Biological response of Chlorella vulgaris to pulsed electric field treatment for improvement of protein extraction

Angesichts des Klimawandels und einer stetig wachsenden Weltbevölkerung können Mikroalgen eine wichtige Rolle als nachhaltige Energie- und Nahrungsquelle der Zukunft spielen. Zur Extraktion wertvoller Inhalts- und Nährstoffe ist ein Zellaufschluss notwendig. Die Elektroimpulsbehandlung (EIB) bietet eine energieeffiziente und schonende Alternative im Vergleich zu mechanischen Zellaufschlussmethoden. Jedoch sind die biologischen Prozesse und zellulären Mechanismen hinter dem Zelltod nach EIB noch wenig untersucht. Aus diesem Grund wurden die einzellige grüne Mikroalge Chlorella vulgaris und das Cyanobakterium Spirulina als Modellorganismen verwendet, um die Wirkung von EIB auf biologische Zellen zu untersuchen. Dafür wurde eine Methode zur Überwachung der Viabilität nach EIB unter Verwendung von Fluoresceindiacetat (FDA) in C. vulgaris etabliert. Im Anschluss wurden die experimentellen EIB-Parameter so eingestellt, dass ein fixes Verhältnis von Zellen nach der Behandlung abstirbt, während der andere Teil überlebt. Mit diesen Werkzeugen war eine quantitative Analyse des Zelltodes nach EIB möglich. Basierend auf den Analyseergebnissen wurde die EIB-Extraktion von Proteinen und dem wertvollen blauen Farbstoff Phycocyanin aus Spirulina unter verschiedenen post-EIB Inkubationsbedingungen untersucht. Zur Optimierung der Elektroextraktionseffizienz in Spirulina wurden die Einflüsse des pH des externen Mediums, der Biomassekonzentration, der Zellaggregation sowie der Energiereduktion untersucht. Das optimierte Elektroextraktionsprotokoll mit höherer Biomassekonzentration und geringerer Behandlungsenergie erfordert eine post-EIB-Inkubation unter kontrollierten Bedingungen (Raumtemperatur, pH 6 oder 8, homogene Suspension), die für die Freisetzung und Stabilität von Phycocyanin entscheidend sind. Mit diesem Wissen besteht eine mögliche biotechnologische Anwendung darin, schonende EIB mit niedrigstem Energieeintrag durchzuführen, was zu einer effizienten Protein- und Phycocyanin-Gewinnung führt. An C. vulgaris konnte gezeigt werden, dass EIB mit niedrigem Energieeintrag auch als abiotisches Stresssignal wirken kann. Dies wurde sichtbar in Form einer gestörten Redox-Homöostase, bei der sowohl die Freisetzung von Wasserstoffperoxid als auch Lipidoxidation gemessen werden konnten. Die Hemmung von Prozessen, die mit dem programmierten Zelltod (PCD) zusammenhängen, zeigten, dass höchstwahrscheinlich Ca-Signalwege, Aktindynamik und Membranversteifung keine notwendige Rolle beim EIB-induzierten Zelltod spielen. Die Freisetzung von Cytochrom f konnte nur im Hochdruckhomogenisations (HPH) Extrakt und nicht nach EIB nachgewiesen werden. Zellsuspensionen mit hoher Zelldichte, die an der Überlebensschwelle gepulst wurden, zeigten nur eine langsame Manifestation des Zelltods. Dies führte zur Entdeckung eines Zelltod-induzierenden Faktors (CDIF). Es konnte nachgewiesen werden, dass durch EIB und HPH-Behandlung der CDIF aus C. vulgaris extrahiert werden kann. Wasserlöslicher Extrakt, der diesen CDIF enthielt, führte zum Absterben von unbehandelten Mikroalgen (insbesondere nur bei C. vulgaris). Weitere Experimente zeigten die Entstehung des CDIF in der stationären Wachstumsphase, Hitzelabilität und Dosisabhängigkeit. Ebenso wie die Empfindlichkeit gegenüber direkter EIB hing die Empfindlichkeit der Empfängerzellen gegenüber dem CDIF vom Zellzyklusstadium ab. Untersuchungen zur Extraktionseffizienz von Proteinen aus C. vulgaris führten zu dem Ergebnis, dass die erforderliche spezifische Energie für maximalen Ertrag der zuvor bestimmten Behandlungsenergie an der Überlebensschwelle entspricht. Alle experimentellen Ergebnisse weisen darauf hin, dass der EIB-induzierte Zelltod und die damit verbundene hohe Extraktionseffizienz nicht nur auf rein physikalische Phänomene zurückzuführen sind, sondern einen biologischen Prozess beinhalten müssen. Das Arbeitsmodell bezüglich des CDIF beinhaltet, dass der Faktor aus zellwandabbauenden Enzymen wie Chitinasen besteht. EIB bei sehr geringem Energieeintrag wirkt als abiotisches Stresssignal. In Kombination mit einer beschädigten Zellintegrität aufgrund von Poren in der Zellmembran führen PCD-Prozesse zu einer enzymatischen Autolyse, bei der der CDIF (Chitinasen) freigesetzt wird. Die Zellwand wird durch den CDIF geschwächt. Wird der CDIF-haltige Extrakt unbehandelten Empfängerzellen zugesetzt, zeigt er zunächst über den Zellwandabbau eine äußere Wirkung. Nach Internalisierung kann der CDIF als internes Signal fungieren, das PCD auslöst

Examination of self-determination theory constructs as mediators of the effect of motivational interviewing on tobacco cessation outcomes

Title from PDF of title page viewed June 17, 2021Dissertation advisor: Kym BennettVitaIncludes bibliographical references (pages 43-61)Thesis (Ph.D.)--Department of Psychology. University of Missouri--Kansas City, 2021Despite an abundance of evidence supporting the efficacy of motivational interviewing for health behavior change, little is known about how it works. This study conducted a secondary analysis of autonomous motivation as a mediator of motivational interviewing’s effects in a recently completed randomized controlled clinical trial comparing motivational interviewing to health education on smoking quit attempts (KC Quest). Results of the parent trial unexpectedly revealed that motivational interviewing was not more effective than health education for inducing quit attempts of smoking cessation. While the mechanism through with the interventions is still unknown it remains feasible that motivational interviewing led to quit attempts and cessation by increasing autonomous motivation while health education was effective through a different mechanism. Interventions consisted of motivational interviewing (n=90) and health education (n=92). The primary outcome was the occurrence of any quit attempt defined as a serious quit attempt of at least 24 hours (Biener & Abrams, 1991; Marlatt, Curry, & Gordon, 1988) by Week 26. The Treatment Self-Regulation Questionnaire (TSRQ), developed from self-determination theory (SDT:Deci & Ryan, 1985), assesses the degree of autonomous self-regulation regarding why people engage or would engage in healthy behavior. Change scores from baseline to week 26 in the Autonomous (AR) and Controlled regulation (CR) subscales were computed for use in the mediation modeling. Log-binomial regression mediation examining each mediator separately revealed neither AR nor CR mediated effects of motivational interviewing or health education on quit attempts. A strength of the KC Quest enrollment was the inclusion of a racially diverse group of participants (67.2% Black) most adversely effected by smoking co-morbidities. Our current study did not detect a difference in smoking outcomes based on motivation mediators among Black participants. An important implication of this study is that while self-regulation failed to explain how, motivational interviewing and health education both increased quit attempts. There is a need for future investigations to examine other SDT constructs, such as relatedness and competence, as potential mediators of smoking interventions.Introduction -- Literature review -- Methods -- Analysis -- Results -- Discussion -- Appendix A. Treatment self-regulation questionnair

Impact of incubation conditions on protein and C-Phycocyanin recovery from Arthrospira platensis post- pulsed electric field treatment

Pulsed electric field (PEF) was conducted for the extraction of proteins/C-Phycocyanins from Arthrospira platensis. The cyanobacterial suspension was treated with 1 μs long pulses at an electric field strength of 40 kV·cm−1 and a treatment energy of 114 kJ·kgsus−1 and 56 kJ·kgsus−1. For benchmarking, additional biomass was processed by high pressure homogenization. Homogeneity of the suspension prior to PEF-treatment influenced the protein/C-phycocyanin extraction efficiency. Stability of C-Phycocyanin during post-PEF incubation time was affected by incubation temperature and pH of the external medium. Biomass concentration severely affect proteins/C-Phycocyanins extraction yield via PEF-treatment. The optimum conditions for extraction of proteins/ C-Phycocyanin was obtained at 23 °C while incubating in pH 8-buffer. The energy demand for PEF-Treatment amounts to 0.56 MJ·kgdw−1 when processing biomass at 100 gdw·kgsus−1. PEF treatment enhances the protein/CPhycocyanin extraction yield, thus, it can be suggested as preferential downstream processing method for the production of C-Phycocyanin from A. platensis biomass

Impact of incubation conditions on protein and C-Phycocyanin recovery from Arthrospira platensis post- pulsed electric field treatment

Pulsed electric field (PEF) was conducted for the extraction of proteins/C-Phycocyanins from Arthrospira platensis. The cyanobacterial suspension was treated with 1 μs long pulses at an electric field strength of 40 kV·cm−1 and a treatment energy of 114 kJ·kgsus−1 and 56 kJ·kgsus−1. For benchmarking, additional biomass was processed by high pressure homogenization. Homogeneity of the suspension prior to PEF-treatment influenced the protein/C-phycocyanin extraction efficiency. Stability of C-Phycocyanin during post-PEF incubation time was affected by incubation temperature and pH of the external medium. Biomass concentration severely affect proteins/C-Phycocyanins extraction yield via PEF-treatment. The optimum conditions for extraction of proteins/ C-Phycocyanin was obtained at 23 °C while incubating in pH 8-buffer. The energy demand for PEF-Treatment amounts to 0.56 MJ·kgdw−1 when processing biomass at 100 gdw·kgsus−1. PEF treatment enhances the protein/CPhycocyanin extraction yield, thus, it can be suggested as preferential downstream processing method for the production of C-Phycocyanin from A. platensis biomass

Phthalates Impair Germ Cell Number in the Mouse Fetal Testis by an Androgen- and Estrogen-Independent Mechanism

Data from experiments conducted almost exclusively in the rat have established that some phthalates have deleterious effects on the fetal testis probably due to their antiandrogenic and/or estrogenic effects, but their mechanisms of action remain unknown. A recent study reported that phthalates also have deleterious effects on human fetal testis with germ cell number, but not steroidogenesis altered. Therefore, we used organ culture of fetal testes at different stages of development to analyze the direct effects of phthalates on both steroidogenesis and gonocyte development and to determine if the effects of MEHP on these functions reported in the rat can be extended to other mammalian species. We defined specific periods of sensitivity of the fetal mouse testis to MEHP for these two functions and showed that the effects of phthalates on steroidogenesis vary with the developmental stage. Conversely, the strong deleterious effects of phthalates on germ cells were constantly present during the active phases of gonocyte development and thus share no relationship with the steroidogenic status. Moreover, all the effects of phthalates were unchanged in testes from mice deficient for estrogen (ERαKO or ERβKO) or androgen (Tfm) receptors. In conclusion, our results demonstrate that phthalates impair mouse fetal germ cell number similarly to other mammalian species, but are neither estrogenic nor antiandrogenic molecules because their effects do not involve, directly or indirectly, ER or AR

Targeting surface nucleolin with multivalent HB-19 and related Nucant pseudopeptides results in distinct inhibitory mechanisms depending on the malignant tumor cell type

<p>Abstract</p> <p>Background</p> <p>Nucleolin expressed at the cell surface is a binding protein for a variety of ligands implicated in tumorigenesis and angiogenesis. By using a specific antagonist that binds the C-terminal RGG domain of nucleolin, the HB-19 pseudopeptide, we recently reported that targeting surface nucleolin with HB-19 suppresses progression of established human breast tumor cells in the athymic nude mice, and delays development of spontaneous melanoma in the RET transgenic mice.</p> <p>Methods</p> <p>By the capacity of HB-19 to bind stably surface nucleolin, we purified and identified nucleolin partners at the cell surface. HB-19 and related multivalent Nucant pseudopeptides, that present pentavalently or hexavalently the tripeptide Lysψ(CH₂N)-Pro-Arg, were then used to show that targeting surface nucleolin results in distinct inhibitory mechanisms on breast, prostate, colon carcinoma and leukemia cells.</p> <p>Results</p> <p>Surface nucleolin exists in a 500-kDa protein complex including several other proteins, which we identified by microsequencing as two Wnt related proteins, Ku86 autoantigen, signal recognition particle subunits SRP68/72, the receptor for complement component gC1q-R, and ribosomal proteins S4/S6. Interestingly, some of the surface-nucleolin associated proteins are implicated in cell signaling, tumor cell adhesion, migration, invasion, cell death, autoimmunity, and bacterial infections. Surface nucleolin in the 500-kDa complex is highly stable. Surface nucleolin antagonists, HB-19 and related multivalent Nucant pseudopeptides, exert distinct inhibitory mechanisms depending on the malignant tumor cell type. For example, in epithelial tumor cells they inhibit cell adhesion or spreading and induce reversion of the malignant phenotype (BMC cancer 2010, <b>10</b>:325) while in leukemia cells they trigger a rapid cell death associated with DNA fragmentation. The fact that these pseudopeptides do not cause cell death in epithelial tumor cells indicates that cell death in leukemia cells is triggered by a specific signaling mechanism, rather than nonspecific cellular injury.</p> <p>Conclusions</p> <p>Our results suggest that targeting surface nucleolin could change the organization of the 500-kDa complex to interfere with the proper functioning of surface nucleolin and the associated proteins, and thus lead to distinct inhibitory mechanisms. Consequently, HB-19 and related Nucant pseudopeptides provide novel therapeutic opportunities in treatment of a wide variety of cancers and related malignancies.</p

Surface Expressed Nucleolin Is Constantly Induced in Tumor Cells to Mediate Calcium-Dependent Ligand Internalization

BACKGROUND: Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in tumorigenesis and angiogenesis. Emerging evidence suggests that the cell-surface expressed nucleolin is a strategic target for an effective and nontoxic cancer therapy. METHODOLOGY/PRINCIPAL FINDINGS: By monitoring the expression of nucleolin mRNA, and by measuring the level of nucleolin protein recovered from the surface and nucleus of cells, here we show that the presence of nucleolin at the cell surface is dependent on the constant induction of nucleolin mRNA. Indeed, inhibitors of RNA transcription or translation block expression of surface nucleolin while no apparent effect is observed on the level of nucleolin in the nucleus. The estimated half-life of surface nucleolin is less than one hour, whereas that of nuclear nucleolin is more than 8 hours. Nucleolin mRNA induction is reduced markedly in normal fibroblasts that reach confluence, while it occurs continuously even in post-confluent epithelial tumor cells consistent with their capacity to proliferate without contact inhibition. Interestingly, cold and heat shock induce nucleolin mRNA concomitantly to enhanced mRNA expression of the heat shock protein 70, thus suggesting that surface nucleolin induction also occurs in response to an environmental insult. At the cell surface, one of the main functions of nucleolin is to shuttle specific extracellular ligands by an active transport mechanism, which we show here to be calcium dependent. CONCLUSION/SIGNIFICANCE: Our results demonstrate that the expression of surface nucleolin is an early metabolic event coupled with tumor cell proliferation and stress response. The fact that surface nucleolin is constantly and abundantly expressed on the surface of tumor cells, makes them a preferential target for the inhibitory action of anticancer agents that target surface nucleolin

Biological signalling supports biotechnology – Pulsed electric fields extract a cell-death inducing factor from Chlorella vulgaris

Compared to mechanical extraction methods, pulsed electric field (PEF) treatment provides an energy-efficient and gentle alternative. However, the biological processes involved are poorly understood. The unicellular green microalga Chlorella vulgaris was used as model organism to investigate the effect of PEF treatment on biological cells. A viability assay using fluorescein diacetate measured by flow cytometry was established. The influence of developmental stage on viability could be shown in synchronised cultures when applying PEF treatment with very low specific energies where one part of cells undergoes cell death, and the other part stays viable after treatment. Reactive oxygen species generation after similar low-energy PEF treatment could be shown, indicating that PEFs could act as abiotic stress signal. Most importantly, a cell-death inducing factor could be extracted. A water-soluble extract derived from microalgae suspensions incubated for 24 h after PEF treatment caused the recipient microalgae to die, even though the recipient cells had not been subjected to PEF treatment directly. The working model assumes that low-energy PEF treatment induces programmed cell death in C. vulgaris while specifically releasing a cell-death inducing factor. Low-energy PEF treatment with subsequent incubation period could be a novel biotechnological strategy to extract soluble proteins and lipids in cascade process

Murine isoforms of retinoic acid receptor gamma with specific patterns of expression.

We have characterized seven murine retinoic acid receptor gamma cDNA isoforms (mRAR-gamma 1 to -gamma 7) generated by alternative splicing of at least seven exons. These isoforms differ from one another in their 5' untranslated region and in two cases (mRAR-gamma 1 and -gamma 2) differ in their N-terminal A region, which is known to be important for differential transactivation by other nuclear receptors. mRAR-gamma 1 and -gamma 2, the predominant isoforms, are differentially expressed in adult tissues and during embryogenesis. Most notably, skin contains almost exclusively mRAR-gamma 1 transcripts. The conservation of the RAR-gamma isoforms from mouse to human together with their patterns of expression suggests that they perform specific functions, which may account for the pleiotropic effect of retinoic acid in embryogenesis and development