Inducible control of plasmid copy number for the production of therapeutic plasmids

TITELBLATT: nur in PRINTAUSGABE! -- Die therapeutische Verwendung von DNA anstelle von Proteinen ist zu einem bedeutenden Gegenstand medizinischer Forschung geworden. Die Entwicklung neuer Therapieformen wie der Gentherapie und DNA-Impfstoffen hat den Bedarf an hochwertiger Plasmid-DNA stark gesteigert. Die Regulation der Plasmidkopienzahl (PCN) ist dabei für eine optimale DNA-Ausbeute ausschlaggebend. Eine zu starke Plasmidreplikation würde nämlich zu einer Überlastung und schließlich zum Zusammenbruch des bakteriellen Stoffwechsels führen. Gegenstand dieser Arbeit war es die Replikation von Plasmiden mit ColE1-ori mittels RNA-RNA-Interaktion sowie tRNA-RNA-Wechselwirkung regulierbar zu machen. Dazu wurde in einem ersten Schritt das RNAI-Gen unter der Kontrolle des T7- Promotors ins bakterielle Escherichia coli (E.coli) Genom integriert. In Schüttelkolbenversuchen wurde anschließend die RNAI-Expression durch die Zugabe von Isopropyl-β-D-thiogalactopyranosid (IPTG) induziert und ihre Auswirkung auf die Replikation verschiedener ColE1-Plasmide untersucht. Es konnte gezeigt werden, dass die Überexpression von RNAI zu einer starken Reduktion der PCN führt. Da es sich beim T7-System allerdings um ein sehr undichtes handelt, wurde dieser Effekt auch im uninduzierten Zustand sichtbar. Deshalb wurde der T7- Promotor in einem weiteren Schritt durch den pLlacO-1 Promotor ersetzt wodurch eine regulierbare RNAI-Expression möglich wurde. Nur im Fall der Induktion durch IPTG wurde die PCN gesenkt. In einem weiteren Ansatz wurde versucht die Plasmidreplikation durch die Überexpression von unbeladenen tRNAs zu steigern. Dazu wurde das tRNAAla-Gen unter die Kontrolle des T7-Promotors gebracht und wiederum ins E.coli Genom integriert. Zwei Punktmutationen in diesem Gen verhinderten dabei die Beladung der tRNA-Moleküle mit Alanin. Auch in diesem Fall wurde die Genexpression in Schüttelkolbenversuchen durch die Zugabe von IPTG induzierte, und deren Auswirkung auf die Replikation von ColE1-Plasmiden untersucht. Laut bereits existierenden Publikationen, sollte es zur Wechselwirkung zwischen unbeladener tRNA und RNAI kommen. Der daraus resultierende Anstieg an freier RNAII sollte in weiterer Folge zu einer Erhöhung der PCN führen. 5 Mit dieser Arbeit wurde gezeigt, dass die PCN von ColE1-Plasmiden über die Expression einer chromosomalen Kopie des RNAI-Gens unter dem pLlacO-1 Promotor reguliert bzw. reduziert werden kann. Die Expression des chromosomalen tRNAAla- Gens bewirkte, entgegen den Erwartungen, eine Senkung der PCN.The application of naked DNA instead of therapeutic proteins has become a main subject of medical investigation. The development of new therapeutic methods like gene therapy and DNA vaccination has caused a growing demand for high quality plasmid DNA (pDNA). For an optimal yield of pDNA, it is important to be able to regulate plasmid copy number (PCN) as extensive plasmid replication exerts metabolic burden on the host cell, and as a consequence leads to growth cessation. In this work, control of PCN of ColE1-type plasmids should be achieved by RNA-RNA interaction as well as by RNA-tRNA interaction. In a first approach the RNAI-gene was inserted into the E.coli chromosome and put under the control of the T7 promoter. By the addition of isopropyl-beta-Dthiogalactopyranoside (IPTG), RNAI-expression was induced in shake flask experiments and the influence on different ColE1-type plasmids was followed. It could be shown that extensive RNAI expression resulted in a dramatic decrease of PCN. As this effect was also observed in the case of no IPTG addition the leaky T7- system was exchanged by the pLlacO-1 promoter. By the use of this promoter, RNAI expression was restricted to IPTG addition. In a second approach regulation of PCN should be achieved by tRNA over expression. It was reported recently, that uncharged tRNAs can interact with RNAI, thereby mediating cleavage of RNAI. Reduction of functional RNAI should further lead to an increase in the number of uninhibited RNAII molecules and thus, in enhanced initiation of plasmid replication. Therefore, a chromosomal copy of the tRNAAla-gene was brought under the control of the T7 promoter. Because of two point mutations charging of tRNA molecules was inhibited. Expression was again induced by the addition of IPTG in shake flask experiments and PCN of ColE1-type plasmids was determined. With this work it could be demonstrated, that PCN of ColE1-type plasmids can be regulated and reduced, respectively by the expression of a chromosomal copy of the RNAI-gene under the control of the pLlacO-1 promoter. The over expression of 3 uncharged tRNAs however, showed an unexpected effect. Contrary to publications, predicting an increase in plasmid replication, expression of tRNA led to a clear reduction of PCN

Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum

Background: Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector® micro-fermentation system. Results: Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl -D-thiogalactopyranoside) and IPTG (isopropyl -D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector® micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a constitutive promoter). Conclusions: We evaluated different inducible promoters, as well as an orthologous expression system, for controlled gene expression in L. plantarum. Furthermore, here we provide proof of concept for a T7 RNA polymerase based expression system for L. plantarum. Thereby we expanded the molecular toolbox for an industrial relevant and generally regarded as safe (GRAS) strain.(VLID)101641

Satori 2023

The Satori is a student literary publication that expresses the artistic spirit of the students of Winona State University. Student poetry, prose, and graphic art are published in the Satori every spring since 1970. The Satori 2023 editors are Gabriel Hathaway, Van Herman, Madeline Schonitzer, Brianna Strohbehn, Page Sutton, Willow Swinbank, and Emily Venné. The Satori 2023 faculty advisor is Dr. Jim Armstrong, Professor of English.https://openriver.winona.edu/satori/1010/thumbnail.jp

Updating vital status by tracking in the community among patients with epidemic Kaposi sarcoma who are lost to follow-up in sub-Saharan Africa.

BACKGROUND: Throughout most of sub-Saharan Africa (and, indeed, most resource-limited areas), lack of death registries prohibits linkage of cancer diagnoses and precludes the most expeditious approach to determining cancer survival. Instead, estimation of cancer survival often uses clinical records, which have some mortality data but are replete with patients who are lost to follow-up (LTFU), some of which may be caused by undocumented death. The end result is that accurate estimation of cancer survival is rarely performed. A prominent example of a common cancer in Africa for which survival data are needed but for which frequent LTFU has precluded accurate estimation is Kaposi sarcoma (KS). METHODS: Using electronic records, we identified all newly diagnosed KS among HIV-infected adults at 33 primary care clinics in Kenya, Uganda, Nigeria, and Malawi from 2009 to 2012. We determined those patients who were apparently LTFU, defined as absent from clinic for ≥90 days at database closure and unknown to be dead or transferred. Using standardized protocols which included manual chart review, telephone calls, and physical tracking in the community, we attempted to update vital status amongst patients who were LTFU. RESULTS: We identified 1222 patients with KS, of whom 440 were LTFU according to electronic records. Manual chart review revealed that 18 (4.1%) were classified as LFTU due to clerical error, leaving 422 as truly LTFU. Of these 422, we updated vital status in 78%; manual chart review was responsible for updating in 5.7%, telephone calls in 26%, and physical tracking in 46%. Among 378 patients who consented at clinic enrollment to be tracked if they became LTFU and who had sufficient geographic contact/locator information, we updated vital status in 88%. Duration of LTFU was not associated with success of tracking, but tracking success was better in Kenya than the other sites. CONCLUSION: It is feasible to update vital status in a large fraction of patients with HIV-associated KS in sub-Saharan Africa who have become LTFU from clinical care. This finding likely applies to other cancers as well. Updating vital status amongst lost patients paves the way towards accurate determination of cancer survival

Pitfalls of Practicing Cancer Epidemiology in Resource-limited Settings: the Case of Survival and Loss to Follow-up after a Diagnosis of Kaposi’s Sarcoma in Five Countries across Sub-Saharan Africa

Background: Survival after diagnosis is a fundamental concern in cancer epidemiology. In resource-rich settings, ambient clinical databases, municipal data and cancer registries make survival estimation in real-world populations relatively straightforward. In resource-poor settings, given the deficiencies in a variety of health-related data systems, it is less clear how well we can determine cancer survival from ambient data. Methods: We addressed this issue in sub-Saharan Africa for Kaposi’s sarcoma (KS), a cancer for which incidence has exploded with the HIV epidemic but for which survival in the region may be changing with the recent advent of antiretroviral therapy (ART). From 33 primary care HIV Clinics in Kenya, Uganda, Malawi, Nigeria and Cameroon participating in the International Epidemiologic Databases to Evaluate AIDS (IeDEA) Consortia in 2009–2012, we identified 1328 adults with newly diagnosed KS. Patients were evaluated from KS diagnosis until death, transfer to another facility or database closure. Results: Nominally, 22 % of patients were estimated to be dead by 2 years, but this estimate was clouded by 45 % cumulative lost to follow-up with unknown vital status by 2 years. After adjustment for site and CD4 count, agelost. Conclusions: In this community-based sample of patients diagnosed with KS in sub-Saharan Africa, almost half became lost to follow-up by 2 years. This precluded accurate estimation of survival. Until we either generally strengthen data systems or implement cancer-specific enhancements (e.g., tracking of the lost) in the region, insights from cancer epidemiology will be limited

Prevalence of major depression in preschool children

The prevalence of preschool major depressive disorder (MDD) was studied in the community. The whole population of children between 3 and 6 years attending preschool nurseries in three areas (one urban, one rural and one suburban) in Spain (n = 1,427) were contacted. Selection was by a two-stage procedure. At stage I, the ESDM 3-6, a screening measure for preschool depression, was used to identify a sample for more intensive interviewing. Sensitivity and specificity of the cut-off point of the ESDM 3–6 had been previously tested in a pilot study (n = 229). During the first stage, 222 preschool children (15.6%) were found to be probable depressives, because they scored 27 or more, the cut-off used. At stage II, the children were interviewed and diagnosed by the consensus of two clinicians, blind to the ESDM 3-6 results. DSM-IV diagnostic criteria were used to define caseness. A total of 16 children (1.12%) met the MDD criteria. The prevalence by areas was urban 0.87%, rural 0.88%, suburban 1.43%. Sex distribution prevalence was 1:1. This study is a contribution to the scarce epidemiology of preschool depression in the community

Pitfalls of practicing cancer epidemiology in resource-limited settings: the case of survival and loss to follow-up after a diagnosis of Kaposi’s sarcoma in five countries across sub-Saharan Africa

Background: Survival after diagnosis is a fundamental concern in cancer epidemiology. In resource-rich settings, ambient clinical databases, municipal data and cancer registries make survival estimation in real-world populations relatively straightforward. In resource-poor settings, given the deficiencies in a variety of health-related data systems, it is less clear how well we can determine cancer survival from ambient data. Methods: We addressed this issue in sub-Saharan Africa for Kaposi’s sarcoma (KS), a cancer for which incidence has exploded with the HIV epidemic but for which survival in the region may be changing with the recent advent of antiretroviral therapy (ART). From 33 primary care HIV Clinics in Kenya, Uganda, Malawi, Nigeria and Cameroon participating in the International Epidemiologic Databases to Evaluate AIDS (IeDEA) Consortia in 2009–2012, we identified 1328 adults with newly diagnosed KS. Patients were evaluated from KS diagnosis until death, transfer to another facility or database closure. Results: Nominally, 22 % of patients were estimated to be dead by 2 years, but this estimate was clouded by 45 % cumulative lost to follow-up with unknown vital status by 2 years. After adjustment for site and CD4 count, age <30 years and male sex were independently associated with becoming lost. Conclusions: In this community-based sample of patients diagnosed with KS in sub-Saharan Africa, almost half became lost to follow-up by 2 years. This precluded accurate estimation of survival. Until we either generally strengthen data systems or implement cancer-specific enhancements (e.g., tracking of the lost) in the region, insights from cancer epidemiology will be limited

Genetic Diversity of EBV-Encoded LMP1 in the Swiss HIV Cohort Study and Implication for NF-Κb Activation

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1