Dynamic SpoIIIE assembly mediates septal membrane fission during Bacillus subtilis sporulation.

SpoIIIE is an FtsK-related protein that transports the forespore chromosome across the Bacillus subtilis sporulation septum. We use membrane photobleaching and protoplast assays to demonstrate that SpoIIIE is required for septal membrane fission in the presence of trapped DNA, and that DNA is transported across separate daughter cell membranes, suggesting that SpoIIIE forms a channel that partitions the daughter cell membranes. Our results reveal a close correlation between septal membrane fission and the assembly of a stable SpoIIIE translocation complex at the septal midpoint. Time-lapse epifluorescence, total internal reflection fluorescence (TIRF) microscopy, and live-cell photoactivation localization microscopy (PALM) demonstrate that the SpoIIIE transmembrane domain mediates dynamic localization to active division sites, whereas the assembly of a stable focus also requires the cytoplasmic domain. The transmembrane domain fails to completely separate the membrane, and it assembles unstable foci. TIRF microscopy and biophysical modeling of fluorescence recovery after photobleaching (FRAP) data suggest that this unstable protein transitions between disassembled and assembled oligomeric states. We propose a new model for the role of SpoIIIE assembly in septal membrane fission that has strong implications for how the chromosome terminus crosses the septum.

Aluminiums for thixoforging and preparatory conditions and parameters necessary before forming: A review.

The thixoforging of aluminium alloys has received in recent years an extensive interest for the manufacture of parts because of its advantages. To choose an aluminium alloy as raw material for a thixoforming process, it is important to know, the product characteristics, respectively the expected mechanical properties, and secondly, the semi-solid interval and the temperature of fusion. Various wrought and foundry aluminium alloys are thoroughly summarized in this review paper, together with a description of the evaluation methods for the heating temperature sensitivity. Furthermore, for thixoforging industrial applications, the heating strategy is very important, and the reheating regime should be systematically analysed. The advantages offered by thixoforging of aluminium alloys are associated to the feedstock material microstructure that results after reheating to the semi-solid range, which is key to understand the rheological behaviour and the final mechanical properties of the thixoformed parts. In the future, a process model needs to be developed to integrate microstructure conditioning during reheating of the material to the semi-solid state.

Origin and fate of the 3' ends of single-stranded DNA generated by conjugal transfer of plasmid R1162.

During conjugation, a single strand of DNA is cleaved at the origin of transfer (oriT) by the plasmid-encoded relaxase. This strand is then unwound from its complement and transferred in the 5'-to-3' direction, with the 3' end likely extended by rolling-circle replication. The resulting, newly synthesized oriT must then be cleaved as well, prior to recircularization of the strand in the recipient. Evidence is presented here that the R1162 relaxase contains only a single nucleophile capable of cleaving at oriT, with another molecule therefore required to cleave at a second site. An assay functionally isolating this second cleavage shows that this reaction can take place in the donor cell. As a result, there is a flux of strands with free 3' ends into the recipient. These ends are susceptible to degradation by exonuclease I. The degree of susceptibility is affected by the presence of an uncleaved oriT within the strand. A model is presented where these internal oriTs bind and trap the relaxase molecule covalently bound to the 5' end of the incoming strand. Such a mechanism would result in the preferential degradation of transferred DNA that had not been properly cleaved in the donor.

Systems-wide analysis revealed shared and unique responses to moderate and acute high temperatures in the green alga Chlamydomonas reinhardtii.

Different intensities of high temperatures affect the growth of photosynthetic cells in nature. To elucidate the underlying mechanisms, we cultivated the unicellular green alga Chlamydomonas reinhardtii under highly controlled photobioreactor conditions and revealed systems-wide shared and unique responses to 24-hour moderate (35°C) and acute (40°C) high temperatures and subsequent recovery at 25°C. We identified previously overlooked unique elements in response to moderate high temperature. Heat at 35°C transiently arrested the cell cycle followed by partial synchronization, up-regulated transcripts/proteins involved in gluconeogenesis/glyoxylate-cycle for carbon uptake and promoted growth. But 40°C disrupted cell division and growth. Both high temperatures induced photoprotection, while 40°C distorted thylakoid/pyrenoid ultrastructure, affected the carbon concentrating mechanism, and decreased photosynthetic efficiency. We demonstrated increased transcript/protein correlation during both heat treatments and hypothesize reduced post-transcriptional regulation during heat may help efficiently coordinate thermotolerance mechanisms. During recovery after both heat treatments, especially 40°C, transcripts/proteins related to DNA synthesis increased while those involved in photosynthetic light reactions decreased. We propose down-regulating photosynthetic light reactions during DNA replication benefits cell cycle resumption by reducing ROS production. Our results provide potential targets to increase thermotolerance in algae and crops.

Evaluation of an airborne alpha and beta particle detection capability using an environmental continuous air monitor system.

We evaluate the ability of the Canberra Alpha Beta Environmental Continuous Air Monitor (ECAM) to detect and quantify airborne radiological contamination. The ECAM essentially consists of a passively-implanted planar silicon (PIPS) detector near a particulate filter through which outside air is pulled. Three years' worth of background measurements on three different systems were assessed and calibrated to compensate for changing conditions and develop an average background response for the systems. The ECAM was also exposed to several radionuclides of interest, including 235U and 239Pu, to measure the response to alpha and beta particle sources. Both standard calibration sources and custom sources consisting of aqueous radioisotope solutions absorbed into clean filters. The ECAM responses to these sources were then scaled to quantities of interest and injected on the averaged background. Various alarm algorithms were evaluated on the source-injected spectra for minimum detectable air concentration for a given false alarm rate. Even in the worst case, the ECAM was able to detect radionuclides of interest at 10% of the Derived Response Level (DRL) for each isotope based on early-phase Protective Action Guides (PAG). Quantification of the radionuclides was also evaluated for the various algorithms, with mixed results, but overall clearly indicating the optimal algorithms for alpha and beta particle alarm and quantification. Finally, a limited evaluation of the beta particle detection efficiency points to a detection energy threshold of approximately 290 keV.

High light and temperature reduce photosynthetic efficiency through different mechanisms in the C4 model Setaria viridis.

C4 plants frequently experience high light and high temperature conditions in the field, which reduce growth and yield. However, the mechanisms underlying these stress responses in C4 plants have been under-explored, especially the coordination between mesophyll (M) and bundle sheath (BS) cells. We investigated how the C4 model plant Setaria viridis responded to a four-hour high light or high temperature treatment at photosynthetic, transcriptomic, and ultrastructural levels. Although we observed a comparable reduction of photosynthetic efficiency in high light or high temperature treated leaves, detailed analysis of multi-level responses revealed important differences in key pathways and M/BS specificity responding to high light and high temperature. We provide a systematic analysis of high light and high temperature responses in S. viridis, reveal different acclimation strategies to these two stresses in C4 plants, discover unique light/temperature responses in C4 plants in comparison to C3 plants, and identify potential targets to improve abiotic stress tolerance in C4 crops.

Phylogenetic analysis identifies many uncharacterized actin-like proteins (Alps) in bacteria: regulated polymerization, dynamic instability and treadmilling in Alp7A.

Actin, one of the most abundant proteins in the eukaryotic cell, also has an abundance of relatives in the eukaryotic proteome. To date though, only five families of actins have been characterized in bacteria. We have conducted a phylogenetic search and uncovered more than 35 highly divergent families of actin-like proteins (Alps) in bacteria. Their genes are found primarily on phage genomes, on plasmids and on integrating conjugative elements, and are likely to be involved in a variety of functions. We characterize three Alps and find that all form filaments in the cell. The filaments of Alp7A, a plasmid partitioning protein and one of the most divergent of the Alps, display dynamic instability and also treadmill. Alp7A requires other elements from the plasmid to assemble into dynamic polymers in the cell. Our findings suggest that most if not all of the Alps are indeed actin relatives, and that actin is very well represented in bacteria.

An XBP-1 dependent bottle-neck in production of IgG subtype antibodies in chemically defined serum-free Chinese hamster ovary (CHO) fed-batch processes.

The optimization of production processes for therapeutic antibodies is a continuing challenge in pharmaceutical biotechnology. Although it could be demonstrated that vector design and host cell engineering can improve transcriptional and translational efficiency and thereby result in generation of high producer cell lines, it is not clear whether introduction of transgenes that regulate protein transport or affect post-translational modifications could further improve such industrial processes. Here, we show that heterologous expression of the transcription factor X-box binding protein-1 (XBP-1) can lead to an increase in endoplasmic reticulum (ER) content and specific therapeutic antibody productivity of Chinese hamster ovary (CHO)-DG44 cells in inoculum suspension cultures. This effect translates into 40% increased overall antibody titers in a fed-batch format where cells are grown in chemically defined serum-free media. Protein-A purified antibody products from mock-transfected cells and XBP-1 transfected cells were found to be of comparable quality with regard to glycosylation pattern and physicochemical characteristics. The data demonstrate the potential of XBP-1 engineering to improve mammalian cell culture production processes to yield high amounts of a therapeutic protein product of desired quality.

135Xe measurements with a two-element CZT-based radioxenon detector for nuclear explosion monitoring.

Measurement of elevated concentrations of xenon radioisotopes (131mXe, 133mXe, 133Xe and 135Xe) in the atmosphere has been shown to be a very powerful method for verifying whether or not a detected explosion is nuclear in nature. These isotopes are among the few with enough mobility and with half-lives long enough to make their detection at long distances realistic. Existing radioxenon detection systems used by the Comprehensive Nuclear-Test-Ban Treaty Organization (CTBTO) suffer from problems such as complexity, need for high maintenance and memory effect. To study the response of CdZnTe (CZT) detectors to xenon radioisotopes and investigate whether it is capable of mitigating the aforementioned issues with the current radioxenon detection systems, a prototype detector utilizing two coplanar CZT detectors was built and tested at Oregon State University. The detection system measures xenon radioisotopes through beta-gamma coincidence technique by detecting coincidence events between the two detectors. In this paper, we introduce the detector design and report our measurement results with radioactive lab sources and 135Xe produced in the OSU TRIGA reactor. Minimum Detectable Concentration (MDC) for 135Xe was calculated to be 1.47 ± 0.05 mBq/m3.

Chromosome segregation in Eubacteria.

It is now clear that bacterial chromosomes rapidly separate in a manner independent of cell elongation, suggesting the existence of a mitotic apparatus in bacteria. Recent studies of bacterial cells reveal filamentous structures similar to the eukaryotic cytoskeleton, proteins that mediate polar chromosome anchoring during Bacillus subtilis sporulation, and SMC interacting proteins that are involved in chromosome condensation. A picture is thereby developing of how bacterial chromosomes are organized within the cell, how they are separated following duplication, and how these processes are coordinated with the cell cycle.

mBLAST: Keeping up with the sequencing explosion for (meta)genome analysis.

Recent advances in next-generation sequencing technologies require alignment algorithms and software that can keep pace with the heightened data production. Standard algorithms, especially protein similarity searches, represent significant bottlenecks in analysis pipelines. For metagenomic approaches in particular, it is now often necessary to search hundreds of millions of sequence reads against large databases. Here we describe mBLAST, an accelerated search algorithm for translated and/or protein alignments to large datasets based on the Basic Local Alignment Search Tool (BLAST) and retaining the high sensitivity of BLAST. The mBLAST algorithms achieve substantial speed up over the National Center for Biotechnology Information (NCBI) programs BLASTX, TBLASTX and BLASTP for large datasets, allowing analysis within reasonable timeframes on standard computer architectures. In this article, the impact of mBLAST is demonstrated with sequences originating from the microbiota of healthy humans from the Human Microbiome Project. mBLAST is designed as a plug-in replacement for BLAST for any study that involves short-read sequences and includes high-throughput analysis. The mBLAST software is freely available to academic users at www.multicorewareinc.com.

MstX and a putative potassium channel facilitate biofilm formation in Bacillus subtilis.

Biofilms constitute the predominant form of microbial life and a potent reservoir for innate antibiotic resistance in systemic infections. In the spore-forming bacterium Bacillus subtilis, the transition from a planktonic to sessile state is mediated by mutually exclusive regulatory pathways controlling the expression of genes required for flagellum or biofilm formation. Here, we identify mstX and yugO as novel regulators of biofilm formation in B. subtilis. We show that expression of mstX and the downstream putative K+ efflux channel, yugO, is necessary for biofilm development in B. subtilis, and that overexpression of mstX induces biofilm assembly. Transcription of the mstX-yugO operon is under the negative regulation of SinR, a transcription factor that governs the switch between planktonic and sessile states. Furthermore, mstX regulates the activity of Spo0A through a positive autoregulatory loop involving KinC, a histidine kinase that is activated by potassium leakage. The addition of potassium abrogated mstX-mediated biofilm formation. Our findings expand the role of Spo0A and potassium homeostasis in the regulation of bacterial development.

Supplementation of serum free media with HT is not sufficient to restore growth properties of DHFR-/- cells in fed-batch processes - Implications for designing novel CHO-based expression platforms.

DHFR-deficient CHO cells are the most commonly used host cells in the biopharmaceutical industry and over the years, individual substrains have evolved, some have been engineered with improved properties and platform technologies have been designed around them. Unexpectedly, we have observed that different DHFR-deficient CHO cells show only poor growth in fed-batch cultures even in HT supplemented medium, whereas antibody producer cells derived from these hosts achieved least 2-3 fold higher peak cell densities. Using a set of different expression vectors, we were able to show that this impaired growth performance was not due to the selection procedure possibly favouring fast growing clones, but a direct consequence of DHFR deficiency. Re-introduction of the DHFR gene reproducibly restored the growth phenotype to the level of wild-type CHO cells or even beyond which seemed to be dose-dependent. The requirement for a functional DHFR gene to achieve optimal growth under production conditions has direct implications for cell line generation since it suggests that changing to a selection system other than DHFR would require another CHO host which - especially for transgenic CHO strains and tailor-suited process platforms - this could mean significant investments and potential changes in product quality. In these cases, DHFR engineering of the current CHO-DG44 or DuxB11-based host could be an attractive alternative.

Evaluation of a combinatorial cell engineering approach to overcome apoptotic effects in XBP-1(s) expressing cells.

Genetic engineering of producer cell lines for production of therapeutic antibodies in order to increase the yield of production processes remains a continuing challenge. Recently it was shown that heterologous expression of the active, spliced form of human X-box binding protein 1 (XBP-1(s)) can increase the amount of secreted protein products in mammalian cell culture processes. However, a prerequisite for the industrial application of any cell engineering approach is the ability to generate monoclonal cell lines that stably express the engineering gene to maintain the desired phenotype. Here, we show a decrease in heterologous human XBP-1(s) expression in CHO production cells producing a therapeutic antibody product monitored over a prolonged period in serial culture. Colony formation assays (CFA) in CHO-K1 cells reveal a general survival disadvantage conferred by XBP-1(s) in this cell type. We aimed to rescue this phenotype by expressing the caspase-inhibitor XIAP (x-linked inhibitor of apoptosis). Using a set of bicistronic expression vectors we engineered an antibody producing CHO cell line with XBP-1(s) and XIAP alone and in combination. Interestingly, co-expression of both genes resulted in the highest specific productivities (Qp) and final titers in a serum-free fed-batch process in chemically defined media. Thus, the combination of secretion and anti-apoptotic engineering provides an interesting approach for future applications in industrial mammalian cell culture.

Transposon assisted gene insertion technology (TAGIT): a tool for generating fluorescent fusion proteins.

We constructed a transposon (transposon assisted gene insertion technology, or TAGIT) that allows the random insertion of gfp (or other genes) into chromosomal loci without disrupting operon structure or regulation. TAGIT is a modified Tn5 transposon that uses Kan(R) to select for insertions on the chromosome or plasmid, beta-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. The resulting gfp insertions maintain target gene reading frame (to the 5' and 3' of gfp) and are integrated at the native chromosomal locus, thereby maintaining native expression signals. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. Several of these latter GFP insertions localize to lacO arrays integrated in the E. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. TAGIT thereby faciliates the isolation of fully functional insertions of fluorescent proteins into target proteins expressed from the native chromosomal locus as well as potentially useful partially functional proteins.

Heterologous expression of the lipid transfer protein CERT increases therapeutic protein productivity of mammalian cells.

Recent studies have demonstrated that the introduction of transgenes regulating protein transport or affecting post-translational modifications can further improve industrial processes for the production of therapeutic proteins in mammalian cells. Our study on improving therapeutic protein production in CHO cells by heterologous expression of the ceramide transfer protein (CERT) was initiated by the recent discovery that CERT is involved in protein kinase D (PKD)-dependent protein transport from the Golgi to the plasma membrane. We generated a set of CHO DG44 cell lines by stable integration of constructs expressing either CERT wild-type or CERT S132A, a mutant conferring increased lipid transfer activity, or a mock plasmid. CHO cells expressing heterologous CERT demonstrated significantly higher specific productivities of the therapeutic protein HSA when grown in inoculum suspension cultures. This effect translated into significantly increased overall HSA titers in a fed-batch format where cells are grown in chemically defined serum-free media. Furthermore, we could show that CERT also enhanced monoclonal antibody secretion in two IgG production cell lines with different basal productivities. The data demonstrate the potential of CERT engineering to improve mammalian cell culture production processes to yield high amounts of a therapeutic protein product of desired quality. To our knowledge, this is the first study showing a bottle neck in recombinant protein secretion at the Golgi complex in mammalian cells.

Bacillus subtilis MinC destabilizes FtsZ-rings at new cell poles and contributes to the timing of cell division.

Division site selection in rod-shaped bacteria depends on nucleoid occlusion, which prevents division over the chromosome and MinCD, which prevent division at the poles. MinD is thought to localize MinC to the cell poles where it prevents FtsZ assembly. Time-lapse microscopy demonstrates that in Bacillus subtilis transient polar FtsZ rings assemble adjacent to recently completed septa and that in minCD strains these persist and are used for division, producing a minicell. This suggests that MinC acts when division proteins are released from newly completed septa to prevent their immediate reassembly at new cell poles. The minCD mutant appears to uncouple FtsZ ring assembly from cell division and thus shows a variable interdivisional time and a rapid loss of cell cycle synchrony. Functional MinC-GFP expressed from the chromosome minCD locus is dynamic. It is recruited to active division sites before septal biogenesis, rotates around the septum, and moves away from completed septa. Thus high concentrations of MinC are found primarily at the septum and, more transiently, at the new cell pole. DivIVA and MinD recruit MinC to division sites, rather than mediating the stable polar localization previously thought to restrict MinC activity to the pole. Together, our results suggest that B. subtilis MinC does not inhibit FtsZ assembly at the cell poles, but rather prevents polar FtsZ rings adjacent to new cell poles from supporting cell division.