A method to investigate sterilization processes and the bacterial inactivation resolved in time and space.

In this study, a Computational Fluid Dynamics (CFD) model was developed to predict all relevant phenomena occurring during a moist heat sterilization process at a high level of temporal and spatial resolution. The developed CFD model was used to simulate the distribution of, e.g., pressure, temperature and residual air within a large-scale industrial steam autoclave (multiphase flow models) which was not published until now. Moreover, the thermodynamic behavior and distribution of fluids and temperatures inside the sterilization load was simulated which were verified with measurements. Based on the obtained sterilization temperature profiles in connection with the sterilization environment (e.g., NCGs, natural convection), the bacterial inactivation could be simulated. A complete moist heat sterilization process was simulated, including all relevant phenomena inside an autoclave chamber and a Peritoneal Dialysis Bag System (PDBS), which represents a complex sterilization item. To verify the simulation results, simulated pressures and temperatures were compared with measurement data for both the autoclave chamber and the PDBS. The results show that the simulated and measured values were in excellent accordance. By using the novel CFD model, the distribution of steam and residual air inside the autoclave chamber, as well as the natural convection inside the sterilization load, could be precisely predicted. To predict the inactivation of Geobacillus stearothermophilus inside different moist heat environments, the CFD model was extended with bacterial inactivation kinetics based on measurement data. The simulation results clearly indicate that our developed CFD model can be used to predict the inactivation kinetics of bacteria, depending on the sterilization temperature profile of the sterilization process as well as the moist heat sterilization environment, and to resolve the kinetics in time and space. Therefore, the developed CFD model represents a powerful tool that might be used in the future to predict, e.g., ″worst case″ locations for any given autoclave and sterilization load or any other relevant process parameter, enabling the operator to develop an effective sterilization process.

Micro-Computed Tomographic Analysis of the Shaping Ability of XP-Endo Shaper in Oval-Shaped Distal Root Canals of Mandibular Molars.

OBJECTIVE: To compare the shaping ability of the XP-endo Shaper (XPS) system to the ProTaper Next (PTN) system in oval-shaped distal root canals. METHODS: From 12 mandibular molars, distal roots with moderately curved single oval canals were randomly assorted to be instrumented with XPS (experimental group) or PTN (control group) and then scanned using micro-computed tomography [Scan 1]. The root canals of the XPS samples were prepared following the manufacturer's instructions using 15 insertions (XPS15) and rescanned [Scan 2]. An additional 10 insertions to the working length were applied, totalling 25 insertions (XPS25), and the roots were rescanned again [Scan 3]. PTN samples were prepared up to the X3 instrument (PTNX3) and rescanned [Scan 2]. The dentine removed and the unprepared areas were assessed. Data were analysed using a t-test with significance at α=0.05. RESULTS: XPS25 was associated with a significantly greater dentine removal than XPS15 over the entire root canal length and in all three-thirds of the root canal (P<0.05). XPS25 significantly removed more dentine than PTNX3 in only the coronal third (P<0.05). XPS25 was also associated with a significantly smaller percentage of unprepared areas than XPS15 overall and in the coronal third (P<0.05). PTNX3 was associated with a significantly larger percentage of unprepared areas than XPS15 and XPS25 overall and in the coronal and middle thirds (P<0.05). CONCLUSION: Ten additional movements with XPS significantly improved instrumentation capacity, reducing the percentage of untouched surface areas but also removing more dentine.

Improving Optical Measurements: Non-Linearity Compensation of Compact Charge-Coupled Device (CCD) Spectrometers.

Charge-coupled device (CCD) spectrometers are widely used as detectors in analytical laboratory instruments and as sensors for in situ optical measurements. However, as the applications become more complex, the physical and electronic limits of the CCD spectrometers may restrict their applicability. The errors due to dark currents, temperature variations, and blooming can be readily corrected. However, a correction for uncertainty of integration time and wavelength calibration is typically lacking in most devices, and detector non-linearity may distort the signal by up to 5% for some measurements. Here, we propose a simple correction method to compensate for non-linearity errors in optical measurements where compact CCD spectrometers are used. The results indicate that the error due to the non-linearity of a spectrometer can be reduced from several hundred counts to about 40 counts if the proposed correction function is applied.

Permanent draft genome of Rhodopirellula sallentina SM41.

The genome of Rhodopirellula sallentina SM41 was sequenced as a permanent draft to supplement the full genome sequence of the type strain Rhodopirellula baltica SH1(T). This isolate is part of a larger study to gain insights into the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the third of a series of five publications reporting in total eight new permanent daft genomes of Rhodopirellula species.

Permanent draft genomes of the three Rhodopirellula baltica strains SH28, SWK14 and WH47.

The genomes of three Rhodopirellula baltica strains were sequenced as permanent drafts to complement the full genome sequence of the type strain R. baltica SH1(T). The isolates are part of a larger study to infer the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the first of a series of five publications reporting in total eight new permanent daft genomes of Rhodopirellula species.

Permanent draft genomes of the Rhodopirellula maiorica strain SM1.

The genome of Rhodopirellula maiorica strain SM1 was sequenced as a permanent draft to complement the full genome sequence of the type strain Rhodopirellula baltica SH1(T). This isolate is part of a larger study to infer the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the fifth of a series of five publications reporting in total eight new permanent daft genomes of Rhodopirellula species.

Permanent draft genomes of the two Rhodopirellula europaea strains 6C and SH398.

The genomes of two Rhodopirellula europaea strains were sequenced as permanent drafts to study the genomic diversity within this genus, especially in comparison with the closed genome of the type strain Rhodopirellula baltica SH1(T). The isolates are part of a larger study to infer the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the second of a series of five publications describing a total of eight new permanent daft genomes of Rhodopirellula species.

Permanent draft genome of Rhodopirellula rubra SWK7.

The genome of Rhodopirellula rubra strain SWK7 was sequenced as a permanent draft to complement the full genome sequence of the type strain Rhodopirellula baltica SH1(T). This isolate is part of a larger study to infer the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the fourth among a series of five publications reporting in a total of eight new permanent draft genomes of Rhodopirellula species.

Genetic diversity of Rhodopirellula strains.

Rhodopirellula baltica SH1(T) is a marine planctomycete with 7,325 genes in its genome. Ten strains of the genus Rhodopirellula were studied in whole genome microarray experiments to assess the extent of their genetic relatedness to R. baltica SH1(T). DNA of strains which were previously affiliated with the species R. baltica (OTU A) hybridized with 3,645-5,728 genes of the type strain on the microarray. Strains SH398 and 6C (OTU B), representing a closely related species with an average nucleotide identity of 88 %, showed less hybridization signals: 1,816 and 3,302 genes gave a hybridization signal, respectively. Comparative genomics of eight permanent draft genomes revealed the presence of over 4,000 proteins common in R. baltica SH1(T) and strains of OTU A or B. The genus Rhodopirellula is characterized by large genomes, with over 7,000 genes per genome and a core genome of around 3000 genes. Individual Rhodopirellula strains have a large portion of strain-specific genes.

Ammonium and attachment of Rhodopirellula baltica.

A dimorphic life cycle has been described for the planctomycete Rhodopirellula baltica SH1(T), with juvenile motile, free-swimming cells and adult sessile, attached-living cells. However, attachment as a response to environmental factors was not investigated. We studied the response of R. baltica to nitrogen limitation. In batch cultures, ammonium limitation coincided with a dominance of free-swimming cells and a low number of aggregates. Flow cytometry revealed a quantitative shift with increasing ammonium availability, from single cells towards attached cells in large aggregates. During growth of R. baltica on glucose and ammonium in chemostats, an ammonium addition caused a macroscopic change of the growth behaviour, from homogeneous growth in the liquid phase to a biofilm on the borosilicate glass wall of the chemostat vessel. Thus, an ammonium limitation-a carbon to nitrogen supply ratio of 30:1-sustained free-living growth without aggregate formation. A sudden increase in ammonium supply induced sessile growth of R. baltica. These observations reveal a response of Rhodopirellula baltica cells to ammonium: they abandon the free-swimming life, attach to particles and form biofilms.

Using sequential injection analysis to improve system and data reliability of online methods: determination of ammonium and phosphate in coastal waters.

This article summarises the advantages of the sequential injection analysis (SIA) for the online determination of nutrients in coastal waters. It concentrates on techniques to improve the reliability of the gained data by continuously monitoring one or more standards and on the advantages of online standard additions and offline determination of manually collected samples with the online SIA system. These measures are advantageous during method development and validation and can be used to verify the system performance on a regular base to reduce the amount of erroneous results. No changes in the flow system are necessary and the sample throughput is only slightly reduced. These techniques have been applied to a SIA system which is able to simultaneously determine ammonium and phosphate at a rate of more than 100 samples per hour each and detection limits (3sigma) of 0.06 muM and 0.05 muM. Results from a campaign in summer 2005 are shown.

Using sequential injection analysis for fast determination of phosphate in coastal waters.

A sequential injection analysis system (SIA) is described which is suited for the fast determination of filterable molybdate reactive phosphate (FRP, 0.2mum) in coastal waters. It processes up to 270 samples per hour with a detection limit (3sigma) of 0.05muM and is used for surface mapping of phosphate in areas with steep concentration gradients like the Wadden Sea. The determination is based on the reaction of phosphate with acidic molybdate to phosphomolybdate, which builds non-fluorescent ion pairs with rhodamine 6G. The remaining rhodamine fluorescence is detected at 550nm with an excitation at 470nm. Syringe pump, valve and detector were controlled by a self made python programme, which was optimised for high speed SIA measurements in monitoring applications.

Effective dephosphorylation of Src substrates by SHP-1.

The protein-tyrosine phosphatase SHP-1 is a negative regulator of multiple signal transduction pathways. We observed that SHP-1 effectively antagonized Src-dependent phosphorylations in HEK293 cells. This occurred by dephosphorylation of Src substrates, because Src activity was unaffected in the presence of SHP-1. One reason for efficient dephosphorylation was activation of SHP-1 by Src. Recombinant SHP-1 had elevated activity subsequent to phosphorylation by Src in vitro, and SHP-1 variants with mutated phosphorylation sites in the C terminus, SHP-1 Y538F, and SHP-1 Y538F,Y566F were less active toward Src-generated phosphoproteins in intact cells. A second reason for efficient dephosphorylation is the substrate selectivity of SHP-1. Pull-down experiments with different GST-SHP-1 fusion proteins revealed efficient interaction of Src-generated phosphoproteins with the SHP-1 catalytic domain rather than with the SH2 domains. Phosphopeptides that correspond to good Src substrates were efficiently dephosphorylated by SHP-1 in vitro. Phosphorylated "optimal Src substrate" AEEEIpYGEFEA (where pY is phosphotyrosine) and a phosphopeptide corresponding to a recently identified Src phosphorylation site in p120 catenin, DDLDpY(296)GMMSD, were excellent SHP-1 substrates. Docking of these phosphopeptides into the catalytic domain of SHP-1 by molecular modeling was consistent with the biochemical data and explains the efficient interaction. Acidic residues N-terminal of the phosphotyrosine seem to be of major importance for efficient substrate interaction. Residues C-terminal of the phosphotyrosine probably contribute to the substrate selectivity of SHP-1. We propose that activation of SHP-1 by Src and complementary substrate specificities of SHP-1 and Src may lead to very transient Src signals in the presence of SHP-1.

c-Jun regulates eyelid closure and skin tumor development through EGFR signaling.

To investigate the function of c-Jun during skin development and skin tumor formation, we conditionally inactivated c-jun in the epidermis. Mice lacking c-jun in keratinocytes (c-jun(Deltaep)) develop normal skin but express reduced levels of EGFR in the eyelids, leading to open eyes at birth, as observed in EGFR null mice. Primary keratinocytes from c-jun(Deltaep) mice proliferate poorly, show increased differentiation, and form prominent cortical actin bundles, most likely because of decreased expression of EGFR and its ligand HB-EGF. In the absence of c-Jun, tumor-prone K5-SOS-F transgenic mice develop smaller papillomas, with reduced expression of EGFR in basal keratinocytes. Thus, using three experimental systems, we show that EGFR and HB-EGF are regulated by c-Jun, which controls eyelid development, keratinocyte proliferation, and skin tumor formation.