Testing of antiseptics in cell culture and bacterial culture

Bei der Behandlung von Wunden ist der Gebrauch von Antiseptika ebenso üblich wie der von Antibiotika. In dieser Arbeit testeten wir die Auswirkungen auf kommerziell erhältliche Präparationen von Octenidin-dihydrochlorid (O: „klassisches“ Octenisept® und O+: Octenisept® verdünnt mit Glucose) in Bezug auf Zellproliferation und Zellmigration in der Zellkultur. Zusätzlich versuchten wir mittels RT-PCR herauszufinden, wie Octenidin die Zellproliferation auf der Ebene der Genexpression steuert. Gemäß den Erwartungen, hohe Konzentrationen von O und O+ waren zytotoxisch. Eine Verdünnung von 1:4096 von O+ bewirkte nicht nur einen Abbruch der Zytotoxizität, sondern auch einen Anstieg der Zellproliferation von 60 % im Vergleich zu Kontrollzellen. Dies galt jedoch nicht für O. Die zytotoxischen Effekte von hohen O+ Konzentrationen waren vermindert, wenn Zellen auf der EZM Komponente CHS kultiviert wurden. Dieses Resultat deutet darauf hin, dass Zellen, die in der EZM eingebettet sind, weniger empfindlich gegenüber O+, aber nicht O, sind. Genexpressionsanalysen zeigten, dass O und O+ Applikation keine signifikante Inhibierung, mit TGF-ß1 als Ausnahme, der Transkription ausgewählter Gene bewirkt. In Kombination mit 20 verschiedenen Antibiotika waren O und O+ waren fähig, die bakterizide Wirkung von vielen der untersuchten Antibiotika zu verbessern. Besonders die gleichzeitige Applikation von O+ und bestimmten Antibiotika zeigte die besten synergistischen Interaktionen gegen S. aureus im Vergleich zu den anderen untersuchten Antiseptika Chlorhexidin, Beta-Isodona® und Taurolin®. Alles in allem hat die Verdünnung von Octenidin mit Glucose einen positiven Effekt auf die Zellproliferation in vitro ohne die bakterizide Wirkung von Octenidin negativ zu beeinflussen. Dies könnte das Anwendungsgebiet von Octenidin erweitern.In wound care use of antiseptics is common as is the use of antibiotics. Therefore, we tested the impact of commercially available preparations of octenidine-hydrochloride (O: “classical” Octenisept® and O+: glucose in combination with Octenisept®). Additionally, we tried to scout the effects of how octenidine did influence cell proliferation and migration at the molecular level using rt-PCR analysis. As expected high concentrations O and O+ were highly cytotoxic but with 1:4096 dilution of O+ the effect was reverted to enhanced cell proliferation (60 % over control cells). This was not observed so with O. Furthermore, the cytotoxic effects of a high O+ concentration were ameliorated when cells were grown on the ECM component CHS. Thus, this result indicates that cells embedded into ECM components are less sensitive to O+ but not so to O. From gene expression analysis data we can reason that none of the investigated genes, except TGF-ß, was inhibited. In combinations with 20 different antibiotics, O and O+ were able to enhance bactericidal potency of the antibiotics in many cases. Especially, concomitant application of O+ and antibiotics had the most positive cooperativity against S. aureus in comparison to other tested antiseptics including Chlorhexidine, Beta-Isodona® and Taurolin®. In conclusion, the dilution of octenidine with glucose has a favorable effect on cell proliferation in vitro without adversely affecting bactericidal properties of octenidine. Together with the potency to enhance microbicidal efficiency of several antibiotics, the dilution of Octenisept® with glucose could extend the field of application of this agent

The potential of calcium hydroxide to reduce storage losses: A four months monitoring study of spruce wood chip piles at industrial scale

The objective of this study was to investigate the effect of an alkaline additive on the storage of wood chips from Norway spruce forest residues. Piles of untreated and calcium hydroxide treated wood chips (250 m3) were set up and investigated for four months. It was demonstrated that adding Ca(OH)2 to moist wood chips decreased the dry matter loss by 6%. This was attributed to the increase of the pH to a level of 8, rendering the habitat less suitable for fungal colonisation. The results suggest the set-up storage strategy as a potential alternative method for preserving wood chips when long term storage is required

The effect of calcium hydroxide on the storage behaviour of poplar wood chips in open-air piles

Biomass degradation by microorganisms may cause major losses during the storage of wood chips for energy production. Poplar wood chips from short rotation coppices are especially prone to degradation with dry matter losses (DML) of up to 25% within a storage period, emphasizing the need for countermeasures. Therefore, we investigated the potential of the addition of alkaline Ca(OH)2 to the wet biomass of poplar wood chips and hypothesised that the establishment of an alkaline environment would reduce the activity of fungi, the primary wood degraders. Three industrial-scale piles (250 m³) with 0, 1.5 and 3% Ca(OH)2 were installed in Güssing, Austria and for four months (April–August 2019) the pile temperature, pH, moisture content, gas evolution (O2, CO2, H2, H2S, CH4) as well as DML were monitored. Ca(OH)2 altered the physicochemical properties of the wood chips but did not prevent biomass losses. However, as compared to literature, the DML were, compared to earlier investigations, also low in the control. In addition, cultivation methods were performed to evaluate the diversity of thermophilic microbes throughout the storage. Numerous filamentous fungi belonging to the phyla Ascomycota and Mucoromycota were isolated, being Rhizomucor pusillus, Aspergillus fumigatus, Thermomyces lanuginosa and Thermoascus aurantiacus the dominant species. Only minor differences in the fungal composition were detected as a result of Ca(OH)2 addition. Instead, clear shifts in colony forming units (CFUs) were detected as a function of progressing storage time, with a decrease of the number of propagules after four months

Establishment of a Laboratory Scale Set-Up with Controlled Temperature and High Humidity to Investigate Dry Matter Losses of Wood Chips from Poplar during Storage

The aim of this work was to improve the understanding of dry matter losses (DML) that occur in wood chips during the initial phase of storage in outdoor piles. For this purpose, a laboratory scale storage chamber was developed and investigated regarding its ability to recreate the conditions that chips undergo during the initial phase of outdoor storage. Three trials with poplar Max-4 (Populus maximowiczii Henry  Populus nigra L.) chips were performed for 6–10 weeks in the storage chamber under controlled temperature and assisted humidity. Two different setups were investigated to maintain a high relative humidity (RH) inside the storage chamber; one using water containers, and one assisted with a humidifier. Moisture content (MC) and DML of the chips were measured at different storage times to evaluate their storage behaviour in the chamber. Additionally, microbiological analyses of the culturable fraction of saproxylic microbiota were performed, with a focus on mesophilic fungi, but discriminating also xerophilic fungi, and mesophilic bacteria, with focus on actinobacteria, in two trials, to gain a view on the poplar wood chip-inhabiting microorganisms as a function of storage conditions (moisture, temperature) and time. Results show that DML up to 8.8–13.7% occurred in the chips within 6–10 storage weeks. The maximum DML were reached in the trial using the humidifier, which seemed a suitable technique to keep a high RH in the testing chamber, and thus, to analyse the wood chips in conditions comparable to those in outdoor piles during the initial storage phase

The Paralogous Histone Deacetylases Rpd3 and Rpd31 Play Opposing Roles in Regulating the White-Opaque Switch in the Fungal Pathogen Candida albicans

Chromatin modifications affect gene regulation in response to environmental stimuli in numerous biological processes. For example, N-acetyl-glucosamine and CO2 induce a morphogenetic conversion between white (W) and opaque (O) cells in MTL (mating-type locus) homozygous and heterozygous (a/α) strains of the human fungal pathogen Candida albicans. Here, we identify 8 histone-modifying enzymes playing distinct roles in the regulation of W/O switching in MTL homozygous and heterozygous strains. Most strikingly, genetic removal of the paralogous genes RPD3 and RPD31, both of which encode almost identical orthologues of the yeast histone deacetylase (HDAC) Rpd3, reveals opposing roles in W/O switching of MTLa/α strains. We show that Rpd3 and Rpd31 functions depend on MTL genotypes. Strikingly, we demonstrate that Rpd3 and Rpd31, which are almost identical except for a divergent C-terminal extension present in Rpd31, exert their functions in distinct regulatory complexes referred to as CaRpd3L and CaRpd31S complexes. Moreover, we identify the Candida orf19.7185 product Ume1, the orthologue of yeast Ume1, as a shared core subunit of CaRpd3L and CaRpd31S. Mechanistically, we show that the opposing roles of Rpd3 and Rpd31 require their deacetylase activities. Importantly, CaRpd3L interacts with the heterodimeric transcriptional repressor a1/α2, thus controlling expression of WOR1 encoding the master regulator of W/O switching. Thus, our work provides novel insight about regulation mechanisms of W/O switching in MTLa/α strains. This is the first example of two highly conserved HDACs exerting opposing regulatory functions in the same process in a eukaryotic cell

The Candida albicans HIR histone chaperone regulates the yeast-to-hyphae transition by controlling the sensitivity to morphogenesis signals.

Morphological plasticity such as the yeast-to-hyphae transition is a key virulence factor of the human fungal pathogen Candida albicans. Hyphal formation is controlled by a multilayer regulatory network composed of environmental sensing, signaling, transcriptional modulators as well as chromatin modifications. Here, we demonstrate a novel role for the replication-independent HIR histone chaperone complex in fungal morphogenesis. HIR operates as a crucial modulator of hyphal development, since genetic ablation of the HIR complex subunit Hir1 decreases sensitivity to morphogenetic stimuli. Strikingly, HIR1-deficient cells display altered transcriptional amplitudes upon hyphal initiation, suggesting that Hir1 affects transcription by establishing transcriptional thresholds required for driving morphogenetic cell-fate decisions. Furthermore, ectopic expression of the transcription factor Ume6, which facilitates hyphal maintenance, rescues filamentation defects of hir1Δ/Δ cells, suggesting that Hir1 impacts the early phase of hyphal initiation. Hence, chromatin chaperone-mediated fine-tuning of transcription is crucial for driving morphogenetic conversions in the fungal pathogen C. albicans

Histone modification by lysine acetylation through writers (KATs) and erasers (KDACs).

<p>Much of the mechanistic knowledge about the role of chromatin modifications in gene expression regulation comes from the nonpathogenic baker’s yeast (for excellent recent reviews, see [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref065" target="_blank">65</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref067" target="_blank">67</a>]). Although the precise mechanisms of the interplay between writers, readers, and erasers remain ill-defined in many cases, it is fair to speculate that histone modifiers may play pivotal roles in the adaption of fungal pathogens to host immune defense. The major nucleosome building blocks, histones H2A, H2B, H3, and H4, are subject to dynamic and reversible posttranslational modifications (PTMs) by several KATs and KDACs functioning as writers and erasers of epigenetic marks. KATs like the Rtt109, which is a fungal-specific writer, and the cognate Hst3 eraser recognize the lysine residue K56 on histone H3. The KAT Esa1 acts primarily on H2A/H2B and H2AZ, with Hda1 and Hos3 acting as erasers (Panel A). By contrast, Hat1 targets mainly, though not exclusively, newly synthesized cytoplasmic histone H4 for the purpose of nuclear nucleosome remodeling during DNA damage repair [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref037" target="_blank">37</a>], as well as other processes demanding nucleosome exchange. The pleiotropic KAT Gcn5 acts mainly on histone H4 and H3. Each N-terminal histone lysine can be recognized by several redundant KATs/KDACs. Histone H3 and H4 are modified by several writers and erasers in <i>C</i>. <i>albicans</i>, creating extensive combinatorial complexity and many possibilities for gene regulation depending on the cellular context. For example, the KDACs, Rpd3/31, Hda1, and the SET3C complex consisting of Set3 and Hos2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref029" target="_blank">29</a>] act mainly on histone H3 and H4 (Panel B). Notably, kinases such as Cst20 (Panel A) and histone methyltransferases such as Dot1 and Set2 show restricted lysine specificities for histone H2B and H3, respectively. Panel C: A number of modulators of KATs/KDACs modulate (inhibit or activate) several KATs/KDACs, whereas others appear enzyme specific. Of note, no activator for KDACs have been identified for fungal KDACs, although several are known for mammalian KDACs [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref056" target="_blank">56</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref083" target="_blank">83</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref087" target="_blank">87</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref096" target="_blank">96</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref104" target="_blank">104</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref112" target="_blank">112</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref121" target="_blank">121</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref141" target="_blank">141</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref155" target="_blank">155</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref157" target="_blank">157</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref162" target="_blank">162</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005938#ppat.1005938.ref165" target="_blank">165</a>]. TSA, trichostatin A; SB, sodium butyrate; SAHA, suberoylanilide hydroxamic acid; VPA, valporic acid; NAM, nicotinamide; CPTH2, Cyclopentylidene-[4-(4-chlorophenyl)thiazol-2-yl)hydrazine; CPTH6, 3-methylcyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazine; NU9056, 5-(1,2-Thiazol-5-yldisulfanyl)-1,2-thiazole; MG149, 2-(4-Heptylphenethyl)-6-hydroxybenzoic acid; CTPB, N-[4-Chloro-3-(trifluoromethyl)phenyl]-2-ethoxy-6-pentadecylbenzamide; TTK21, N-(4-Chloro-3-trifluoromethyl-phenyl)-2-n-propoxy- benzamide; HDPHs, histone dephoshorlyases; HMTs, histone methyltransferases; KATs, lysine acetyltransferases; KDACs, lysine deacetylases. Red boxes, fungal-specific modifications; grey circles, evolutionary conserved lysines in histone tails; orange ellipses, writer KATs; yellow ellipses, eraser KDACs; blue ellipses, histone dephosphorylases; cyan ellipses, histone kinases; green ellipses, histone methyltransferases.</p

Molecular monitoring of the poplar wood chip microbiome as a function of storage strategy

One of the most challenging aspects of using wood chips as renewable energy source is the loss of biomass related to storage. Therefore, we installed three outdoor industrial-scale piles (250 m³) of poplar wood chips and monitored the bacterial and fungal communities by next-generation sequencing over a storage period of 120 d. Two of the three piles were supplemented with calcium dihydroxide (Ca(OH)2) (1.5%, 3% w/w) in order to test its potential as alkaline stabilization agent to preserve woody biomass during storage. Shifts in the microbial community composition occurred almost entirely in the beginning of the storage experiment, which we attribute to the temperature rise of up to 60 °C within the first week of storage. Later, however, we found little changes. Independent of Ca(OH)2 concentration, a consortium of lignocellulolytic and thermotolerant microorganisms dominated the stored wood chip microbiota emphasizing their role as key players during wood decomposition. Although the addition of Ca(OH)2 altered the physicochemical properties of wood chips, it did not prevent loss of biomass. Especially the pH was increased in Ca(OH)2 treated piles. However, only minor differences in the microbial communities’ composition were detected following Ca(OH)2 addition, highlighting the microbes tolerance towards and adaptation to changing environmental conditions

Pour un développement territorial durable : l'aménagement des terroirs villageois au Niger : cas du projet Dallol Bosso Sud (1989-1994)

Depuis le milieu des années 1980, sur le continent africain, l'approche «aménagement des terroirs villageois» a émergé comme une réponse concrète aux problèmes de développement et de gestion des ressources naturelles au sein des communautés villageoises. Cette approche, qui est désormais inscrite comme option officielle de développement rural dans tous les pays du Sahel mérite d'être évaluée. Notre thèse se veut une contribution à l'évaluation de cette approche «d'aménagement des terroirs villageois». Elle porte sur la stratégie de développement territorial durable au Niger et centrée sur l'analyse et l'interprétation des résultats du premier projet du genre appliqué dans la région du Dallol Bosso Sud (1989-1994). Notre recherche emprunte une démarche interdisciplinaire et s'appuie sur les théories du développement territorial, du développement durable, de l'éco-aménagement et de l'écodéveloppement. Nous en parlerons comme étant une approche de développement territorial durable. C'est l'objet de la première partie de la thèse axée sur l'analyse théorique de cette notion de développement territorial durable. La deuxième partie porte sur l'étude empirique du projet nigérien d'aménagement des terroirs villageois du Dallol Bosso Sud. L'enquête terrain a été réalisée entre mars et juin 1999 auprès des acteurs-clés impliqués dans le projet. Cette enquête a porté sur l'appréciation et l'analyse des résultats et des impacts du projet (sociaux, économiques, écologiques et politiques) et sur l'appropriation (socio-politique, technique, financière) de la démarche du projet par les populations concernées. L'analyse a été réalisée à la fois à partir d'entretiens, d'observation directe sur terrain et par l'approche documentaire. Nos résultats révèlent que l'approche «aménagement des terroirs villageois» est porteuse d'orientations de développement territorial durable, n s'agit en effet d'une approche globale, multidimensionnelle et centrée sur la réalisation simultanée de plusieurs activités qui ont des impacts sociaux, économiques, écologiques, politiques et spatiaux très concrets pour les populations et leurs territoires. L'étude démontre par contre que la population n'a pas pu s'approprier suffisamment les différentes techniques, technologies et méthodes proposées par le projet à cause de l'imbrication malheureuse de certains facteurs conjoncturels et structurels. Notre recherche va ainsi au-delà de certaines idées reçues, voulant incriminer les populations dans un refus d'appropriation alors que les enjeux véritables sont de l'ordre des défis d'un métissage culturel majeur entre des manières d'être et de produire différenciées