Poznoantično višinsko naselje Burgbichl pri Irschnu v dolini Drave na avstrijskem Koroškem je bilo do sedaj raziskano v dveh arheoloških akcijah. Burgbichl leži na križišču ene glavne poti v ...jugovzhodnih Alpah – poti po Dravski dolini – in prečne poti, ki poteka iz Italije čez prelaz Gailpass (Gailbergsattel). Ker hrib kasneje ni bil poseljen, so antične zgradbe izredno dobro ohranjene: na severu obkroža približno hektar veliko poselitveno območje masiven obodni zid. Delno so bili izkopani deli vhoda in obzidja, velika stanovanjska stavba, dve obrtni območji, vodni zbiralnik in zgodnjekrščanska cerkev. V cerkvi s prizidkom na vzhodu sta ugotovljeni dve gradbeni fazi. Pomemben je dokaz o uporabi marmorja na več mestih: za prag, stopnico in okrasitev prezbiterija. V osrednjem delu prezbiterija je jama za relikviarij. Doslej sta bila odkrita dva grobova – eden v grobnici.
Pri poznoantičnih najdbah iz 5. in 6. st. gre za tipičen spekter, znan s sočasnih najdišč, z množico grobe keramike, posameznimi uvoženimi kosi keramike, novci in fibulami. Posebnost je koščena pasna spona, okrašena s koncentričnimi krožci s pikami. Na več mestih na hribu so vidni ostanki starejše uporabe prostora, tudi med najdbami je veliko predmetov iz rimskega imperialnega obdobja. Razumevanje teh starejših najdb v povezavi z očitno aktivnostjo na najdišču v obdobju od 1. do 3. st. bo vsekakor med glavnimi cilji prihodnjih raziskav.
Poznoantično višinsko naselje Burgbichl pri Irschnu v dolini Drave na avstrijskem Koroškem je bilo do sedaj raziskano v dveh arheoloških akcijah. Burgbichl leži na križišču ene glavne poti v ...jugovzhodnih Alpah – poti po Dravski dolini – in prečne poti, ki poteka iz Italije čez prelaz Gailpass (Gailbergsattel). Ker hrib kasneje ni bil poseljen, so antične zgradbe izredno dobro ohranjene: na severu obkroža približno hektar veliko poselitveno območje masiven obodni zid. Delno so bili izkopani deli vhoda in obzidja, velika stanovanjska stavba, dve obrtni območji, vodni zbiralnik in zgodnjekrščanska cerkev. V cerkvi s prizidkom na vzhodu sta ugotovljeni dve gradbeni fazi. Pomemben je dokaz o uporabi marmorja na več mestih: za prag, stopnico in okrasitev prezbiterija. V osrednjem delu prezbiterija je jama za relikviarij. Doslej sta bila odkrita dva grobova – eden v grobnici. Pri poznoantičnih najdbah iz 5. in 6. st. gre za tipičen spekter, znan s sočasnih najdišč, z množico grobe keramike, posameznimi uvoženimi kosi keramike, novci in fibulami. Posebnost je koščena pasna spona, okrašena s koncentričnimi krožci s pikami. Na več mestih na hribu so vidni ostanki starejše uporabe prostora, tudi med najdbami je veliko predmetov iz rimskega imperialnega obdobja. Razumevanje teh starejših najdb v povezavi z očitno aktivnostjo na najdišču v obdobju od 1. do 3. st. bo vsekakor med glavnimi cilji prihodnjih raziskav.
The late Republican structures (variously called ‘casas fuertes’, ‘castella’, ‘fortins’ or ‘recintos-torres’) presented in the following are examples from building complexes whose distribution ...appears to concentrate in groups in modern Portugal in the Upper and southern Lower Alentejo and the northern Algarve coastline (Figure 1). They date mainly to the C1st BC and AD – perhaps in some cases to the early the C2nd AD – and are characterised by a square, or almost square-shaped central building (Figure 2), and secondary structures usually lower down the slope. The central buildings have thick external walls, the only openings in which are a door
Governance of the endogenous gene regulatory network enables the navigation of cells towards beneficial traits for recombinant protein production. CRISPRactivation and interference provides the basis ...for gene expression modulation but is primarily applied in eukaryotes. Particularly the lack of wide-ranging prokaryotic CRISPRa studies might be attributed to intrinsic limitations of bacterial activators and Cas9 proteins. While bacterial activators need accurate spatial orientation and distancing towards the target promoter to be functional, Cas9-based CRISPR tools only bind sites adjacent to NGG PAM sequences. These circumstances hampered Cas9-guided activators from mediating the up-regulation of endogenous genes at precise positions in bacteria. We could overcome this limitation by combining the PAM independent Cas9 variant SpRY and a CRISPRa construct using phage protein MCP fused to transcriptional activator SoxS. This CRISPRa construct, referred to as SMS, was compared with previously reported CRISPRa constructs and showed up-regulation of a reporter gene library independent of its PAM sequence in Escherichia coli. We also demonstrated down-regulation and multi-gene expression control with SMS at non-NGG PAM sites. Furthermore, we successfully applied SMS to up-regulate endogenous genes, and transgenes at non-NGG PAM sites, which was impossible with the previous CRISPRa construct.
Genome-based Escherichia coli expression systems are superior to conventional plasmid-based systems as the metabolic load triggered by recombinant compounds is significantly reduced. The efficiency ...of T7-based transcription compensates for low gene dosage (single copy) and facilitates high product formation rates. While common Gene Bridges' λ-red mediated recombination technique for site directed integration of genes into the host genome is very efficient, selection for positive clones is based on antibiotic resistance markers and removal thereof is often time consuming. For the generation of industrial production strains, flexibility in terms of integration site is not required, yet time from gene design to a stable clone is a quite relevant parameter. In this study, we developed a fast, efficient and antibiotic-free integration method for E. coli as production strain. We combined the λ-red recombination system with the site-directed homing endonuclease I from Saccharaomyces cerevisiae (I-SceI) for selection. In a first step, λ-red proteins are performing genome integration of a linear, antibiotic marker-free integration cassette. The engineered host strain carries the I-SceI restriction sequence at the attTn7 site, where the integration event happens. After homologous recombination and integration at the target site, site-specific genome cleavage by endonuclease I-SceI is induced, thereby killing all cells still containing an intact I-SceI site. In case of positive recombination events, the genomic I-SceI site is deleted and cleavage is no longer possible. Since plasmids are designed to contain another I-SceI restriction site they are destroyed by self-cleavage, a procedure replacing the time-consuming plasmid curing. The new plasmid-based "All-In-One" genome integration method facilitates significantly accelerated generation of genome-integrated production strains in 4 steps.
Introduction
Postmortem multi-detector computed tomography (PMCT) has become an important part in forensic imaging. Modern reconstruction techniques such as iterative reconstruction (IR) are ...frequently used in postmortem CT angiography (PMCTA). The image quality of PMCTA depends on the strength of IR. For this purpose, we aimed to investigate the impact of different advanced IR levels on the objective and subjective PMCTA image quality.
Material and methods
We retrospectively analyzed the coronary arteries of 27 human cadavers undergoing whole-body postmortem CT angiography between July 2017 and March 2018 in a single center. Iterative reconstructions of the coronary arteries were processed in five different level settings (0%; 30%; 50%; 70%; 100%) by using an adaptive statistical IR method. We evaluated the objective (contrast-to-noise ratio (CNR)) and subjective image quality in several anatomical locations.
Results
Our results demonstrate that the increasing levels of an IR technique have relevant impact on the image quality in PMCTA scans in forensic postmortem examinations. Higher levels of IR have led to a significant reduction of image noise and therefore to a significant improvement of objective image quality (+ 70%). However, subjective image quality is inferior at higher levels of IR due to plasticized image appearance.
Conclusion
Objective image quality in PMCTA progressively improves with increasing level of IR with the best CNR at the highest IR level. However, subjective image quality is best at low to medium levels of IR. To obtain a “classic” image appearance with optimal image quality, PMCTAs should be reconstructed at medium levels of IR.
Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli (E. coli) and the high degree of similarity within this protein class, a generic platform ...technology is still not available. Indeed, feasible production of new Fab candidates remains challenging. In this study, a setup that enables direct characterization of host cell response to Fab expression by utilizing genome‐integrated (GI) systems is established. Among the multitude of factors that influence Fab expression, the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest (GOI) are varied. The resulting 32 production clones are characterized in carbon‐limited microbioreactor cultivations with yields of 0–7.4 mg Fab per gram of cell dry mass. Antigen‐binding region variations have the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performs better than the BL21(DE3) strain. Translocation mechanism variations mainly influence leader peptide cleavage efficiency. Plasmid‐free systems, with a single copy of the GOI integrated into the chromosome, reach Fab yields in the range of 80–300% of plasmid‐based counterparts. Consequently, the GI Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions.
The production of antibody fragments (Fab) in Escherichia coli is challenging. In this study, periplasmic Fab expression systems with varying influence factors on growth and yield are investigated in carbon‐limited microbioreactor cultivations. Systems with one genome‐integrated gene copy are benchmarked against conventional plasmid‐based systems. It turns out that genome‐integrated clones facilitate direct characterization of Fab‐driven effects on host cell metabolism.
Abstract
Background
The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a ...single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ
70
promoters, which were recognized by the
Escherichia coli
host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing
lac
operators (
lacO
) into the constitutive T5 and A1 promoter sequences.
Results
We showed that, in genome-integrated
E. coli
expression systems that used σ
70
promoters, the number of
lacO
sites must be well balanced. Promoters containing three and two
lacO
sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single
lacO
site and the
lac
repressor element,
lacI
Q
, on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level.
Conclusions
Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the
lac
repressor protein, LacI. We reasoned that the affinity of LacI for the
lacO
sites of the GOI must be lower than the affinity of LacI to the
lacO
sites of the endogenous
lac
operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in
lac
repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel
E. coli
expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.
Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their ...lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.
High Five cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of high titers of ...baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five cells is successfully demonstrated at bioreactor scale, resulting in a higher maximum viable cell concentration (5.1 × 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100–200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins.
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