directions. Various known affectors of physiological activity and ROS production, including Ca2+, ADP, lipoylation status or pH, were investigated. The human complexes were also reconstituted with the most prevalent human pathological mutant of the E3 component, G194C and characterized; isolated human E3 with the G194C substitution was previously reported to have an enhanced ROS generating capacity. It is demonstrated that: i. PDHc, similarly to OGDHc, is able to generate ROS and this feature is displayed by both the E. coli and human complexes, ii. Reconstituted hPDHc generates ROS at a significantly higher rate as compared to hOGDHc in both the forward and the reverse reactions when ROS generation is calculated for unit mass of their common E3 component, iii. The E1 component or E1-E2 subcomplex generates significant amount of ROS only in hOGDHc; iv. Incorporation of the G194C variant of hE3, the result of a disease-causing mutation, into reconstituted hOGDHc and hPDHc indeed leads to a decreased activity of both complexes and higher ROS generation by only hOGDHc and only in its reverse reaction.

Dissolved carbon dioxide determines the productivity of a recombinant hemagglutinin component of an influenza vaccine produced by insect cells.

Dissolved carbon dioxide (dCO2 ) accumulation during cell culture has been recognized as an important parameter that needs to be controlled for successful scale-up of animal cell culture because above a certain concentration there are adverse effects on cell growth performance and protein production. We investigated the effect of accumulation of dCO2 in bioreactor cultures of expresSF+(®) insect cells infected with recombinant baculoviruses expressing recombinant influenza virus hemagglutinins (rHA). Different strategies for bioreactor cultures were used to obtain various ranges of concentrations of dCO2 (<50, 50-100, 100-200, and >200 mmHg) and to determine their effects on recombinant protein production and cell metabolic activity. We show that the accumulation of dCO2 at levels > 100 mmHg resulted in reduced metabolic activity, slowed cell growth, prolonged culture viability after infection, and decreased infection kinetics. The reduced rHA yields were not caused by the decrease in the extracellular pH that resulted from dCO2 accumulation, but were most likely due to the effect of dCO2 accumulation in cells. The results obtained here at the 2 L scale have been used for the design of large-scale processes to manufacture the rHA based recombinant vaccine Flublok™ at the 2500 L scale Biotechnol. Bioeng. 2015;112: 2267-2275. © 2015 Wiley Periodicals, Inc.

Probing of Carrier Recombination in n- and p-Type 6H-SiC Using Ultrafast Supercontinuum Pulses

Excess carrier dynamics in 6H-SiC substrates with n- and p-type moderate doping were detected using femtosecond pump-probe measurements with supercontinuum probing. Band-to-band recombination and carrier trapping were determined as the main recombination processes in both materials. Spectral fingerprints corresponding to each of these recombination components were obtained using the global and target analysis. It was shown that, in spite of background doping, the band-to-band recombination in 6H-SiC is dominated by the excess electron absorption component and the carrier trapping is dominated by the excess hole absorption.

Experimental design of complement component 5a-induced acute lung injury (C5a-ALI): a role of CC-chemokine receptor type 5 during immune activation by anaphylatoxin

Excessive activation of the complement system is detrimental in acute inflammatory disorders. In this study, we analyzed the role of complement-derived anaphylatoxins in the pathogenesis of experimental acute lung injury/acute respiratory distress syndrome (ALI/ARDS) in C57BL/6J mice. Intratracheal administration of recombinant mouse complement component (C5a) caused alveolar inflammation with abundant recruitment of Ly6-G(+)CD11b(+) leukocytes to the alveolar spaces and severe alveolar-capillary barrier dysfunction (C5a-ALI; EC(50[C5a]) = 20 ng/g body weight). Equimolar concentrations of C3a or desarginated C5a (C5a(desArg)) did not induce alveolar inflammation. The severity of C5a-ALI was aggravated in C5-deficient mice. Depletion of Ly6-G(+) cells and use of C5aR1(-/-) bone marrow chimeras suggested an essential role of C5aR1(+) hematopoietic cells in C5a-ALI. Blockade of PI3K/Akt and MEK1/2 kinase pathways completely abrogated lung injury. The mechanistic description is that C5a altered the alveolar cytokine milieu and caused significant release of CC-chemokines. Mice with genetic deficiency of CC-chemokine receptor (CCR) type 5, the common receptor of chemokine (C-C motif) ligand (CCL) 3, CCL4, and CCL5, displayed reduced lung damage. Moreover, treatment with a CCR5 antagonist, maraviroc, was protective against C5a-ALI. In summary, our results suggest that the detrimental effects of C5a in this model are partly mediated through CCR5 activation downstream of C5aR1, which may be evaluated for potential therapeutic exploitation in ALI/ARDS.

Effects of a recombinant complement component C3b functional fragment α2MR (α2-macroglobulin receptor) additive on the immune response of juvenile orange-spotted grouper (Epinephelus coioides) after the exposure to cold shock challenge

The effects of Ec-α2MR (Epinephelus coiodes-α2-macroglobulin receptor) on growth performance, enzymatic activity, respiratory burst, MDA level, total antioxidant capacity, DPPH radical scavenging percentage and immune-related gene expressions of the juvenile orange-spotted grouper were evaluated. The commercial diet supplemented with α2MR additive was used to feed the orange-spotted grouper for six weeks. Although a slight increase was observed in the specific growth rate, survival rate and weight gain, no significance was observed among different group. After the feeding trial, the groupers were exposed to cold stress. Respiratory burst activity and MDA level decreased significantly in α2MR additive group by comparing with the control and additive control group, while a sharp increase of ACP activity, ALP activity, total antioxidant capacity and DPPH radial scavenging percentage was observed in α2MR additive group. qRT-PCR analyses confirmed that the up-regulated mRNA expressions of C3, TNF1, TNF2, IL-6, CTL, LysC, SOD1 and SOD2 were observed in α2MR additive group at 20 °C. These results showed that α2MR additive may moderate the immune response in grouper following cold shock challenge.

Recombination current in AlGaAs/GaAs superlattice solar-cells grown by molecular beam epitaxy

We investigate the effect of the recombination current of p–i–n junction solar-cells. We develop a simple evaluation method of the recombination and diffusion current component of the solar-cells based on the measured three characteristic values: short circuit current, open circuit voltage, and fill factor without the knowledge in the details of the device structure. The advantage of the developed technique is its simplicity and wide applicability to various p–i–n junction solar-cells. We apply the method to GaAs bulk and AlGaAs/GaAs superlattice solar-cells. Obtained parameters well reproduce the whole current-voltage characteristics. The diode current is almost dominated by the recombination current at the maximum-output voltage for both GaAs bulk and superlattice cells. The higher contribution of the recombination current in the superlattice solar-cell is due to the quality of the AlGaAs barriers and the AlGaAs/GaAs interfaces. This result indicates that the good crystalline quality is important to enhance the efficiency of the solar-cells.

The Ratio Between Cor a 1- and Hazelnut-Specific IgE Predicts Negative Challenge Outcome in Children

Background: Hazelnut represents one of the most important food allergens. Yet, sensitization to hazelnut is often randomly detected during routine diagnostic workup without a clear-cut history of allergy. The aim of this study was to assess the diagnostic usefulness of recombinant hazelnut components and whole hazelnut extract for the prediction of clinical reactivity in sensitized children. Methods: Fifty-three consecutive children were investigated who underwent oral food challenge (OFC) due to positive in vitro test results and/or cutaneous reactions after hazelnut ingestion. Determination of specific immunoglobulin E (sIgE) against allergen extracts and recombinant hazelnut components had been performed in all patients prior to OFC. Results: OFC revealed hazelnut allergy in 19 children (36%). At a mean total IgE of 518 kU/L, sIgE to hazelnut extract was significantly higher in allergic than in tolerant children (38 vs. 13 kU/L). In contrast, no significant difference in sIgE levels against allergen components was detected. A ratio of rCor a 1 to hazelnut extract sIgE levels >1 predicted hazelnut tolerance with a sensitivity and a negative predictive value of 100%. The corresponding specificity (47%) and positive predictive value (51%) were low. Conclusions: In the present study, a ratio of rCor a 1 to hazelnut extract sIgE >1 predicted hazelnut tolerance during OFC in children with suspected hazelnut allergy. However, further investigations in larger populations are warranted to investigate the transferability of these results to other patient populations. Until then, OFC remain the diagnostic gold standard for the definitive diagnostic workup of suspected food allergy.

ATP Binding is Prerequisite to the Helical Structure of Human Rad51 Presynaptic Filament

Human Rad51 is a key protein component of homologous recombination, the error free repair process of double strand DNA breaks. It is a 37 kDa protein of two domains. The full structure of the protein is not known in atomic details, X-ray crystallography has been successful only in the case of the larger C-domain in complex with a regulatory protein, BRCA2. The structure of the N-domain has been determined by NMR. It is known that in the repair process, the recombinases form a helical filament around ssDNA overhangs - the presynaptic filament (PSF) - at the double strand breaks, the structure of which is of vital significance in the proceeding of the repair. It is also known that the recombinases possess ATPase activity, and ATP is needed for the successful repair. While in the case of the bacterial-, archeal,- and yeast - homologues of HsRad51, data were presented concerning the parameters of their ATP-dependent PSF structures in crystalline conditions, such results are non-existent for HsRad. In this study our goal was to provide evidence for the purely structural role of ATP in forming the PSF structure by HsRad51. Therefore, the hydrolysis of ATP has been excluded by adding Ca to the solutions instead of Mg. The topology of HsRad structures without and with added ATP/Ca and 75mer ssDNA has been determined by transmission electron microscopy. The formations were labeled by the fluorescent dye ANS, and pressure perturbation fluorescence spectroscopy has been applied to characterize the strength of interaction at protomeric interfaces in the Rad51 filaments. We found that the formation of the ordered filamentous structure clearly requires the presence of ATP/Ca, and that the interface binding strength is the highest in the presynaptic filament of helical structure.

The global distribution of Banana bunchy top virus reveals little evidence for frequent recent, human-mediated long distance dispersal events.

Banana bunchy top virus (BBTV; family Nanoviridae, genus Babuvirus) is a multi-component single-stranded DNA virus, which infects banana plants in many regions of the world, often resulting in large-scale crop losses. We analyzed 171 banana leaf samples from fourteen countries and recovered, cloned, and sequenced 855 complete BBTV components including ninety-four full genomes. Importantly, full genomes were determined from eight countries, where previously no full genomes were available (Samoa, Burundi, Republic of Congo, Democratic Republic of Congo, Egypt, Indonesia, the Philippines, and the USA [HI]). Accounting for recombination and genome component reassortment, we examined the geographic structuring of global BBTV populations to reveal that BBTV likely originated in Southeast Asia, that the current global hotspots of BBTV diversity are Southeast Asia/Far East and India, and that BBTV populations circulating elsewhere in the world have all potentially originated from infrequent introductions. Most importantly, we find that rather than the current global BBTV distribution being due to increases in human-mediated movements of bananas over the past few decades, it is more consistent with a pattern of infrequent introductions of the virus to different parts of the world over the past 1,000 years.

Observation of “fast” and “slow” decay processes in oxygen-doped hydrogenated amorphous silicon nitride thin films

Oxygen-doped amorphous silicon nitride thin films were fabricated in a plasma enhanced chemical vapor deposition system by using H2 diluted gases under a substrate temperature of 250 °C. An intense photoluminescence was achieved under 325 nm laser excitation and the luminescence is reduced to about 70% of the initial emission intensity after 1hr laser irradiation, which is improved compared with that deposited at low substrate temperature. Time-resolved photoluminescence behaviors were characterized and two recombination processes were observed, one is a “fast” nanosecond PL decay component which was explained in terms of the relaxation process to the localized O-Si-N related states, and the other is a “slow” microsecond radiative recombination component via the localized states associated with O-Si-N bands.

Protein synthesis using a reconstituted cell-free system.

Most cell-free protein-synthesis systems are based on cell extracts, which often contain undesirable activities. Reconstituted systems, by contrast, are composed of a defined number of purified and recombinant components with minimal nuclease and protease activities. This unit describes the use of a particular commercial reconstituted system, PURExpress. This system allows in vitro synthesis of proteins from mRNA and circular and linear DNA templates, as well as co-translational labeling of proteins. Unique to this system, all recombinant protein components of the system are His-tagged, allowing purification of the synthesized untagged protein by removing the rest of the system's components. Newly synthesized proteins can often be visible on an SDS-PAGE gel and directly assayed for their functions without labeling and purification. Certain components of the system, such as ribosomes or release factors, can be omitted for specific applications. Such "delta" versions of the system are well suited for studies of bacterial translation, assays of ribosome function, incorporation of unnatural amino acids, and ribosome display of protein libraries.