Extended Incubation Time Research Articles

Characterization of the single-stranded DNA binding protein pVVGJΦ of VGJΦ phage from Vibrio cholerae

pVVGJΦ, a single-stranded DNA binding protein of the vibriophage VGJΦ was subject to biochemical analysis. Here, we show that this protein has a general affinity for single-stranded DNA (ssDNA) as documented by Electrophoretic Mobility Shift Assay (EMSA). The apparent molecular weight of the monomer is about 12.7kDa as measured by HPLC–SEC. Moreover, isoelectrofocusing showed an isoelectric point for pVVGJΦ of 6.82 pH units. Size exclusion chromatography in 150mM NaCl, 50mM sodium phosphate buffer, pH 7.0 revealed a major protein species of 27.0kDa, suggesting homodimeric protein architecture. Furthermore, pVVGJΦ binds ssDNA at extreme temperatures and the complex was stable after extended incubation times. Upon frozen storage at −20°C for a year the protein retained its integrity, biological activity and oligomericity. On the other hand, bioinformatics analysis predicted that pVVGJΦ protein has a disordered C-terminal, which might be involved in its functional activity. All the aforementioned features make pVVGJΦ interesting for biotechnological applications.

Bioinspired Crystallization of CaCO3 Coatings on Electrospun Cellulose Acetate Fiber Scaffolds and Corresponding CaCO3 Microtube Networks

This article describes the mineralization behavior of CaCO(3) crystals on electrospun cellulose acetate (CA) fibers by using poly(acrylic acid) (PAA) as a crystal growth modifier and further templating synthesis of CaCO(3) microtubes. Calcite film coatings composed of nanoneedles can form on the surfaces of CA fibers while maintaining the fibrous and macroporous structures if the concentration of PAA is in a suitable range. In the presence of a suitable concentration of PAA, the acidic PAA molecules will first adsorb onto the surface of CA fibers by the interaction between the OH moieties of CA and the carboxylic groups of PAA, and then the redundant carboxylic groups of PAA can ionically bind Ca(2+) ions on the surfaces of CA fibers, resulting in the local supersaturation of Ca(2+) ions on and near the fiber surface, which can induce the nucleation of CaCO(3) on the CA fibers instead of in bulk solution. Calcite microtube networks on the macroscale can be prepared by the removal of CA fibers after the CA@CaCO(3) composite is treated with acetone. When the CA fiber scaffold is immersed in CaCl(2) solution with an extended incubation time, the first deposited calcite coatings can act as secondary substrate, leading to the formation of smaller calcite mesocrystal fibers. The present work proves that inorganic crystal growth can occur even at an organic interface without the need for commensurability between the lattices of the organic and inorganic counterparts.

Mycoplasma Bovis Real-Time Polymerase Chain Reaction Assay Validation and Diagnostic Performance

Mycoplasma bovis is an important bacterial pathogen in cattle, producing a variety of clinical diseases. The organism, which requires specialized culture conditions and extended incubation times to isolate and identify, is frequently associated with concurrent infection with other pathogens which can potentially be more easily identified. Real-time polymerase chain reaction (real-time PCR) is a valuable diagnostic technique that can rapidly identify infectious agents in clinical specimens. A real-time PCR assay was designed based on the uvrC gene to identify M. bovis in diagnostic samples. Using culture as the gold standard test, the assay performed well in a variety of diagnostic matrices. Initial validation testing was conducted on 122 milk samples (sensitivity: 88.9% [95% confidence interval (CI): 68.4-100%], specificity: 100%); 154 lung tissues (sensitivity: 89.0% [95% CI: 83.1-94.9%], specificity: 97.8% [95% CI: 93.5-100%]); 70 joint tissue/fluid specimens (sensitivity: 92.3% [95% CI: 82.1-100%], specificity: 95.5% [95% CI: 89.3-100%]); and 26 nasal swabs (sensitivity: 75.0% [95% CI: 45.0-100%], specificity: 83.3% [95% CI: 66.1-100%]). Low numbers of other sample matrices showed good agreement between results of culture and PCR. A review of clinical cases from 2009 revealed that, in general, PCR was used much more frequently than culture and provided useful diagnostic information in conjunction with clinical signs, signalment, and gross and histopathologic lesions. Diagnostic performance of the real-time PCR assay developed as a testing method indicates that it is a rapid, accurate assay that is adaptable to a variety of PCR platforms and can provide reliable results on an array of clinical samples.

Specificity and kinetics of norovirus binding to magnetic bead-conjugated histo-blood group antigens

To characterize the specificity and effect of pH and ionic strength on the kinetics of virus binding to histo-blood group antigens (HBGA)-conjugated magnetic beads. HBGAs from porcine gastric mucin (PGM) have been conjugated to magnetic beads (PGM-MB) for concentration of NoV. A GII.4 virus was used for the detailed binding kinetics study and a panel of genogroup I (GI) NoVs, genogroup II (GII) NoVs and recombinant NoVs (rNoVs) were used for specificity and binding efficiency assays. We determined that NoV can be captured after 15min of incubation with PGM-MB, and virus recovery efficiency is decreased after extended incubation times. rNoV binding as measured by ELISA and NoV recovery as measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), were both enhanced significantly at acidic pH conditions. rNoV binding to PGM as measured by ELISA was increased up to 66%. While real-time RT-PCR analyses suggest that NoV could be concentrated as much as 1000-fold at neutral pH, up to 3·4-fold further increase of NoV recovery was achieved by adjusting the pH of the sample to 3·0-4·2. Variation between GI and GII viral binding to the PGM-MB at basic pH was observed. All five GI rNoVs tested and 6 of 9 GII rNoVs were captured by PGM. All eight GI strains tested were concentrated by PGM-MB, ranging from 28-fold (GI.4) to 1502-fold (GI.1). Eleven of 13 GII strains were concentrated from 30-fold (GII.5) to 1014-fold (GII.4, lab strain) by PGM-MB. GI and GII rNoVs viral capsid proteins were recovered with high salt conditions, but results were inconsistent for whole virus recovery. All GI and 85% of GII NoVs tested could be captured and concentrated by PGM-MB method. The binding occurred rapidly and was enhanced at low pH. These results facilitated development of a prototype method for sensitive detection of NoV in samples requiring larger volumes.

Ribosomes Lacking Protein S20 Are Defective in mRNA Binding and Subunit Association

The functional significance of ribosomal proteins is still relatively unclear. Here, we examined the role of small subunit protein S20 in translation using both in vivo and in vitro techniques. By means of lambda red recombineering, the rpsT gene, encoding S20, was removed from the chromosome of Salmonella enterica var. Typhimurium LT2 to produce a ΔS20 strain that grew markedly slower than the wild type while maintaining a wild-type rate of peptide elongation. Removal of S20 conferred a significant reduction in growth rate that was eliminated upon expression of the rpsT gene on a high-copy-number plasmid. The in vitro phenotype of mutant ribosomes was investigated using a translation system composed of highly active, purified components from Escherichia coli. Deletion of S20 conferred two types of initiation defects to the 30S subunit: (i) a significant reduction in the rate of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in association with the 50S subunit. Both of these impairments were partially relieved by an extended incubation time with mRNA, fMet-tRNAfMet, and initiation factors, indicating that absence of S20 disturbs the structural integrity of 30S subunits. Considering the topographical location of S20 in complete 30S subunits, the molecular mechanism by which it affects mRNA binding and subunit docking is not entirely obvious. We speculate that its interaction with helix 44 of the 16S ribosomal RNA is crucial for optimal ribosome function.

5.07] Recovery fluorescence after 5-ALA-induced PpIX-photodynamic therapy

Objective Loss of fluorescence is used to determine the endpoint of irradiation in PDT with 5-ALA of non-melanoma skin cancers (NMSC) as actinic keratosis (AK) and superficial basal cell carcinomas (sBCC). Based on earlier research, we investigated the appearance of recovery fluorescence (recurrence of any fluorescence after initial therapeutic irradiation) with regard to the type of lesion. As penetration of any applicable drug into skin is time-related and the production of PpIX from 5-ALA depends on the transformation of the externally applied precursor (5-ALA) by the diseased cells, thicker lesions should behave different from smaller lesions with regard to the time frame. Material and methods In 20 patients with AK, Bowen's disease (MB) or BCC-lesions 5-ALA/Tylose-gel (20%) was applied under occlusion and light shielding dressings for 2 h. Then the remaining gel was removed and incubation with light shielding was continued for another two hours. After a total of four hours fluorescence detection with a D-light-system (Storz) was performed and signal wavelength, relative intensity and the contrast to surrounding tissues were measured (AVASpec). Irradiation with 635 nm up to a dose of 37–74 J/cm2 with LED (Aktilite) in AK and 70–125 J/cm2 with diode laser (Biolitec) in BCC was performed under protective (non-bleaching) yellow ambient light. Irradiation was maintained until intermittent fluorescence monitoring showed the complete loss of signal. The lesions were covered again with light shielding dressings for 24 h and fluorescence was re-evaluated. If fluorescence re-occurred, another irradiation was performed until no recurrent fluorescence could be detected each 24 h. Anesthesia was maintained by cooled air stream and if requested, either by regionally or locally applied xylocain. Results AK displayed less frequent recovery fluorescence than deeper lesions (MB, BCC) at day two and none at day three. In most patients with MB or BCC a significant recovery-fluorescence was found. All patients with recovery-fluorescence received further irradiation with 635 nm-light until PpIX-specific fluorescence was eliminated. Local necrosis was more pronounced in patients who had received repeated irradiations; nevertheless some recurrences were seen during a follow-up of at least six months than in patients who were treated conventionally with one irradiation (e.g. in BCC). Conclusion The finding of a recovery-fluorescence suggests: (a) excess amount of 5-ALA in the lesions, (b) deeper penetration of 5-ALA with extended incubation times, (c) local persistence of viable cells being able to produce PpIX after first irradiation, (d) a need for an individualized light and drug dosimetry including prolonged incubation times, repeated irradiation and fluorescence control during topically ALA-induced PpIX-PDT.

Satratoxin G interaction with 40S and 60S ribosomal subunits precedes apoptosis in the macrophage

Satratoxin G (SG) and other macrocyclic trichothecene mycotoxins are potent inhibitors of eukaryotic translation that are potentially immunosuppressive. The purpose of this research was to test the hypothesis that SG-induced apoptosis in the macrophage correlates with binding of this toxin to the ribosome. Exposure of RAW 264.7 murine macrophages to SG at concentrations of 10 to 80 ng/ml induced DNA fragmentation within 4 h that was indicative of apoptosis. To relate these findings to ribosome binding of SG, RAW cells were exposed to different toxin concentrations for various time intervals, ribosomal fractions isolated by sucrose density gradient ultracentrifugation and resultant fractions analyzed for SG by competitive ELISA. SG was found to specifically interact with 40S and 60S ribosomal subunits as early as 5 min and that, at high concentrations or extended incubation times, the toxin induced polysome disaggregation. While co-incubation with the simple Type B trichothecene DON had no effect on SG uptake into cell cytoplasm, it inhibited SG binding to the ribosome, suggesting that the two toxins bound to identical sites and that SG binding was reversible. Although both SG and DON induced mobilization of p38 and JNK 1/2 to the ribosome, phosphorylation of ribosomal bound MAPKs occurred only after DON treatment. SG association with the 40S and 60S subunits was also observed in the PC-12 neuronal cell model which is similarly susceptible to apoptosis. To summarize, SG rapidly binds small and large ribosomal subunits in a concentration- and time-dependent manner that was consistent with induction of apoptosis.

Transmembrane Form Agrin-induced Process Formation Requires Lipid Rafts and the Activation of Fyn and MAPK

Overexpression or clustering of the transmembrane form of the extracellular matrix heparan sulfate proteoglycan agrin (TM-agrin) induces the formation of highly dynamic filopodia-like processes on axons and dendrites from central and peripheral nervous system-derived neurons. Here we show that the formation of these processes is paralleled by a partitioning of TM-agrin into lipid rafts, that lipid rafts and transmembrane-agrin colocalize on the processes, that extraction of lipid rafts with methyl-beta-cyclodextrin leads to a dose-dependent reduction of process formation, that inhibition of lipid raft synthesis prevents process formation, and that the continuous presence of lipid rafts is required for the maintenance of the processes. Association of TM-agrin with lipid rafts results in the phosphorylation and activation of the Src family kinase Fyn and subsequently in the phosphorylation and activation of MAPK. Inhibition of Fyn or MAPK activation inhibits process formation. These results demonstrate that the formation of filopodia-like processes by TM-agrin is the result of the activation of a complex intracellular signaling cascade, supporting the hypothesis that TM-agrin is a receptor or coreceptor on neurons.

Dynamic Histone H1 Isotype 4 Methylation and Demethylation by Histone Lysine Methyltransferase G9a/KMT1C and the Jumonji Domain-containing JMJD2/KDM4 Proteins

The linker histone H1 generally participates in the establishment of chromatin structure. However, of the seven somatic H1 isotypes in humans some are also implicated in the regulation of local gene expression. Histone H1 isotype 4 (H1.4) represses transcription, and its lysine residue 26 (Lys26) was found to be important in this aspect. H1.4K26 is known to be methylated and acetylated in vivo, but the enzymes responsible for these post-translational modifications and the regulatory cues that promote H1.4 residence on chromatin are poorly characterized. Here we report that the euchromatic histone lysine methyltransferase G9a/KMT1C mediates H1.4K26 mono- and dimethylation in vitro and in vivo and thereby provides a recognition surface for the chromatin-binding proteins HP1 and L3MBTL1. Moreover, we show evidence that G9a promotes H1 deposition and is required for retention of H1 on chromatin. We also identify members of the JMJD2/KDM4 subfamily of jumonji-C type histone demethylases as being responsible for the removal of H1.4K26 methylation.

Surface Characteristics of Orthodontic Materials and Their Effects on Adhesion of Mutans streptococci

To test the hypothesis that there are no significant differences in the adhesion of mutans streptococci (MS) to various orthodontic materials based on their surface characteristics. Surface roughness (SR) and surface free energy (SFE) characteristics were investigated for nine different orthodontic materials (four orthodontic adhesives, three bracket raw materials, hydroxyapatite blocks, and bovine incisors) using confocal laser scanning microscopy and sessile drop method. Each material, except the bovine incisors, was incubated with whole saliva or phosphate-buffered saline for 2 hours. Adhesion assays were performed by incubating tritium-labeled MS with each material for 3 or 6 hours. Orthodontic adhesives had higher SFE characteristics and lower SR than bracket materials. Orthodontic adhesives showed a higher MS retaining capacity than bracket materials, and MS adhesion to resin-modified glass ionomer and hydroxyapatite was highest. Extended incubation time increased MS adhesion, while saliva coating did not significantly influence MS adhesion. SFE, specifically its dispersive and polar components, was positively correlated with MS adhesion, irrespective of saliva coating. The hypothesis is rejected. This study suggests that SFE characteristics play an important role in the initial MS adhesion to orthodontic materials.

Evaluation of three automated chromogenic FVIII kits for the diagnosis of mild discrepant haemophilia A

In some mild haemophilia A patients (discrepant phenotype), coagulation FVIII levels by one-stage assay (FVIII-1st) are more than double those by classical two-stage coagulation assay (FVIII-2st), and may fall within the normal range. Our aim was to assess automated two-stage chromogenic FVIII assays (FVIII-chr) for diagnosis of mild discrepant haemophilia A. Three chromogenic FVIII kits (Biophen, Coamatic and Dade-Behring) were evaluated, using recommended and extended incubation times. Samples were tested from patients with discrepant haemophilia (n = 7) and equivalent mild haemophilia (agreement between FVIII-1st and FVIII-2st, n = 4). For equivalent haemophilia, FVIII-chr were consistent with FVIII-1st and FVIII-2st for all kits at all incubation times. For discrepant haemophilia, using recommended incubation times, mean FVIII-chr using Biophen reagents was 22 IU/dl (range 13-31), with Coamatic 26 (17-34) and with Dade-Behring 41 (33-47), compared with 36 (27-44) for FVIII-1st and 8 (6-9) for FVIII-2st. FVIII-chr decreased as incubation time was increased with Biophen and Coamatic, but decreased less with Dade-Behring. FVIII-chr using the Dade-Behring kit gave similar results to FVIII-1st and is not suitable for diagnosis of mild discrepant haemophilia A. FVIII-chr by Biophen and Coamatic kits is suitable for diagnosis of these patients, especially with an extended incubation time.

미국산과 인도산 옥수수의 steam flaking 처리가 공정라인별 mycotoxin 함량의 변화와 in vitro 발효 특성에 미치는 영향

본 연구는 미국산 옥수수와 인도산 옥수수의 steam flaking 처리가 in vtro 가스발생량과 미생물 성장량 그리고 곰팡이 독소 aflatoxin <TEX>$B_1$</TEX>과 ochratoxin A의 농도에 미치는 영향을 구명하기 위하여 수행되었다. 실험 설계는 4개의 처리구, (1) USCW (미국산 무처리 옥수수), (2) USCF (미국산 steam flaking 옥수수 ), (3) IDCW (인도산 무처리 옥수수) 그리고 (4) IDCF (인도산 steam flaking 옥수수), 처리구당 4반복으로 6개의 발효시간대(3, 6, 9, 12, 18 및 24)를 두고 in vitro 실험을 수행하였다. 공시한 옥수수중 aflatoxin <TEX>$B_1$</TEX>이나 ochratoxin A와 같은 곰팡이 독소의 함량은 항구, hopper, 사일로 그리고 가공 전까지 보관기간이 길어짐에 따라 증가하는 경향을 보였다. 공정라인별로 곰팡이 독소를 측정한 결과 입고 시 인도산 옥수수(IDCW)와 미국산 옥수수 (USCW)의 aflatoxin <TEX>$B_1$</TEX> 수치는 각각 11.71 ppb와 1.78 ppb으로 나타났지만 steam flaking 후의 aflatoxin <TEX>$B_1$</TEX> 함량은 USCW 구와 IDCW구에서 전혀 검출되지 않아(0.00 ppb) steam flaking 처리가 곰팡이 Aspergillus flavus를 감소시킬 수 있는 것으로 조사되었다. 그러나 이러한 경향은 ochratoxin A 함량에서 관찰되지 않았다. In vitro 실험에서 gas 발생량은 원산지별로는 미국산 옥수수 (USCW & USCF)가 인도산 옥수수 (IDCW & IDCF) 보다 유의적으로 높았으며 가공 처리별로는 steam flaking 처리한 옥수수가 알곡 옥수수보다 발효 3시간대를 기준으로 <TEX>$1.5{\sim}2%$</TEX> 정도 높았다. 배양액 중의 pH 는 <TEX>$6.05{\sim}6.54$</TEX>의 범위로서 미생물이 성장하기에 적정한 pH를 유지하였으며 처리 구간에 유의적인 차이는 찾아 볼 수 없었으나 USCF구의 pH가 다른구에 비해 다소 낮았다. pH는 배양 12 시간까지 감소하였으며 이 시간 중에 가스 발생량은 급격히 증가하였다. In vitro 미생물 성장량도 발효 18시간까지 증가하다가 그 이후 시간대에서는 성장량이 증체를 보이거나 오히려 감소하는 경향이었다. 결론적으로 원산지별로는 in vitro 실험결과와 곰팡이 독소 함량을 기준으로, 미국산 옥수수가 인도산 옥수수보다는 품질이 훨씬 높았으며, 수입산 옥수수의 steam flaking 처리는 invitro 가스발생량 및 미생물 성장량을 개선 시킬 뿐만 아니라, aflatoxin <TEX>$B_1$</TEX>이나 ochratoxin A와 같은 곰팡이 독소를 감소시키는 역할을 하였다. The objective of this study was to examine the effects of steam flaking treatment of corn grains imported from USA and India on in vitro gas production, microbial growth and contents of aflatoxin <TEX>$B_1$</TEX> and ochratoxin A. Each treatment was composed of total 4 treatments including (1) USCW (US com-whole type), (2) USCF (US corn-flaked type), (3) IDCW (India corn-whole type) and (4) IDCF (India orn-flaked type) with 4 replications <TEX>$\times$</TEX> 6 incubation times (3, 6, 9, 12, 18 and 24 hr). Mycotoxin (aflatoxin <TEX>$B_1$</TEX> & ochratoxin A) contents in test corns tended to increase gradually with increasing logistics periods from the harbor, hopper, silo to processing line. The contents of aflatoxin <TEX>$B_1$</TEX> in India corn (IDCW) and US corn (USCW) were 11.71 and 1.78 ppb, respectively when measured at the hopper. After steam flaking, both contents of aflatoxin <TEX>$B_1$</TEX> in USCW and IDCW were 0.00 ppb. It means that Aspergillus flavus could be decreased by steam flaking. However, this trend was not observed in ochratoxin content. The gas production rate of USA corns (USCW & USCF) was significantly (p<0.05) higher than India corns (IDCW & IDCF), and that of steam flaked corns (USCF & IDCF) was higher <TEX>$1.5{\sim}2%$</TEX> than whole corns (USCW & IDCW) after 3 hr incubation in in vitro experiment. pH value was optimally maintained for microbial growth during whole incubation times with the value of 6.05 to 6.54, and was not significantly different between treatments, but USCF was somewhat lower than other treatments. pH value decreased following 12 hr of incubation but gas production increased rapidly during the same period. In addition, in vitro microbial growth rates also increased with up to 18 hr of incubation period, thereafter experienced a decrease with extended incubation time. In conclusion, US corn was superior to India corn by origin based on the results of in vitro and mycotoxin contents. And steam flaking process of imported corns tended to decrease mycotoxin contents such as aflatoxin <TEX>$B_1$</TEX> and ochratoxin A as well as improve in vitro gas production and microbial growth rates.

Displacement of the Occluding Loop by the Parasite Protein, Chagasin, Results in Efficient Inhibition of Human Cathepsin B

Cathepsin B is a papain-like cysteine protease showing both endo- and exopeptidase activity, the latter due to a unique occluding loop that restricts access to the active site cleft. To clarify the mode by which natural protein inhibitors manage to overcome this obstacle, we have analyzed the structure and function of cathepsin B in complexes with the Trypanosoma cruzi inhibitor, chagasin. Kinetic analysis revealed that substitution of His-110e, which anchors the loop in occluding position, results in 3-fold increased chagasin affinity (Ki for H110A cathepsin B, 0.35 nm) due to an improved association rate (kon, 5 x 10(5) m(-1)s(-1)). The structure of chagasin in complex with cathepsin B was solved in two crystal forms (1.8 and 2.67 angstroms resolution), demonstrating that the occluding loop is displaced to allow chagasin binding with its three loops, L4, L2, and L6, spanning the entire active site cleft. The occluding loop is differently displaced in the two structures, indicating a large range of movement and adoption of conformations forced by the inhibitor. The area of contact is slightly larger than in chagasin complexes with the endopeptidase, cathepsin L. However, residues important for high affinity to both enzymes are mainly found in the outer loops L4 and L6 of chagasin. The chagasin-cathepsin B complex provides a structural framework for modeling and design of inhibitors for cruzipain, the parasite cysteine protease and a virulence factor in Chagas disease.

Isolation of novel bacteria, including a candidate division, from geothermal soils in New Zealand

We examined bacterial diversity of three geothermal soils in the Taupo Volcanic Zone of New Zealand. Phylogenetic analysis of 16S rRNA genes recovered directly from soils indicated that the bacterial communities differed in composition and richness, and were dominated by previously uncultured species of the phyla Actinobacteria, Acidobacteria, Chloroflexi, Proteobacteria and candidate division OP10. Aerobic, thermophilic, organotrophic bacteria were isolated using cultivation protocols that involved extended incubation times, low-pH media and gellan as a replacement gelling agent to agar. Isolates represented previously uncultured species, genera, classes, and even a new phylum of bacteria. They included members of the commonly cultivated phyla Proteobacteria, Firmicutes, Thermus/Deinococcus, Actinobacteria and Bacteroidetes, as well as more-difficult-to-cultivate groups. Isolates possessing < 85% 16S rRNA gene sequence identity to any cultivated species were obtained from the phyla Acidobacteria, Chloroflexi and the previously uncultured candidate division OP10. Several isolates were prevalent in 16S rRNA gene clone libraries constructed directly from the soils. A key factor facilitating isolation was the use of gellan-solidified plates, where the gellan itself served as an energy source for certain bacteria. The results indicate that geothermal soils are a rich potential source of novel bacteria, and that relatively simple cultivation techniques are practical for isolating bacteria from these habitats.

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

A purification step prior to commercial sensitive immunoassay is necessary to achieve clinical usefulness when quantifying serum 17β-estradiol in prepubertal children

To test the clinical usefulness of sensitive commercial immunoassays for determination of low 17beta-estradiol concentrations in children. The lower limit of detection and clinical usefulness (functional sensitivity) of three commercial estradiol immunoassays were validated by use of 500 sera from prepubertal and pubertal children and 55 pooled sera. The three immunoassays consisted of two modified direct immunoassays; one RIA (Spectria Estradiol RIA) and one time-resolved fluoroimmunoassay (AutoDELFIA Estradiol), both with increased serum volume in relation to antibody concentration and extended incubation time. In the third method, serum was purified and concentrated using diethyl ether extraction prior to measurement by the modified Spectria Estradiol RIA. The lower limits of detection and clinical usefulness were 9 and 30 pmol/l for the direct RIA, 11 and 50 pmol/l for the AutoDELFIA, and 4 and 6 pmol/l for serum determined by extraction RIA. When measuring the serum pool originating from girls at breast stages 1-2, the direct RIA and AutoDELFIA resulted in significantly higher 17beta-estradiol concentrations when compared with the extraction RIA (+58 and +267%, P<0.001). We found a significant difference in 17beta-estradiol concentrations between girls at breast stages 1 (median 6 pmol/l) and 2 (median 16 pmol/l), when quantified by the extraction RIA (P<0.0001) but no difference when quantified with the direct RIA (median values 12 and 14 pmol/l respectively). For determination of low serum 17beta-estradiol concentrations in children, an extraction step prior to commercial immunoassay is needed to achieve clinically useful results.

ApoA-I cleaved by transthyretin has reduced ability to promote cholesterol efflux and increased amyloidogenicity

A fraction of plasma transthyretin (TTR) circulates in HDL through binding to apolipoprotein A-I (apoA-I). Moreover, TTR is able to cleave the C terminus of lipid-free apoA-I. In this study, we addressed the relevance of apoA-I cleavage by TTR in lipoprotein metabolism and in the formation of apoA-I amyloid fibrils. We determined that TTR may also cleave lipidated apoA-I, with cleavage being more effective in the lipid-poor prebeta-HDL subpopulation. Upon TTR cleavage, discoidal HDL particles displayed a reduced capacity to promote cholesterol efflux from cholesterol-loaded THP-1 macrophages. In similar assays, TTR-containing HDL from mice expressing human TTR in a TTR knockout background had a decreased ability to perform reverse cholesterol transport compared with similar particles from TTR knockout mice, reinforcing the notion that cleavage by TTR reduces the ability of apoA-I to promote cholesterol efflux. As amyloid deposits composed of N-terminal apoA-I fragments are common in the atherosclerotic intima, we assessed the impact of TTR cleavage on apoA-I aggregation and fibrillar growth. We determined that TTR-cleaved apoA-I has a high propensity to form aggregated particles and that it formed fibrils faster than full-length apoA-I, as assessed by electron microscopy. Our results show that apoA-I cleavage by TTR may affect HDL biology and the development of atherosclerosis by reducing cholesterol efflux and increasing the apoA-I amyloidogenic potential.

Histochemical studies on the accumulation of reactive oxygen species (O 2− and H 2O 2) in the incompatible and compatible interaction of wheat— Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O 2 −) and hydrogen peroxide (H 2O 2), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O 2 − and H 2O 2 was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H 2O 2 accumulation in guard cells. In the incompatible interaction, a rapid increase of O 2 − and H 2O 2 generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H 2O 2 accumulation further increased, whereas those exhibiting O 2 − accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O 2 − and H 2O 2 generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H 2O 2 accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H 2O 2 accumulation was observed in mesophyll cells surrounding the rust lesion.

Nokardioseinfektion einer Gefäßendoprothese

A 61-year-old woman presented with purple-red subcutaneous nodules on her right leg. She had experienced fever up to 40 degrees C for the past 10 days and felt generally weak over the last two months. Four months earlier, a vascular graft had been implanted in her right femoral artery. Based on the diagnosis of skin infection due to implantation of an infected prosthesis, she was hospitalized and treated with an antibiotic regime. During the initial antibiotic treatment, the symptoms deteriorated, and she developed joint, hepatic and CNS abscesses. Finally, a microbiologic culture with an extended incubation time revealed the diagnosis of an infection with the slowly growing bacterium Nocardia ssp. Hereupon the patient was treated purposefully with Meropenem over six months. This treatment resulted in complete recovery.

Development and comparisons of efficient gas-cultivation systems for anaerobic carbon monoxide-utilizing microorganisms

We describe a system for the cultivation of gaseous substrate utilizing microorganisms that overcomes some of the limitations of fixed volume culture vessels and the costs associated with sparging. Cali-5-Bond™ gas-sampling bag was used as the culture vessel. The bags contain approximately six times more mass of CO than the 40 mL vials at 1 atm of pressure and performed equally to the 40 mL vials in terms of their ability to maintain the composition of the gas over extended incubation times. Experiments using Clostridium ljungdahlii and CO as the sole carbon and energy source in both the gas sampling bag cultivation system and the traditional vial system demonstrated that this culture had a 15× increase in optical density in 24 h of incubation. The gas-sampling bags offer a viable alternative to gas sparging while overcoming the limitations of fixed volume culture vessels.