A Homologous Recombination System to Generate Epitope-Tagged Target Genes in Chaetomium thermophilum: A Genetic Approach to Investigate Native Thermostable Proteins

Chaetomium thermophilum is an attractive eukaryotic model organism which, due to its unusually high temperature tolerance (optimal growth at 50–52 °C), has a thermostable proteome that can be exploited for biochemical, structural and biotechnological applications. Site directed gene manipulation for the expression of labeled target genes is a desirable approach to study the structure and function of thermostable proteins and their organization in complexes, which has not been established for this thermophile yet. Here, we describe the development of a homologous recombination system to epitope-tag chromosomal genes of interest in Chaetomium thermophilum with the goal to exploit the derived thermostable fusion proteins for tandem-affinity purification. This genetic approach was facilitated by the engineering of suitable strains, in which factors of the non-homologous end-joining pathway were deleted, thereby improving the efficiency of homologous integration at specific gene loci. Following this strategy, we could demonstrate that gene tagging via homologous recombination improved the yield of purified bait proteins and co-precipitated factors, paving the way for related studies in fundamental research and industrial applications.     

Examination of biogenic amines in grapevine musts originating from vineyards treated with different viticultural practices

Viticultural practices as defoliation (DF) and cluster tipping (CT) (hereinafter referred to as the abbreviation) are often used for crop regulation to enhance quality. Reduced yield can be accompanied not only with more favourable sugar-acid composition but also with changes of other chemical compounds in the berries, for example, biogenic amines as well. The latter play a significant role in human health. The experiment has been carried out at three different growing sites of Hungary in 2014. DF was done at full bloom, CT occurred when the berry reached pea size. Berry sampling for biogenic amine analysis was carried out several times during the ripening period. In our trial, we examined the effect of the above-mentioned treatments, the sites and the sampling dates on the amount of berries’ biogenic amine content. We concluded that DF and CT changed the amount of each type of amines in the berries. According to our measurements the treatments and the site did affect the amount and composition of biogenic amines, but the sampling time not. The treatments were most effective in the case of histamine which is crucial for triggering allergic reactions.     

Turnover rate and kinetic mechanism for the reaction of hydrodechlorination of 1,1-dichlorotetrafluoroethane (CF3-CFCl2) over a polycrystalline Pd foil

The kinetics (turnover rate, activation energy, reaction order) of catalytic hydrodechlorination of 1,1-dichlorotetrafluoroethane or CFC 114a (CF₃-CFCl₂) were determined on a ~ 0.5 cm² palladium polycrystalline foil as a catalyst. The reaction rates were measured in the temperature range of 80-200°C at a total pressure of 770 Torr. Using a reaction cell that was connected to an ultrahigh vacuum surface characterization chamber the composition of the metal surface was monitored before and after reaction. The products formed in a parallel reaction network and the turnover rates at 150°C, 50 Torr CFC 114a, 100 Torr H₂, and 0.1 Torr HCl were 2.1 s^-1 for CF₃-CFH₂, 3.0× 10^-1 s^-1 for CF₃-CFClH, and 6.4×10^-2 s^-1 for CF₃-CH₃. For the two most abundant products, the reaction order is 1 in CF₃-CFCl₂, 0.5 in H₂ and -1 in the reaction product HCl. These results suggest that the rate determining step for the reaction is the associative chemisorption of CF₃-CFCl₂ on the palladium surface. The palladium surface was free of adsorbates after the reaction with the exception of sulfur impurity that may accumulate during reaction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Primary vitrectomy without scleral buckling for rhegmatogenous retinal detachment

BACKGROUND: Pars planta vitrectomy has evolved as an alternative method in the treatment of more complicated rhegmatogenous retinal detachments. We report a series of patients who underwent primary vitrectomy with gas tamponade without the use of additional scleral buckling. METHODS: A retrospective study of 53 patients with a follow-up of 6-45 months (mean 17.8 months) was carried out. Preoperative findings included unusual, multiple or large breaks, vitreous haemorrhage, proliferative vitreoretinopathy and bullous retinal detachment. Preoperative visual acuity was between light perception and 1.0, with 30% (16/53) of patients with 0.4 or better. RESULTS: Retinal reattachment was achieved in 64% of cases (34/53) with one and in 92% (49/53) with one or more operations. Final visual acuity was between light perception and 1.0, with 41% (22/53) of patients with 0.4 or better. Cataract formation occurred in 86% (37/43) of all patients with a clear lens preoperatively. Macular pucker was noted in 11% (6/53) and postoperative proliferative vitreoretinopathy causing redetachment in 6% (3/53). CONCLUSION: With primary vitrectomy, a high final anatomical success rate with few intraoperative complications can be achieved in more complicated forms of rhegmatogenous retinal detachment. The major drawback of the procedure is the high incidence of post-operative cataract formation.     

Enhanced linear dynamic range property of Franz-Keldysh effectwaveguide modulator

The spurious-free dynamic range (SFDR) of an InGaAsP-InP Franz-Keldysh effect (FKE) waveguide modulator is studied. Low-biasing the FKE modulator is shown to enhance the SFDR by 22 dB with a 3-dB optical power penalty. Polarization-independent RF operation is also obtained with low bias. At 2 mA photocurrent, the simulated narrowband and broadband SFDR are, respectively, 127 dB and 104 dB in a 1-Hz bandwidth. The measured result projects a narrowband SFDR of 129 dB-Hz ^4/5 at this photocurrent     

Physics and performance of calorimetric particle detectors with dielectric absorbers and superconducting phase transition thermometers

We present results obtained with detectors consisting of dielectric absorber crystals and superconducting phase transition thermometers made of Ir/Au bilayers. With a 31 g sapphire crystal and an Ir/Au thermometer operated at 45 mK we have obtained an energy resolution of 220 eV (FWHM) for collimated 5.9 ke V X-rays. To explain the measured pulse shapes we have developed a model which includes the effect of non-thermal phonons. Results obtained previously with a 18 g Si absorber and an Ir/Au thermometer with broad transition allowed checking this model over a wide temperature range. A heater experiment performed with this calorimeter provides further support of our model.     

Comparison of 12 assays for detecting hCG and related molecules in urine samples from Down syndrome pregnancies

Urine is a new medium for Down syndrome testing. In an effort to determine the best type of human chorionic gonadotropin (hCG)-related immunoassay for urine testing, we examined 14 Down syndrome and 91 unaffected pregnancy urine samples with 12 established assays. The assays included (a) those that detect hCG beta-core fragment only; (b) those that detect beta-core fragment with less than 18 per cent free beta-subunit cross-reactivity; (c) that which equally detects free beta-subunit and beta-core fragment; and (d) those that detect hCG, free beta-subunit, or combinations thereof. The seven type a and b assays had the highest sensitivity for Down syndrome. The median MOM for Down syndrome was 5.93 (range 4.73-7.53). At a 10 per cent false-positive rate, the median observed detection rate was 93 per cent (range 79-100 per cent) and the median predicted detection rate was 85 per cent (range 69-96 per cent). The assays that did not mainly detect beta-core fragment (types c and d) had poorer screening performance. The median MOM for Down syndrome was 2.70 (range 2.16-3.63 MOM). At a 10 per cent false-positive rate, the median observed detection rate was 50 per cent (range 36-64 per cent) and the median predicted detection rate was 37 per cent (range 21-62 per cent). We infer that the assays that only detect beta-core fragment, or beta-core fragment with minor free beta-subunit cross-reactivity (types a and b), are the better urine-based tests for Down syndrome screening.