Exploitation of the twin-arginine translocation pathway and promoter control for biopharmaceutical production in E. coli strains

The industrial production of proteins in bacteria has found its limits due to the size and requirements of post-translational folding of many proteins. However, bacteria are still a preferred expression host and various tools are applied to broaden the expression spectrum of these microorganisms. We investigated methods to decrease costs and improve protein expression in Escherichia coli and applied tools such as genetic engineering, change of promotor and signal peptides, and aiding disulphide bond formation. Here we examined the ability of Escherichia coli strain MC4100 encoding the TatABC protein secretion pathway of Agrobacterium tumefaciens to export complex proteins into the periplasm. We have shown that the mutant strain can export native Tat proteins but rejects non-native proteins of biopharmaceutical interest which are exported over Tat in Escherichia coli wildtype. Furthermore, we examined a sodium nitrate inducible promoter on its expression strength and periplasmic export, and we conducted research into the expression and export to the periplasm of the novel biodegrading enzyme PETase. Additionally, we show that the narG-CC promoter is capable of high-level protein expression of GFP but does not facilitate periplasmic expression of hGH and an scFv. Furthermore, we show that PETase can be exported into the periplasm by the Sec pathway but not the Tat pathway. The combined research shows that Escherichia coli is still a key player in the production of recombinant proteins and that proteins that require cytoplasmic post-translational folding can be exported under the correct circumstances, although finding the best tools to improve the expression can be a challenge

New SSHRC Scholarships in the Social Sciences and Humanities

Ce numéro du Bulletin a été réalisé par une température bien variable : tantôt glaciale, tantôt plutôt clémente. Il sert à vous rappeler qu'il se passe pas mal de choses, ces temps-ci, dans le domaine de l'histoire, à commencer, bien sûr avec le nouveau site web sur la Nouvelle-France.The Government of Canada has recently established the Canada Graduate Scholarships program (CGS). When fully implemented by 2006, the program will support 2,000 doctoral students and 2,000 master’s students

Imaging of vascular remodeling after simulated thoracoabdominal aneurysm repair

ObjectiveA better understanding of the response of the spinal cord blood supply to segmental artery (SA) sacrifice should help minimize the risk of paraplegia after both open and endovascular repair of thoracoabdominal aortic (TAA) aneurysms.MethodsTwelve female juvenile Yorkshire pigs were randomized into 3 groups and perfused with a barium–latex solution. Pigs in group 1 (control) had infusion without previous intervention. Pigs in group 2 were infused 48 hours after ligation of all SAs (T4-L5) and those in group 3 at 120 hours after ligation. Postmortem computed tomographic scanning of the entire pig enabled overall comparisons and measurement of vessel diameters in the spinal cord circulation.ResultsWe ligated 14.5 ± 0.8 SAs: all filled retrograde to the ligature. Paraplegia occurred in 38% of operated pigs. A significant increase in the mean diameter of the anterior spinal artery (ASA) was evident after SA sacrifice (P < .0001 for 48 hours and 120 hours). The internal thoracic and intercostal arteries also increased in diameter. Quantitative assessment showed an increase in vessel density 48 hours after ligation of SAs, reflected by an obvious increase in small collateral vessels seen on 3-dimensional reconstructions of computed tomographic scans at 120 hours.ConclusionsRemodeling of the spinal cord blood supply—including dilatation of the ASA and proliferation of small collateral vessels—is evident at 48 and 120 hours after extensive SA sacrifice. It is likely that exploitation of this process will prove valuable in the quest to eliminate paraplegia after TAA aneurysm repair

Einfluss verschiedener Inkohlungsbedingungen auf die Qualität von HTC-Biokohle als Bodenhilfsstoff – Hinweise durch Pflanzenwachstum und NMR-Spektroskopie

Bei der hydrothermalen Karbonisierung (HTC) wird Biomasse unter großem Druck (ca. 20 bar) und hoher Temperatur (180 – 230 °C) über mehrere Stunden mit Wasser erhitzt. Dabei entsteht ein torf- bis braunkohleähnliches Produkt, die HTC-Biokohle. Durch diesen Prozess können organische Abfälle, wie Klärschlamm, ohne Vortrocknung verwertet werden. Ähnlich der Biokohle aus Pyrolyse (Verschwelung bei > 350°C) wird auch für die HTC-Biokohle positive Effekte auf die Bodenfruchtbarkeit vor allem für sandige nährstoffarme Böden erwartet (erhöhte Nährstofffixierung und Wasserhaltefähigkeit). Ziel war es festzustellen, wie sich verschiedene Inkohlungsbedingungen (Temperatur, Verweildauer im Reaktor) der HTC-Biokohle auf das Pflanzenwachstum auswirken und mit Hilfe von NMR-Spektroskopie aufzuklären, welche funktionellen organischen Gruppen (die typisch für Klassen phytotoxischer Substanzen sind) bei gehemmten Wachstum vorhanden sind. In einem Säulenversuch mit Eiszapfen (Raphanus sativus spec. sativus) und lehmigen Sand als Ausgangsmaterial, wurden 4 verschieden hergestellte HTC-Biokohlen und eine Pyrolyse-Biokohle getestet. Bestimmt wurde der Ertrag und der Stickstoffgehalt in den Pflanzen, die Ammonium und Nitratkonzentration im Sickerwasser, sowie die Nährstoffe N, P, K, Mg und der pH-Wert im Boden. Der Ertrag unterschied sich stark je nach eingesetzter Biokohle. Pyrolyse-Biokohle und HTC-Biokohle welche bei erhöhter Temperatur hergestellt wurde, führten zu höheren Erträgen, während die anderen HTC-Biokohlen zu geringeren Erträgen im Vergleich zur Kontrolle führten. Die Qualität der HTC-Biokohlen ist folglich erheblich von den Prozessbedingungen während der Inkohlung abhähngig Es werden phytotoxische Substanzen vermutet, die bei höherer Temperatur abgebaut werden können. Die NMR-Spektren werden aktuell ausgewertet. Diese Eingrenzung ist eine Vorstufe zur Identifikation von phytotoxischen Einzelsubstanzen und dient gleichzeitg zum Verständnis, wie der HTC-Prozess die chemische Struktur des Ausgansmaterials umwandelt

Asteroid 2008 TC3, not a polymict ureilitic but a polymict C1 chondrite parent body? Survey of 249 Almahata Sitta fragments

On October 7, 2008, the asteroid 2008 TC3 exploded as it entered the Earth’s atmosphere, producing significant dust (in the atmosphere) and delivering thousands of stones in a strewn field in Sudan, collectively known as the Almahata Sitta (AhS) stones. About 600 fragments were officially recovered in 2008 and 2009. Further rocks were collected since the fall event by local people. From these stones, 249 were classified at the Institut für Planetologie in Münster (MS) known as MS‐xxx or MS‐MU‐xxx AhS subsamples. Most of these rocks are ureilitic in origin (168; 67%): 87 coarse‐grained ureilites, 60 fine‐grained ureilites, 15 ureilites with variable texture/mineralogy, four trachyandesites, and two polymict breccias. We identified 81 non‐ureilitic fragments, corresponding to 33% of the recovered samples studied in Münster. These included chondrites, namely 65 enstatite chondrites (43 EL; 22 EH), 11 ordinary chondrites (OC), one carbonaceous chondrite, and one unique R‐like chondrite. Furthermore, three samples represent a unique type of enstatite achondrite. Since all AhS stones must be regarded as individual specimens independent from each other, the number of fresh ureilite and enstatite chondrite falls in our meteorite collections has been increased by several hundred percent. Overall, the samples weigh between <1 and 250 g and have a mean mass of ~15 g. If we consider—almost 15 years after the fall—the mass calculations, observations during and after the asteroid entered the atmosphere, the mineralogy of the C1 stones AhS 91A and AhS 671, and the experimental work on fitting the asteroid spectrum (e.g., Goodrich et al., 2019; Jenniskens et al., 2010; Shaddad et al., 2010), the main portion of the meteoroid was likely made of the fine‐grained (carbonaceous) dust and was mostly lost in the atmosphere. In particular, the fact that C1 materials were found has important implications for interpreting asteroid 2008 TC3's early spectroscopic results. Goodrich et al. (2019) correctly suggested that if scientists had not recovered the “water‐free” samples (e.g., ureilites, enstatites, and OC) from the AhS strewn field, 2008 TC3 would have been assumed to be a carbonaceous chondrite meteoroid. Considering that the dominating mass of the exploding meteoroid consisted of carbonaceous materials, asteroid 2008 TC3 cannot be classified as a polymict ureilite; consequently, we state that the asteroid was a polymict carbonaceous chondrite breccia, specifically a polymict C1 object that may have formed by late accretion at least 50–100 Ma after calcium–aluminum‐rich inclusions