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CAZymes from the thermophilic fungus Thermoascus aurantiacus are induced by C5 and C6 sugars
Biotechnology for Biofuels volume 14, Article number: 169 (2021)
Filamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood.
A fed-batch system with plant biomass-derived sugars d-xylose, l-arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus. The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and l-arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways.
This work has identified two additional CAZyme inducers for T. aurantiacus, l-arabinose and cellobiose, along with d-xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures.
Carbohydrate active enzymes (CAZymes) are vital for the conversion of plant polysaccharides to biofuels and bio-based chemicals [1]. Cellulose and hemicellulose are the most abundant polysaccharides in plant biomass and thus are an immense untapped carbon pool for biotechnological applications. Through using CAZymes such as cellulases and xylanases, lignocellulosic plant biomass can be deconstructed into simple sugars that can be further converted into biofuels and bioproducts [2].
The thermophilic fungus Thermoascus aurantiacus is a notable host for thermostable CAZyme production [3]. The enzymes of this fungus were found to be more heat stable and effective at deconstructing lignocellulose than enzymes from other thermophilic fungi and demonstrated the release of sugars from pre-treated biomass at comparable levels to the commercial enzymatic mixture CTec2 at 50 °C. Notably, the enzymatic mixture from T. aurantiacus lost only half of its activity during saccharification of pre-treated switchgrass at 70 °C, while CTec2 was inactive at this temperature [4]. Moreover, T. aurantiacus secretes large amounts of a lytic polysaccharide monooxygenase (LPMO), which depolymerizes cellulose and has been extensively studied and shown to be the most abundant protein secreted by T. aurantiacus [5]. Recently, procedures for genetic transformation, sexual crossing and gene editing using CRISPR/Cas9 have been established for this self-crossing organism, which opens many possibilities to harness the ability of T. aurantiacus to secrete highly active and thermostable CAZymes [6, 7]. However, little is known about CAZyme induction in T. aurantiacus, which is vital for designing improved strain engineering and bioprocess strategies.
Cellulase and xylanase induction has been intensively studied in a few filamentous fungi [8,9,10,11]. Trichoderma reesei has historically been the most prominent fungus for commercial cellulase production [12, 13]. The cellulases of T. reesei are induced in the presence cellulose as well as simple sugars like cellobiose, lactose and sophorose. While cellobiose induces cellulases in a variety of filamentous fungi, including the model fungus Neurospora crassa, cellulase induction by lactose and sophorose is limited to a few fungi besides T. reesei [14]. For N. crassa and T. reesei, xylose induces xylanase production; however, xylose was found to induce both cellulases and xylanases in the industrially used host Aspergillus niger [15]. While less is known about the inducing effect of l-arabinose, another major plant cell wall sugar, it was reported that this sugar induced xylanases in N. crassa and T. reesei [16,17,18]. These findings indicate that sugars related to plant cell wall degradation have very different effects on CAZyme secretion, even in closely related fungal species.
Induction of CAZymes by T. aurantiacus is not well understood. It was initially reported that T. aurantiacus secreted the highest level of cellulases when grown on alkali pre-treated bagasse and xylan while weak cellulase activity was found on Avicel cellulose [8]. To identify further inducers for T. aurantiacus, batch cultivations with d-xylose, cellobiose and l-arabinose were performed. High xylanase secretion of T. aurantiacus was reported for batch-cultures of xylan and non-metabolizable methyl β-d-xylopyranoside (MXP), but not xylobiose and d-xylose [10, 19]. Recently, experiments using controlled feeding of d-xylose to T. aurantiacus cultured in shake flasks and bioreactors demonstrated substantial cellulase and xylanase induction that was higher than batch cultures with xylan [11]. Studies performing similar fed-batch experiments using T. reesei found strong CAZyme secretion induction with sugars related to plant cell wall degradation [17, 20]. Therefore, continuous feeding helps to circumvent two challenges associated with batch cultures: (1) the high initial amount of sugar first causes carbon catabolite repression (CCR) [21]; the sugar is then consumed rapidly, leading to no further induction; and (2) the presence of impurities in highly purified plant polysaccharides (e.g., cellulose contaminated with residual xylan) that can lead to fundamentally different outcomes compared to using purified sugar substrates at low concentration [11]. Lastly, fed-batch studies can be of great utility for -omics experiments to refine bioprocess and genetic engineering strategies for generating high enzyme secreting fungal strains [22,23,24].
The goal of this study was to test CAZyme inducers of T. aurantiacus by fed-batch cultivation to screen for enzymatic activities and to reveal how gene expression is affected by these inducers.
Arabinose and cellobiose induce CAZymes in T. aurantiacus
Previously, fed-batch induction experiments led to the identification of d-xylose as an inducer of CAZymes in T. aurantiacus [11]. During this previous fed-batch study, beechwood xylan and a variety of celluloses (Avicel, Sigmacell, bacterial cellulose) also induced CAZyme production in T. aurantiacus. Since both beechwood xylan and the plant-derived celluloses (Avicel, Sigmacell) contained xylose, the inductive effects of other sugars besides xylose in these substrates could not be deconvoluted [24]. Therefore, we chose two additional lignocellulose-derived sugars to test as inducers: l-arabinose, which constitutes ~ 10% of beechwood xylan, and cellobiose, which is a common cellulase inducer and the product of cellobiohydrolase [11]. T. aurantiacus was grown first in a seed culture in d-glucose medium supplemented with soy meal peptone for 48 h and shifted to two sets of shake flasks; one set containing each individual sugar (0.5% w/v xylose, cellobiose and l-arabinose, referred to as batch culture set) and another set of flasks where the same amount of those sugars were continuously fed (fed-batch set) over a 3-day period (Fig. 1). The batch culture set also included triplicate beechwood xylan (0.5% w/v) cultures to compare the purified sugars with a complex substrate that previously found to be a good CAZyme inducer.
SDS-PAGE of culture filtrates demonstrated that the characteristic CAZyme bands previously observed for T. aurantiacus (cellobiohydrolase (~ 54 kDa), endoglucanase/xylanase (~ 33 kDa), and lytic polysaccharide monooxygenase (~ 25 kDa) [3, 4] were clearly visible for the fed-batch cultures, which had higher total protein titers than batch cultures (Fig. 1A and B). The three sugar feeds induced different enzymatic activities. High xylanase activities were found during C5 sugar feeding, while cellobiose fed-batch led to a small increase in xylanase activity in the culture broth (Fig. 1C). Interestingly, cellobiosidase activity followed the same trend of high activity during C5 sugar feeding (Fig. 1D) while endoglucanase activities, measured through the CMCase assay, were in a comparable range for both the cellobiose and C5 fed-batch cultures (Fig. 1E). In contrast, the cellobiose fed-batch and batch cultured displayed higher β-glucosidase activity than the C5 sugar cultures (Fig. 1F). The ability of the supernatants from the fed-batch cultures to release glucose from Avicel was also tested. In these experiments, the supernatant from the cellobiose-grown cultures had 72% higher glucose release than the xylose-grown cultures and 114% higher glucose release than the arabinose-grown cultures (Fig. 1G).
Development of minimal medium for T. aurantiacus protein production
The fed-batch experiments identified d-xylose, l-arabinose and cellobiose as inducers of CAZyme production. Gene expression experiments were designed to study gene expression patterns under these growth conditions to describe the cellular responses to these sugars. However, the T. aurantiacus induction experiments described above were performed with soy meal peptone, a complex nitrogen source containing sugars. Peptones can pose challenges when using systems biology tools, since considerable differences among manufacturers and also batch effects for the same product can be found [25]. While peptones were found to enable high protein production of T. aurantiacus, using a defined medium appeared more favorable for gene expression experiments. Therefore, a minimal medium with a defined nitrogen source was designed in this study. Previously, T. aurantiacus cultivations have been performed on media containing complex nitrogen sources such as yeast extract or peptones [4, 8, 9, 19, 26,27,28,29,30,31]. In only one study, T. aurantiacus was cultivated in Vogel's minimal glucose medium [10]. This medium supported poor growth in our hands, so the McClendon's medium, which has been used for the above described CAZyme production was adapted to employ (NH4)2SO4 as the sole nitrogen source for a defined minimal medium to study T. aurantiacus induction.
T. aurantiacus growth on d-glucose with McClendon's medium with soy meal peptone as the nitrogen source was compared with growth with (NH4)2SO4 (Fig. 2A and B). Growth of T. aurantiacus with (NH4)2SO4 as the nitrogen source in liquid medium was < 30% of the growth with soy meal peptone as measured by mycelial biomass. Growth with (NH4)2SO4 resulted in a drop in pH after 3 days from the initial of pH 5.5–2.5, while growth on soy meal peptone after 3 days resulted in pH of 6. The poor growth on (NH4)2SO4 was attributed to the drop in pH, which is consistent with previous studies demonstrating optimal growth and enzyme production by T. aurantiacus at pH ≥ 5 (12). To prevent the drop in pH during cultivation, the (NH4)2SO4-containing medium was buffered with 25 mM sodium citrate. The citrate buffer maintained the pH around five for the submerged T. aurantiacus culture and doubled the fungal growth compared to adding (NH4)2SO4 alone. This minimal medium was tested in a fed-batch experiment with d-xylose, cellobiose and l-arabinose as described before (Fig. 2). Although the overall protein production was lower in the minimal medium than in medium amended with soy meal peptone, the enzyme activity patterns were consistent between both tests. Therefore, this improved minimal medium was used to assess gene expression patterns during induction.
Differential gene expression analysis of T. aurantiacus
The gene expression study was performed using the fed-batch system described above. T. aurantiacus was grown in d-glucose minimal medium and then shifted to shake flasks containing minimal medium, where d-xylose, cellobiose and l-arabinose were added through continuous feeding. Additionally, a starvation condition (only minimal medium without a carbon source) and a CAZyme-repressing condition (high d-glucose minimal medium) were added as controls.
A Venn diagram was generated to uncover genes differently expressed in d-glucose medium and the sugar fed-batch conditions compared to no carbohydrate medium to investigate the effects of those sugars on gene expression (Fig. 3). All up-regulated genes of the d-xylose, cellobiose, l-arabinose and d-glucose treatments compared to no carbohydrate medium (logFC > 1, pval < 0.05) were used for this analysis. All numbers in parenthesis are T. aurantiacus protein IDs from the JGI Mycocosm database unless otherwise stated.
Genes specific for the l-arabinose fed-batch condition were a putative xylulokinase (Prot. ID: 63574) an arabinofuranosidase (Prot. ID: 51505) and an α-xylosidase (Prot. ID: 58732) together with a sugar transporter (Prot. ID: 44260) that is most closely related to the d-xylose transporter xtrD (AN0250) of A. nidulans [32].
For the d-xylose feed condition, no genes specific for xylan and C5 sugar metabolism were found except for a putative α-glucuronidase (Prot. ID: 36875) and an F-box protein orthologue (Prot. ID: 43769) of the A. nidulans gene fbxA. Deletion of fbxA caused reduced secretion of xylanases in its native host and impaired CCR, which was indicated by resistance of the fbxA mutant to the d-glucose analog 2-deoxy-d-glucose [33]. Additionally, an unknown sugar transporter (Prot. ID: 55223) was up-regulated. A putative transcriptional regulator with highest similarity to the A. nidulans sexual development regulator nsdD was found here, which was recently found to be vital for cellulase and xylanase in P. oxalicum [34].
When the genes specific for both C5 sugar feeds were investigated, we found two of the most highly secreted enzymes of T. aurantiacus: the xylanase (Prot. ID: 1236) and endoglucanase (Prot. ID: 65156) which have been identified earlier by proteomics [4]. Also, a putative β-xylosidase (Prot. ID: 64461), xylitol dehydrogenase (Prot. ID: 66263) and d-xylose dehydrogenase (Prot. ID: 63693) were identified, indicating expression of a variety of genes related to xylan hydrolysis and the d-xylose and l-arabinose catabolism of T. aurantiacus. The metabolism of those two C5 sugars has been extensively investigated for the related fungus A. niger [35]. Orthologous of all necessary enzymes for d-xylose and l-arabinose catabolism were identified in T. aurantiacus, except for the l-arabinose reductase, the first enzyme needed for l-arabinose assimilation (Additional file 1: Figure S1). All the genes in the T. aurantiacus d-xylose/l-arabinose assimilation pathway were highly up-regulated during l-arabinose feed, while d-xylose caused up-regulation of the same genes except the putative l-xylulose reductase orthologue. Another pathway that appeared to be up-regulated during the C5 sugar feeds was the unfolded protein response (UPR) (Additional file 1: Figure S2). Several components of this pathway, namely a bipA [36] orthologue (Prot. ID: 46710) was up-regulated in the l-arabinose condition and clxA [37] (Prot. ID: 65748) was highly expressed in the l-arabinose and d-xylose condition. We found that other UPR-related genes, such as the orthologue of the regulator that activates UPR, hacA [38] (Prot. ID: 7916), and the protein disulfide-isomerase pdiA [39] (Prot. ID: 64656), were highly expressed during d-xylose and l-arabinose feeding relative to all other conditions (Additional file 1: Figure S2). UPR genes are often expressed during fungal enzyme secretion to counteract protein folding stress [40].
Intriguingly, the cellobiose feed condition did not display any plant cell wall degradation related CAZymes that were specific for this condition. However, genes up-regulated during d-xylose, cellobiose and l-arabinose feed were the main secreted cellobiohydrolase (Prot. ID: 41785) and a β-glucosidase (Prot. ID: 38776). Therefore, the expression of the main secreted xylanase, endoglucanase and cellobiohydrolase together with several xylan degrading enzymes happened in the C5 sugar conditions, while the fungus in the cellobiose condition appeared to only express the cellobiohydrolase at high levels. Lastly, a serine carboxypeptidase (Prot. ID: 57314) was identified up-regulated in all sugar feeds, which had highest sequence similarity to protH of A. niger. This enzyme is predicted to be secreted, which could be involved in degradation of secreted CAZymes.
Expression analysis of glycoside hydrolases, auxiliary family enzymes and carbohydrate esterases
The analysis of expression patterns was extended to compare expression of related CAZyme proteins. The carbohydrate active enzymes (CAZy) database classifies enzymes relevant for the degradation, modification or creation of glycosidic bonds [41]. The JGI mycocosm protein portal contains those CAZyme annotations for T. aurantiacus, where 323 CAZy genes are annotated in the T. aurantiacus genome [42]. Enzymes belonging to the glycoside hydrolases (GH), auxiliary activity (AA) and carbohydrate esterases (CE) have been found to be most important for plant cell wall deconstruction [14]. Five CAZymes (Prot. ID: 3070, AA 9; 41785, GH7; 65156, GH5; 1236, GH10; 46699, GH3) have been previously identified in T. aurantiacus ATCC 26904 from liquid cultures grown on plant biomass by proteomics analysis [3]. Virtually nothing is known about what other genes of this fungus respond to plant polysaccharides and their breakdown products. To uncover the expression trends of those unknown genes, we generated a heat map of the expression trends of the T. aurantiacus GH, AA and CE families.
The GH heat map (Fig. 2a) contained two main clusters: cluster 1 showed GHs that were highly up-regulated on d-glucose or d-glucose and other conditions, while cluster 2 contained GHs that exhibited low expression on d-glucose. Cluster 1 contained diverse types of GHs with only few predicted types of GH related to cellulose, xylan and pectin deconstruction. Conversely, the main four secreted GH described above were all found in cluster 2 (Fig. 4a). Cluster 2 also contained most of the cellulase, xylanase and pectinase genes and was divided into two sub-clusters (Cluster 2.1 and 2.2). Cluster 2.1 contained GHs that were highly up-regulated in no carbohydrate medium and to different degrees up-regulated during the sugar feed conditions. Thus, cluster 2.1 comprised GH that most likely resemble starvation responsive genes. The β-glucosidase that was previously identified in the T. aurantiacus supernatant [3]was also found to be strongly expressed during starvation and to a lesser extend during the sugar feed conditions. However, Cluster 2.2 contained GHs that were highly expressed under the sugar feed conditions and showed lower expression during starvation. This cluster appeared to resemble the genes induced by the sugar feed conditions and contains the previously identified endoglucanase, cellobiohydrolase and xylanase (marked). Also, most of cluster 2.2 genes are putative orthologues of A. niger GHs linked to plant cell wall deconstruction (Fig. 4a). Aspergillus niger orthologues of the GH51 arabinofuranosidase afbA, was found to released l-arabinose from arabinan and an unknown putative GH5 (T.ID: 44708) with highest similarity to exgC in A. nidulans were found highly expressed on d-xylose. Genes highly expressed on l-arabinose showed high similarity to the α-glucuronidase aguA, α-xylosidase axlB and the β-xylosidase xlnD. In addition to the high expression of the main secreted GH5, 7 and 10, we also found another putative GH5 that had highest similarity to exgA and was highly expressed on both C5 sugars. A putative β-glucosidase with highest similarity to bgl4 was the only GH highly expressed under all three inducing conditions. Few GHs showed elevated expression on cellobiose with only one gene exclusively expressed under this condition predicted β-glucuronidase (Prot. ID: 61003) that had the highest similarity to two uncharacterized A. nidulans genes: AN7089 and AN3992. Genes highly expressed during cellobiose feed in cluster 2.2 were mostly cellulases (GH1, GH3, GH5 and GH7). This is in accordance with our finding that cellobiose feed led to only weak xylanase activity in the culture supernatant while activity was high for glucose release from Avicel (Fig. 1).
Similar trends were found for the AA heat map (Fig. 4b), where cluster 1 contained AAs that were highly expressed on d-glucose with almost no expression on all other conditions. Cluster 2 was divided in two sub-clusters. Cluster 2.1 contained AA that were highly expressed during starvation including four predicted laccases, whose expression is often associated with starvation also in other fungi, and one putative unknown LPMO (Prot. ID: 2027). Cluster 2.2 contained genes that displayed low expression on d-glucose and no carbohydrate medium, and high expression during the sugar feed conditions. Here, the well-studied T. aurantiacus LPMO was found highly expressed during d-xylose and l-arabinose and to a less extent on cellobiose. Two predicted laccases showed a similar trend. Interestingly, one further laccase (Prot. ID: 39797) was highly expressed on d-glucose. This gene has a high similarity to A. niger mcoH and Trichophyton benhamiae conidial pigment biosynthesis oxidase. Therefore, this laccase might be required for pigmentation and could serve as a color locus for strain engineering efforts to identify targeted integrations of DNA constructs, using the newly developed transformation procedure for T. aurantiacus [6].
The CE expression heat map (Fig. 4c) revealed that all genes displayed virtually no expression on d-glucose, with the exception of a putative chitin-deacetylase (Prot. ID: 45413). Most CEs showed high expression on both d-xylose and l-arabinose and low expression on cellobiose, consistent with their deconstruction of bonds in hemicellulose. None of these genes had high similarities to genes characterized in Aspergilli. The CEs that might likely be of interest for lignocellulose deconstruction are those four that showed low expression on d-glucose and no carbon and thus seemed to be specifically expressed on one or both C5 sugars (Fig. 4c, black frame).
The understanding of fungal CAZyme induction is fundamental to enable the efficient conversion of lignocellulose into biofuels and bio-based products. In this study, we compared the CAZyme-inducing effect of d-xylose, cellobiose and l-arabinose through a fed-batch system at shake-flask scale using a peristaltic pump. Here, it was found that continuously feeding d-xylose, cellobiose and l-arabinose caused elevated secretion of cellulases of T. aurantiacus, which was not observed when the same amount of sugar was added in the beginning of the experiment. Notably, we found that while stimulating high enzyme secretion, individual enzymatic activities depended on which sugar was fed to the medium. Xylanase activity was almost absent in the cellobiose feed cultures, while this activity was highly induced in the C5 sugar feed cultures. While overall CMCase activity was similar among all three sugar feeds, we found elevated cellobiosidase activity during C5 sugar feeding and elevated β-glucosidase activity during cellobiose feeding. Cellobiose feeding also resulted in culture supernatants with an increased ability to release sugars from Avicel. The reason for lower enzyme secretion during batch cultivations may be that simple sugars can cause CCR when present at high amounts, which is partly mediated through the carbon catabolite repressor Cre-1/Cre1/CreA (22, 23, 45, 46)[21, 22, 43, 44]. Typically, secretion of CAZyme for the breakdown of complex sugars, such as polysaccharides, is repressed until all simple sugars are reduced to low levels. However, once the sugars are depleted, there will be no further induction in batch media. Complex lignocellulosic substrates, however, are broken down over longer periods of time and the sugar release thus happens more slowly and continuously, similar to a fed-batch process. Fed-batch has been found to improve many aspects of enzyme production in bioreactors with filamentous fungi compared to batch cultivations, indicating the importance of regulated substrate levels [20, 45]. We argue that fed-batch tests are vital for understanding the potential of individual inducer molecules, which is not feasible when using complex carbon sources such as cellulose or xylanase.
The high level of CAZyme production when feeding purified sugars illuminates previous studies of fungal gene expression and enzymatic activity, which reported that simple sugars such as d-xylose, cellobiose and l-arabinose during batch fermentations weakly induced cellulases and xylanases compared to complex polysaccharides such as xylan and lignocellulosic plant biomass in T. aurantiacus [9], A. niger [15] and Talaromyces versatilis [46]. These studies might have found stronger induction effects of the simple sugars on cellulases and xylanases expression when applying continuous feeding as we observed for T. aurantiacus. Nevertheless, it is widely established that complex substrates induce a broader variety of CAZymes [14]. Feeding mixtures of sugars or hydrolysates may generate more efficacious enzyme formulations without adding solid substrates to the fermentation medium [46,47,48].
The gene expression study established that cellulase and xylanase induction in T. aurantiacus follows a pattern similar to the related fungus A. niger, where d-xylose was found to induce production of these enzymes [15]. Putative T. aurantiacus orthologues of most of the characterized cell wall degrading enzymes from A. niger were highly up-regulated during d-xylose and l-arabinose feeding. These highly expressed T. aurantiacus genes have high similarity to A. niger orthologues related to deconstruction of cellulose (exgA, eglC, cbhA/cbhB and bgl4), xylan (xynA, axlB and xlnD) and arabinan (abfA). Notably, for the cellobiose feed, expression of all the respective T. aurantiacus genes was substantially lower compared to the C5 sugar feeds, with only the putative bgl4 orthologue showing equally high expression. Only putative orthologues of exgA, bgl4, eglC and cbhA/cbhB showed increased expression during cellobiose feed. These observations indicated that cellobiose mainly induced cellulase genes. We also found that the prominent T. aurantiacus LPMO [5] showed highest expression during C5 sugar feeds and intermediate expression during cellobiose feeding.
The effect of d-xylose feeding was different for the T. reesei CL847 mutant where this sugar strongly induced xylanase activity but very little cellulase activities while cellobiose and lactose strongly induced cellulase activities [20]. Furthermore, feeding a mixture of d-xylose and cellobiose or lactose led to reduction in cellulase activities compared to not feeding d-xylose. In contrast, feeding a mixture of d-xylose, d-glucose and cellobiose did not decrease cellulase production of the T. reesei M3-1 mutant compared to not adding d-xylose to the feed, while xylanase secretion was substantially promoted by adding d-xylose [17]. This contrary observation could be due to the different mutant strains used in the respective studies. However, unlike industrial hosts such as Trichoderma sp., d-xylose and l-arabinose alone can be used to produce cellulases and xylanases simultaneously at high amounts in T. aurantiacus.
The genetic regulation of T. aurantiacus CAZyme production at this point remains unknown. In A. niger and T. reesei it was found that both types of enzymes were induced by the same orthologues (XlnR/Xyr1) [14, 15]. Notably, XlnR in A. niger was found to induce expression of xlnD and aguA when grown on d-xylose and xylan [15]. Both putative CAZyme orthologues were also highly expressed in T. aurantiacus when grown on d-xylose. This finding, together with the recent demonstration that XlnR overexpression in T. aurantiacus caused a substantial increase in xlyanase production compared to the wild type strain [6], suggests that XlnR has a similar role in T. aurantiacus as in A. niger. Additonally, two further orthologues (Prot. IDs 43769 and 600557) of the f-box protein gene fbxA of A. nidulans and the sexual development regulator nsdD were identified through Venn diagram binning in the xylose condition in T. aurantiacus; fbxA was found to be essential for xylanase expression in A. nidulans [33]. Recently, the nsdD orthologue was deleted in P. oxalicum, which caused a substantial reduction of cellulase and xylanase secretion [34]. Thus, 600557 might have a similar function in T. auranticus and could be another target for strain improvement. Further transcription factors that have been found to mediate CAZyme production were orthologues of carbon catabolite repressor Cre1/Cre-1/CreA [43, 44, 49], the cellulase/xylanase regulators XlnR/Xyr1, Clr-1/ClrA, Clr2/ClrB and ClbR [14, 15, 48, 50], the amylase regulator AmyR/Col26 [51,52,53], the nitrogen- and starvation regualtor PacG/Vib1[52] and further regulators involved in pectin and rhamnose utilization GaaR/Pdr-1/RhaR [54,55,56]. Putative orthologues for all of those genes were found in T. aurantiacus and their dynamics could be uncovered using our data set. We found that the putative T. aurantiacus orthologues of all of the genes listed above except ClbR and CreA showed statistically significant lower expression on d-glucose (pval < 0.05) compared to most other feed and starvation conditions (Additional file 1: Figure S3). The comparably high expression of those regulators on the no carbohydrate medium compared to the sugar feed conditions is not surprising, since some transcription factors such as CRE1 and XLNR were found to be regulated at the protein level through post-translational modifications in other fungi [57,58,59,60,61,62]. Nevertheless, CCR seemed to affect those regulators in a similar way, potentially mediated by the CreA orthologue, as it has been described for A. niger, N. crassa and T. reesei. Therefore, we hypothesize that overexpression of the respective T. aurantiacus genes showing high similarity to known transcriptional CAZyme activators will likely have substantial effects on expression of cellulases, xylanases and pectinases. Furthermore, we found that several gene orthologues related to UPR in A. niger were found to be highly expressed during d-xylose and l-arabinose feed in T. aurantiacus. The observation of upregulation of genes associated with UPR is consistent with previous observations in A. niger of stress responses under conditions where high levels of protein are secreted [60, 61]. In N. crassa, disruption of UPR-related genes did not effect the transcription of CAZyme genes when grown on cellulose, but disrupted the secretion of their gene products [62]. While it is not certain at this point whether this pathway is stimulated by C5 sugars or the onset of high enzyme secretion induced by those sugars, the UPR pathway may be a promising target for future strain engineering efforts. Lastly, we identified several candidate genes in T. aurantiacus that had highest similarity to genes used by A. niger to assimilate d-xylose and l-arabinose. While experimental studies of their activities are required to draw final conclusions, their high up-regulation in T. aurantiacus during growth on l-arabinose and d-xylose makes them the suitable targets for studying C5 sugar catabolism in this fungus.
This study has identified three lignocellulose-derived sugars, d-xylose, l-arabinose and cellobiose, as CAZyme inducers for T. aurantiacus. Gene expression analysis indicates that the C5 sugars induce the production of a broad spectrum of CAZymes, including cellulases and xylanases while cellobiose primarily induces cellulases. The observation of broad CAZyme induction by C5 sugars is similar to what has previously observed for CAZyme induction in A. niger.
All chemicals were purchased from MilliporeSigma (St. Louis, MO, USA) unless otherwise noted.
Strains and culture conditions
Thermoascus aurantiacus ATCC 26904, which was obtained from the American Type Cell Culture Collection and maintained on potato dextrose agar by inoculating fresh plates with a piece of a grown culture. Cultures were incubated for 2 days at 50 °C to enhance growth and were then shifted to 45 °C to prevent desiccation of the plates. All incubators (Complete Culture Control Incubator CCC 2.5d, Boekel Scientific, Feasterville-Trevose, PA, USA) contained several water reservoirs to keep the air moist and prevent plate desiccation. New fungal cultures were periodically inoculated from cryostocks.
Induction experiments
From a 6-day-old agar plate culture, T. aurantiacus ascospores were harvested by pouring 5 ml of autoclaved water on the plate surface and gently scraping the plate with disposable cell spreaders (Heathrow Scientific, Vernon Hills, IL, USA). The spores were transferred to a sterile 15 ml Falcon tube (Cellstar®, Greiner Bio-One, Monroe, NC, USA) and continuously vortexed and were then used as an inoculum (106 spores/ml) for 300 ml glucose medium pre-cultures [McClendon's salts (4), 2% glucose (w/v) and 0.8% soy meal peptone]. The pre-cultures were incubated for 48 h at 50 °C in a rotary shaker (Multitron, American Laboratory Trading, San Francisco, CA, USA). After 48 h, all pre-cultures were combined and filtered in vacuo under sterile conditions over a glass fiber filter-based system until the mycelia were dry. The mycelia (1.5 g) were weighed into baffled 250-ml Erlenmeyer culture flasks containing 50 ml of culture medium that did not have a carbon substrate. The carbon sources (d-xylose, cellobiose, l-arabinose) were autoclaved separately and added to 1% w/v for the batch cultures. The batch cultures were sealed with foam stoppers and incubated for 72 h at 50 °C at 180 rpm. For the fed-batch induction experiments, sterile solutions of the sugar were added by a peristaltic pump [BT100-1L Multi-Channel Peristaltic Pump (Langer Instruments Corp., Boonton, NJ, USA)] according to a previously established protocol [11].
Thermoascus minimal medium development
Thermoascus aurantiacus cultures for minimal medium development were performed by comparing growth with 50 ml of modified McClendon's medium (McClendon's salts, 2% glucose and 0.8% soymeal peptone) to a medium where the peptone has been replaced by 25 mM (NH4)2SO4 or 25 mM (NH4)2SO4 and 25 mM trisodium citrate. The cultures were inoculated with 106 spores in 250-ml baffled Erlenmeyer flasks, and incubated on a rotary shaker at 50 °C and 180 rpm. The peptone cultures were incubated for 48 h and the (NH4)2SO4 cultures were incubated for 72 h. The pH determination was performed with a calibrated pH meter (SYMPHONY SB70P, VWR, Radnor, PA, USA) and the dry weight of the mycelia was measured by filtering the content of an entire culture over a previously weighed filter paper with a vacuum system and washing it several times with ddH2O. The mycelia were dried for 48 h at 65 °C and weighed. Induction of T. aurantiacus cellulases and xylanases was performed by fed-batch induction with the peristaltic pump as described above with the culture medium containing 25 mM (NH4)2SO4 and 30 mM trisodium citrate instead of 0.8% soy meal peptone.
Cultivations for gene expression analysis
For gene expression analysis, T. aurantiacus pre-cultures were grown as described above with the minimal medium containing 25 mM (NH4)2SO4 and 30 mM trisodium citrate and the mycelia from these pre-cultures isolated and inoculated into fed-batch shift experiments as described above, The peristaltic pump was calibrated to release 50 mg/l/h of either d-xylose, l-arabinose or cellobiose during 16 h to four-shake flask replicates for each sugar. In addition to these cultures, four cultures were set up as a no carbohydrate control and four cultures containing 2% (w/v) d-glucose served as a control treatment for carbon catabolite repressing conditions. After 16 h, the feed was stopped and 10 ml of each culture was rapidly filtered over a filter paper, peeled off and snap-frozen with liquid nitrogen. These frozen mycelia were ground with a mortar and a pestle and stored at − 80 °C. The RNA extraction was performed with Maxwell RSC system (Promega, Madison, WI, USA) and the Plant RNA kit (Promega, Madison, WI, USA) according to the fungal RNA-extraction protocol. Briefly, aliquots of extracted mycelium were transferred into designated tubes and 400 μl of homogenization solution and 200 μl of Lysis buffer were added and vortexed and the extraction was initiated. The purified RNA was eluted in nuclease-free water. RNA quality was assessed with a NanoDrop spectrometer and the quality with the RNA 6000 Nano kit (Agilent, Waldbronn, Baden-Württemberg, Germany). RNA concentration was determined with the Qubit RNA HS Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) and sent to the UC Davis DNA Technologies Core for sequencing. With these RNA samples, the library preparation, sequencing and the preliminary differential gene expression calculations were performed by UC Davis DNA Technologies Core through the 3′Tag-Seq method (https://dnatech.genomecenter.ucdavis.edu/tag-seq-gene-expression-profiling/) (https://doi.org/10.1186/s12864-018-5393-3). The libraries were sequenced using a HiSeq 4000 instrument (Illumina).
Gene expression analysis
The Tag-Seq reads were aligned to the T. aurantiacus genome (https://genome.jgi.doe.gov/portal/pages/dynamicOrganismDownload.jsf?organism=Theau1) with STAR v184.108.40.206b [63] to generate a table containing the counts per gene. Furthermore, 500 bases were added to the coordinates of the 3′ end of each transcript (or until the start of the next gene) to improve annotation (from only 21% of the reads aligned, to 77%). Genes with mean expression levels lower than one were filtered which resulted in a table of 8591 genes (instead of the original 8798 genes). This minimal filtering step ensured that genes with low expression such as transcriptional regulators remained in the data set. The differential expression analysis was performed with the DESeq2 package in R [64]. Heat maps were created using [pheatmap(sampleDistMatrix)] with the pheatmap package and the Morpheus online tool (https://software.broadinstitute.org/morpheus/). For generating the Venn diagram, the genes were grouped in several Venn bins using the software supplied at: http://bioinformatics.psb.ugent.be/webtools/Venn/. For the heatmaps, the list of annotated CAZyme genes in T. aurantiacus was matched with the corresponding filtered expression values for each condition (see Additional file 2). To obtain the Z-score for each condition, the mean calculated for each condition was subtracted from each value and then divided by the standard deviation. Hierarchical clustering with the Spearman's rank correlation as a metric was used to cluster the rows (genes) and columns (conditions).
Protein analysis
Culture filtrates for protein analysis were recovered by transfer of the culture broth to a spin column, which was centrifuged at 8000 rpm for 3 min. The filtrate from the column was then used for protein assays.
The protein profile was investigated with SDS-PAGE gels (8–16% Tris–glycine mini gel; Invitrogen, Carlsbad, CA, USA). Accordingly, 15 μl of the filtered culture broth was mixed in a 5:1 ratio with loading dye [Laemmli buffer/2-mercaptoethanol (4:1)] and boiled for 5 min at 95 °C. The mixture was loaded together with 5 μl of the Novex sharp prestained protein standard molecular weight markers (Thermo Fisher Scientific, Waltham, MA USA) and separated for 65 min at 140 V. The gel was then stained with SimplyBlue safe stain (Thermo Fisher Scientific, Waltham, MA USA).
Protein concentrations were measured with the Bradford Protein Assay (Bio-Rad, Hercules, CA, USA). The assay reagent was diluted 1:5 and 200 ml were added to a 96-well plate together with 5 μl of culture filtrate. Bovine gamma globulin (Thermo Fisher Scientific, Waltham, MA USA) was added as a standard (0–2 g/l) [11]. The absorbance measurement was performed at 595 nm.
CMCase and xylanase activity was measured with the DNS method [65] with 1% (w/v) sodium carboxymethylcellulose (CMC) or beechwood xylan as substrate in 50 mM sodium acetate buffer pH 5. For the assay, culture filtrates were diluted either 1:20 or to 50 μg protein/ml and 10 μl were added in triplicates to a 96-well PCR plate together with 70 μl of the polysaccharide substrates. Monomeric sugar standards (d-glucose for CMCase and d-xylose for xylanase) ranging from 0 to 12.5 uM was added in duplicates to each plate. All assays were performed in PCR cyclers for 30 min at 65 °C. The reactions were stopped via adding 80 μl of the DNS reagent and boiling for 5 min at 95 °C. The amount of released sugars was measured at λ = 540 nm. One unit of enzyme activity (U/ml) was defined as amount of released sugar (nmol) per time (min) per volume of culture supernatant (ml). Assays for p-nitrophenyl β-d-cellobioside (pNPC) and p-nitrophenyl β-d-glucopyranoside (pNPG) were performed as previously described [66].
Glucose release from Avicel (2%) was measured by diluting filtrates (40 μl) from d-xylose (0.90 mg/ml), l-arabinose (0.61 mg/ml) and cellobiose (0.56 mg/ml) cultures into 200 μl of 50 mM NaOAc buffer pH 5.0 containing 0.6 mM ascorbate and incubating the reactions at 50 °C and 180 rpm for 24 h. After 24 h, glucose was measured using a YSI 2500 (YSI Incorporated, Yellow Springs, Ohio, USA).
Availability of data and materials
The RNA-seq datasets generated and/or analyzed during the current study are available in the NCBI Sequence Read Archive (SRA) in Bioproject PRJNA681608 under Biosamples SAMN19237569–SAMN19237583.
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This work was supported by the German Academic Exchange Service (DAAD) through a granted stipend to R.G. (Jahresstipendien für Doktorandinnen und Doktoranden, Studienjahr 2018/19 under the Program Number 57380837). Additional funding for student internships was provided by the Austrian Marshall Plan Foundation (L.F., R.G.). Portions of this work were funded by the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, Bioenergy Technologies Office. Portions of this work were performed at the DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE‑AC02‑05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.
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RG, SWS, AF and TS designed experiments; RG, RM, LF, CH and SH performed research; RG and SWS wrote the manuscript. All authors read and approved the final manuscript.
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Additional file 1: Figure S1.
Overview of the putative D-xylose and L-arabinose assimilation pathway in T. aurantiacus. The table contains a heat map of the putative pathway genes shown below. The predicted orthologues of A. niger are shown on the right hand side. Enzymes and intermediates of the pathway are shown below, where high expression is indicated during L-arabinose (Ara) or D-xylose (Xyl) feed or NA if the predicted gene could not be identified for T. aurantiacus. Red indicates high and blue low gene expression. Figure S2. Comparison of genes related to the unfolded protein response in T. aurantiacus under different growth conditions. Bars (mean) and error bars (standard deviation) were calculated from 3 biological replicates, asterisks indicate statistical significance compared to the no carbohydrate condition (pval < 0.05). Figure S3. Regulation trends of T. aurantaicus genes that are putative orthologues of transcriptional CAZy regulators in A. niger. Numbers represent T. aurantaicus protein IDs from the JGI MycoCosm database (43) and gene names refer to the A. niger genes showing the highest similarity based on BLAST searches using gene sequences. Conditions are indicated as follows: Xyl, Cel and Ara = D-xylose, cellobiose and L-arabinose feed, Glu = high D-glucose medium and NC = no carbohydrate medium. Each bar represents the average of 3 biological replicates and error bars are standard deviations of those replicates. A statistical significant difference to the glucose codnitons is marked with asterisks below each bar (pval < 0.05).
Additional file 2.
Tables for differential expression by T. aurantiacus induced with C5 and C6 sugars. Data is shown for genes whose differential expression has an adjusted pval < 1.
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Gabriel, R., Mueller, R., Floerl, L. et al. CAZymes from the thermophilic fungus Thermoascus aurantiacus are induced by C5 and C6 sugars. Biotechnol Biofuels 14, 169 (2021). https://doi.org/10.1186/s13068-021-02018-5
DOI: https://doi.org/10.1186/s13068-021-02018-5


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Running over five nights between Aug. 19 and 25, "Masc: Trans Men, Butch Dykes, and Gender-nonconforming Heroes in Cinema" brings stories of queer, masculine identity from the Criterion Channel to the big screen with films such as the documentary "Chavela" (2017), exploring the celebrated Costa Rican–Mexican singer Chavela Vargas, whose approach to music and life resonated with queer audiences, and "Pariah" (2011), filmmaker Dee Rees' debut feature about a young, queer Black woman.
As part of the ongoing Archive Television Treasures series, remarkable cult-classic TV gets a showing with "Ernie Kovacs: Television of the Absurd" (July 22), a tribute to the legendary comedian, actor and writer, and "Honey West: TV Private Eye '65" (Aug. 26), the trailblazing series starring Golden Globe Award–winning actress Anne Francis as a detective and inspired by Britain's spy show "The Avengers."
The series "Telegrams from the Edge: The Message Picture in the Age of Noir" will run from July 14 to Aug. 6, presenting a sample of Hollywood message films from the 1950s and '60s in the film noir and pulp genres.
The first night of "Flaming Creatures: Jack Smith, Barbara Rubin and the Cinematic Orgiastic" (July 15–16) marks the 60th anniversary of the double-bill theatrical premiere of Smith's "Flaming Creatures" (1962), in which images and bodies slide over one another in a tumult of freedom and feeling, and Ken Jacobs' "Blonde Cobra" (1963) at the Bleecker Street Cinema in New York. The second evening of the program features three works inspired by "Flaming Creatures": Ron Rice's Chumlum (1963), Jud Yalkut's "Kusama's Self-Obliteration" (1967) and Barbara Rubin's "Christmas on Earth" (1963).
To view the full summer 2023, including additional programs and details, visit the UCLA Film & Television Archive events page.
In-person screenings take place at the Billy Wilder Theater at the Hammer Museum, 10899 Wilshire Blvd. (map). All programs are free through June 2024, thanks to a gift from an anonymous donor.

subset: commoncrawl


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The Meaning Of Steve Lacy's "Bad Habit" Lyrics
The Grammy-nominated song is an anthem for those too scared to make the first move.
One of 2022's biggest hits is also one of the oddest in recent memory. After years of making genre-defying, web-savvy soul music as a member of The Internet, Steve Lacy broke out as a solo artist with "Bad Habit," which landed him major Grammy nominations for Record of the Year and Song of the Year. The lead single from his solo sophomore album Gemini Rights is an adventurous R&B anthem containing so many quotable lines wrapped in numerous hypnotic melodies that TikTok users immediately latched onto the song, helping it reach No. 1 on the Billboard Hot 100.
While "Bad Habit" sounds smooth and self-assured sonically, the lyrics are almost the ultimate embodiment of the expression "would've, could've, should've" (only Taylor Swift has him beat, given her 2022 track with that same title). Lacy expresses his feelings of regret right at the outset. "Made a move, could've made a move," he sings, before asking, "Is it too late to pursue?" In the chorus, Lacy mourns that he was romantically interested in someone but couldn't build up the guts to make a move, explaining that it's always been a pattern that he can't break. "I bite my tongue, it's a bad habit," he sings. "Kinda mad that I didn't take a stab at it / Thought you were too good for me, my dear / Never gave me time of day, my dear." The catchy post-chorus makes his intentions loud and clear, pleading, "I wish I knew, I wish I knew you wanted me."
Lacy perhaps gets the most pensive and reflective as the music completely drops out and the object of his eye returns and suddenly becomes available, leaving nothing but his own disbelief. "You can't surprise a Gemini," he sings. "I'm everywhere, I'm cross-eyed, and now that you're back, I can't decide." Maybe there's room for a happy ending after all.
During a recent interview on CBS Sunday Morning, Lacy played a snippet of an early version of "Bad Habit," which sounded much more stripped back and acoustic-based than the final production, putting the lyrics of conflict and regret in the foreground. As he told Billboard in July 2022, "Bad Habit" evolved over the course of a year, and the drums and chorus were very different at first. Even his friend Tyler, the Creator got involved, encouraging him to change the post-chorus. "At first it was, 'I wish I knew you / I wish I knew you wanted me.' And then he was like, 'It'd actually be more memorable if you cut that word ['you'] out,' so shout out to Tyler," he said.
Although Lacy may not have the most public experience with being too hesitant to pursue a crush, he doesn't have much advice for those in the same boat, as he told Grammy.com. "When it comes to shooting your shot, that's a good question, but I'd say just do whatever feels good to you," he said. Read the full lyrics of "Bad Habit" below if you need some inspiration.
I wish I knew, I wish I knew you wanted me
I wish I knew, I wish I knew you wanted me
What you, ooh, uh, what you do?
Made a move, coulda made a move
If I knew I'd be with you
Is it too late to pursue?
I bite my tongue, it's a bad habit
Kinda mad that I didn't take a stab at it
Thought you were too good for me, my dear
Never gave me time of day, my dear
It's okay, things happen for reasons that I think are sure, yeah
I wish I knew, I wish I knew you wanted me
I wish I knew (oh), I wish I knew you wanted me
I wish I knew (yeah), I wish I knew you wanted me (oh)
I wish I knew, I wish I knew you wanted me
Say to me (please just say to me)
If you still want it
I wish you wouldn't play with me, I wanna know (oh no)
Uh, can I bite your tongue like my bad habit?
Would you mind if I tried to make a pass at it?
Were you not too good for me, my dear?
Funny you come back to me, my dear
It's okay, things happen for reasons that I can't ignore, yeah
I wish I knew, I wish I knew you wanted me
I wish I knew (wish I knew), I wish I knew you wanted me (oh)
You can't surprise a Gemini, I'm everywhere, I'm cross-eyed, and
Now that you're back, I can't decide, if I decide if you're invited
You always knew the way to wow me
Fuck around, get tongue-tied, and I turn it on, I make it rowdy
Then carry on, but I'm not hidin'
You grabbin' me hard 'cause you know what you found is biscuits, is gravy, babe, ah-ah
You can't surprise a Gemini, but you know it's biscuits, is gravy, babe
I knew you'd come back around 'cause you know it's biscuits, it's gravy, babe
Let's f*ck in the back of the mall, lose control, go stupid, go crazy, babe
I know I'll be in your heart 'til the end
You'll miss me, don't beg me, babe


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The hunt is on.
We've got a pair of new scavenger hunts ready for you to take a shot at this week.
That includes our Apollo DAO Sunchaser Hunt…
…and our Lido Raindrop Hunt.
Check them out below along with the rest of this week's bounties. As always, feel free to reach out with any questions or comments on Discord.
👻 Aave
[Easy] DeFi Saver Flash Loan Dominance 💪
Bounty: up to .67 AAVE
What percent of flash loan traffic is driven by Defi Saver? Hint: These will be ds_proxy contracts
[Hard] Aave Staking Reward Flows 👛
Bounty: up to 1.35 AAVE
Analyze how Aave rewards (staking) are spent on mainnet -- are they reinvested into positions? Used to pay down loans? Simply liquidated/swapped for ETH? Link the Aave Rewards question from last round
[Hard] Preferred C-Ratio 📊
Bounty: up to 1.35 AAVE
What is the collateralization-ratio (c-ratio) that people like to keep their loans at? What is the distribution of this perferred value across addresses that volume that different borrowing addresses work with (small accounts vs. whales)? How does this ratio change with the price action of the collateral?
🌎 Terra
[Elite] Historic bAssets Price Movements 📈
Bounty: up to 18 LUNA
What is the most bLuna has dipped in a X second window (based on bLuna price oracle timing)? Focus on the events of the Black swan event in mid-May. Make the default size of the window 30 seconds. - How much has bLuna drifted from its Luna peg? - How much has bEth dipped in a 30-second window - How much has bEth drifted from the Ethereum Price
[Hard] Staking APY 🤑
Bounty: up to 9 LUNA
Select a time period (minimum 30 days) and break down staking returns into their component parts. How much of overall staking yield comes from airdrops historically? How do you expect this to change between now and year's end?
Example template: station.terra.money
[Hard] Bridges, Side by Side 🌉
Bounty: up to 9 LUNA
For each address that has used the Ethereum <> Terra bridge, identify the pairs of addresses, and compare activity by week on Ethereum vs Terra. To contend for the grand prize: provide a Luna vs ETH distribution-of-wealth breakdown by address.
[Hard] Airdrop Summary 🪂
Bounty: up to 9 LUNA
For the last 90 days, summarize the following about airdrops being claimed by addresses in the Terra ecosystem: 1) how many airdrops total were claimed? 2) how much money overall was claimed? 3) how often were airdrops claimed?
[Hard] Governance Participation 🗳
Bounty: up to 9 LUNA
Summarize participation in governance activities by address. What is the distribution of level of governance activity among Terra addresses? How is this distribution related to how large (in holdings) the addresses are?
[Hard] Protocol Stats 🔢
Bounty: up to 9 LUNA
Over the last 90 days, characterize the number, type, and frequency of Projects/Protocols used by addresses in the Terra ecosystem (bonus points for tangential/related stats!)
[Easy] Currencies
Bounty: up to 5.45 LUNA
Over the last 90 days, for each active address on Terra, characterize the # Currencies Transacted with, as well as frequency and volume of those transactions [address x currency x stat]
[Easy] Ideal Airdrop Recipients: Part One 🪂
Bounty: up to 5.45 LUNA
As you have seen, many of the recent bounties are related to airdrops on Terra. This is because a number of Terra protocols are interested in the question: "who is the ideal recipient for airdrops? To whom do I want to airdrop my tokens, so that they become loyal and active members of my project?" We've provided some metrics that may help identify "valuable addresses", i.e. Terrans who are active, loyal members of the Terra ecosystem. This question is asking you to share the metrics you think are important. Share at least two metrics (more is fine) that you think are important to identify ideal airdrop recipients, and provide a brief description of how you would find these metrics with on-chain data.
Note: Since every Terra project is different, feel free to talk generally, or to focus on a particular upcoming Terra project and talk about what metrics that project is likely to care about.
[Easy] Orion Money 💸
Bounty: up to 1.5 LUNA
Make a complete tutorial on Orion Money.
Learn more here: https://learnterra.io/bounties/
💧 Lido
Daily bETH Volume 🔥
Bounty: up to 80.99 LDO
What is the daily volume of bETH bridged from Ethereum to Terra using Lido? Has there been an increase leading up to Columbus-5? What is the daily volume of bETH bridged from Terra back to Ethereum? (We have data from bridging through Lido, but not from eth to terra via the https://bridge.terra.money/)
stETH Usage 🥩
Bounty: up to 80.99 LDO
What are users doing with stETH from Lido? (Possible use cases to explore: https://blog.lido.fi/steth-defi-usecases/ )
Daily stETH Metrics 📅
Bounty: up to 80.99 LDO
Lido's staked ETH (stETH) metrics questions: Daily volume of stETH? Average stake size? Number of stakers over time? Average time staked? Percentage of stETH converted to bETH and bridged to Terra?
Free Square Question 🤑
Bounty: up to 215.98 LDO
Provide any interesting insights on Lido. These will be judged by a council that includes other community members and the Flipside team!
🎼 Harmony
CentOS/RHEL/AL2: Support Installation For Harmony 🎵
Bounty: Up to 3426.29 ONE
Description: Package and distribute the Harmony binaries for Linux's CentOS/RHEL/AL2 distribution. For example, CentOS's yum install harmony-one
should initiate installation of Harmony binaries alongside all the dependencies. Also allow the installation to be pinned to a certain version.
Ubuntu/Debian: Support Installation For Harmony 🎶
Bounty: Up to 3426.29 ONE
Description: Package and distribute the Harmony binaries for Linux's Ubuntu/Debian distribution. For example, Ubuntu/Debian's apt install harmony-one
should initiate installation of Harmony binaries alongside all the dependencies. Also allow the installation to be pinned to a certain version.
🏆Bounty Submission of the Week 🏆
Welcome back to our Bounty Submission of the Week!
In this week's edition, we're giving our Aave bounties some more love.
This week's winner is a look at Aave rewards from @CryptWizards. This time, our analyst sought to find out who's getting the most rewards from Aave.
The answer?
According to @CryptWizards, it's a whole lot of users. As we can see in the chart of the top 20 addresses receiving rewards above, there's no one whale dominating rewards on Aave — and that's by design.
"We can easily see that what Aave protocol wants to do is not let one party with large amount dominated the whole eco system," @CryptWizards notes, "but using the rewards/incentive program try best to distributed the rewards evenly to different participants."
See the whole submission from @CryptWizards right here.
Good luck with your bounties this week🤞🍀 Make sure you join our community on Discord to keep up with all things bounties and get answers to any questions you have about solving our queries:
Sharing is caring! Make sure you subscribe and share the Bounty Brief with a friend that wants to get involved in the space:


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Game Details
Release name: My Train Arrives
Size: 680 Mb
Genre: SimulationStrategyIndieTrainsSingleplayer
Developer: Kochkin Dmitry
Publisher: Kochkin Dmitry
Release Date: 10 Sep, 2019
ALL REVIEWS: Positive (27)
♂ Support the software developers and buy this game
About This Game
My Train Arrives is a railway construction simulation game. Build railroads, open new routes for trains. Enjoy the pleasant atmosphere of the game.
System Requirements
- Requires a 64-bit processor and operating system
- OS: Windows 7/8/10
- Processor: Intel Core 2 Duo E4500 @ 2.2GHz or AMD Athlon 64 X2 5600+ @ 2.8GHz
- Memory: 2 GB RAM
- Graphics: ATi Radeon HD 2400 or NVIDIA GeForce 7600
- Storage: 1 GB available space
How To Install The Game
1) Download the game using a Torrent program or Direct program
2) After downloading the game, go to the downloaded game folder
3) Extract the files with WinRar or 7-Zip
4) Now start the installation with " Setup.exe ", choose the directory to install
5) Wait until the installation is complete
6) Now just start the game as an administrator with the shortcut that was created on the desktop
7) Play!
General Notes:
– Block the game's exe in your firewall to prevent the game from
trying to go online ..
– If you install games to your systemdrive, it may be necessary
to run this game with admin privileges instead


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Institution: Inter-American Development Bank
Country: Belize
Project: Program for Digital Innovation to Boost Economic Development in Belize.
Sector: Finance and Public Service
Abstract: The project's implementation includes the procurement of several contracts that falls in the following categories: goods, consulting (individuals and firms) and non-consulting services for digital transformation services, digital transformation of government services and demand for digital technology and innovation.
Loan/Credit/Grant No.: 5647/0C-BL
Borrower: Government of Belize
The Government of Belize has received financing in the amount equivalent to ten million US Dollars (US $10M) from the Inter-American Development Bank (IDB), and it intends to apply part of the proceeds to payments for goods, consulting (individuals and firms) and non-consulting services to be procured under the project Program for Digital Innovation to Boost Economic Development in Belize. Bidding will be governed by the Inter-American Development Bank's eligibility rules and procedures.
The main objective of this project is to promote digitalization of firms and government services in order to promote growth and increase efficiency in Belize's economy. The specific objectives are to: (i) promote firms' growth through increased adoption of digital solutions; (ii) promote firms' efficiency through digitization of licensing processes; and (iii) reduce costs for citizen through the digitization of civil registration services. The project contains two (2) components.
Component 1 Digital transformation for firm growth and efficiency- stimulate private sector demand for digital technology and digital innovation, enhance local digital capacities; and digitalize government services for firms.
Component 2 Digital transformation of government services for citizens -support the optimization and digitalization of government processes related to citizens registration services.


text:
Grow-It-LED Excalibur King Series 740W Full Spectrum LED Grow Light
The Excalibur King Series 740 Watt Veg LED Grow Light is a commercial grade LED grow light designed to replace DE HPS fixtures up to 1000 watts. Grow-it-LED has designed a system that consistently outperforms the competition at a competitive price. Excalibur King series LED grow lights offer multiple spectrum options, allowing you to have excellent flexibility for your indoor garden or grow operation.


text:
État de publication: Publiée (2019 Mai )
Titre du livre: Narrative, literacy and other skills : Studies in intervention
Éditeur: John Benjamins Publishing
Lieu: Amsterdam, Pays-Bas
Intervalle de pages: 63-88
Résumé: Reading comprehension refers to the ability to construct a global representation of the macrostructure of a text and to establish links between its different components. To achieve this, however, the reader must also manage the microstructure of the text, in particular, processing the written code in such a way as to identify the written words, while making inferences to construct their meaning. Based on this perspective, this chapter presents data coming from two pedagogical contexts whose aim was to promote emergent literacy in kindergarten. Results show that children made significant progress in their processing of the written code, measured through their level of representation of the alphabetic system, an emergent writing task and a reading test. Progress in narrative structuring remained however more modest.
Mots clé(s):
Théorie de l'activité:


text:
4 foolproof ways to protect your home (and your bank account)
4 foolproof ways to protect your home (and your bank account)
4 foolproof ways to protect your home (and your bank account)
You might want to take another look at your home security. You live in a nice neighborhood, you have a great alarm system, and your door is always locked. So what else could there be? a lot of!
Below you can find some tips on how to prevent unexpected situations:
Photo by Nathy dog on Unsplash
1) Install a good security system.
This is your first line of defense against intrusions. Make sure to choose a system with features like motion sensors and video surveillance.
One of the best ways to protect your home is to install quality door locks. Cheap locks are easy to break, so it's important to invest in something that can withstand a break-in. If you have any windows that are accessible from the outside, be sure to install window rails or locks to keep burglars out.
When it comes to home security, one of the best things you can do is build a fence. A good fence will keep burglars and intruders out. Make sure to choose a tall, sturdy fence and be sure to keep it well maintained.
Plants are a natural deterrent to burglars because they don't want to be seen near well-protected property. Choose tall, dense plants, such as bamboo or ivy, to form a natural barrier around your home.
2) Get home warranty.
In the event of a disaster, a home warranty will help you pay for repairs.
A home warranty is an insurance policy that covers the repair or replacement of major appliances and systems in your home. They're a great way to protect yourself from unexpected disasters like a broken refrigerator or a burst water main. Most home warranties work by helping pay for repairs to major appliances, plumbing, and electrical systems. They usually also cover the cost of replacing the roof, floors and siding.
3) Provide a safe place for your valuables.
If you have jewelry, rare coins, or other valuables, it's important to have a safe place to store them. A fire safe is the best way to protect your belongings in the event of a fire.
Your best bet is to install the safe in a closet or somewhere that is not easily visible from the outside. Be sure to keep your safe password secret and store your keys in a safe place.
4) Have a plan.
If disaster does happen, you'll be glad you have a plan.
In the event of a fire, make sure everyone knows where the safe place is and how to get there. If you are not home when the disaster strikes, make sure someone is there to let the firefighters in.
If you are away, make sure you have a friend or relative who can help in an emergency.
Photo by Gerd Altmann Photo from Pixabay
Disasters can strike at any time, so it's important to be prepared. By protecting your home from the inside out, you can help protect yourself and your belongings from accidental damage. Has a good security system, quality locks, safe storage for valuables, and a home warranty to help you in the event of a disaster. Most importantly, have a plan!
Keep it up Artkraft
Find more articles in our categories DIY Home & DIY Projects et Tips & Crafts .
Thanks for visiting we hope our article 4 foolproof ways to protect your home (and your bank account)
, don't forget to share the article on Facebook, instagram and whatsapp with the hashtags ☑ #foolproof #ways #protect #home #bank #account ☑!


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A long time ago in the days of the Amiga, a game called Hero Quest was released, which itself was a conversion of the great table top game. It was one of the best Isometric Amiga games of it's time, although it did have a number of system ports, even the Spectrum! Fast forward to now and we have DarkQuest, from Brain Seal Entertainment which is inspired by Hero Quest. You can even hear the slight variation music sound track in DarkQuest from the original title. This is a turn based fantasy rpg where you assume the role of a muscle bound barbarian on his quest to destroy the evil sorcerer. The graphics are of high quality and i'm sure many Hero gamers will get that nostalgia vibe.
An evil sorcerer has appeared in the lands, he has built a dungeon in the nearby village of faladir, where his minions use it as a base to raid the surrounding villages to bring terror and death to all its citizens. A hero by the name Zantor dares to defy the powers of the sorcerer and seeks to destroy the dungeon and put an end to this.
You'll be pleased to know that the game is currently available for Android and iOS, with a PC version available through the Humble Store.
A Steam Greenlight campaign is still ongoing which needs your votes to appear on Steam


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Yacht Overview
Make: Boston Whaler
Model: 280 Outrage
Year: 2014
Length: 28′
Located In: Coconut Grove, FL
Engine/Fuel Type: unleaded
Hull Material: Fiberglass
EZ Navigation
MOTIVATED SELLER! BRING OFFERS This is a very clean 28′ center console. She's a one-owner boat with just 550 hours on the twin Mercury 250 Verados and she's loaded with the following features and equipment:
- Dual-screen Raymarine Chart Plotters/Displays
- Raymarine Radar
- Raymarine Autopilot
- RayMarine VHF Radio
- Fusion Stereo
- Outriggers
- Plenty of Rod Holders and Rod Storage
- Integrated Bait Well, Bait Prep, & Tackle Storage
- Windlass Anchor
- Trim Tabs
This boat is turn-key and ready for immediate delivery.
Call for details and schedule your private viewing at Grove Harbour Marina.
Full Specs
Beam: 9.33 ft
Bridge Clearance: 8.75 ft
Dry Weight: 6,100 lb
Deadrise: 23 °
Engine Brand: Mercury
Engine Model: L6 DTS Verado
Engine/Fuel Type: unleaded
Engine Power: 250|horsepower
Engine Type: Outboard 4 Stroke
Engine Brand: Mercury
Engine Model: L6 DTS Verado
Engine/Fuel Type: unleaded
Engine Power: 250|horsepower
Engine Type: Outboard 4 Stroke
Fresh Water Tanks: 1 (28 Gallon)
Fuel Tanks: 1 (186 Gallon)
Holding Tanks: 1 (6.5 Gallon)
Number of heads: 1
Manufacturer Provided DescriptionThe 280 Outrage is a superior fishing, entertaining and cruising vessel. Other boat manufacturers can't come close to matching the premium craftsmanship of the 280 Outrage, starting with the boat's state-of-the-art center console. It features a tempered-glass windshield with power-actuated vent, cutting-edge Mercury SmartCraft instrumentation and Raymarine displays, and a heavy-duty integrated hard-top system with floodlights and life jacket storage. An enclosed head lends comfort, and the boat's wide beam and plush seating enhance group pleasure cruises. The 280 Outrage is a bona fide sport fishing machine, too, with serious angling features such as the deluxe leaning post with bait prep area, a 40-gallon livewell, and three massive fishboxes. For those looking for a dedicated tender for their luxury yacht, the 280 Outrage answers the call. Better than a RIB, more than a dinghy, this boat serves as a tender and a memory-making platform for fishing runs and family cruises.
Deck / Cockpit
- 28" interior freeboard
- 8" stainless steel cleats - bow (2), spring line with hawse pipes (2), stern with hawse pipes (2), anchor locker (1)
- Acrylic transom door with stainless steel latch
- Aft coaming bolsters
- Aft tackle storage center (port side)
- Bow and stern lifting eyes
- Cockpit and bow courtesy lights
- Drink holders - bow (4), hawse pipes (4), leaning post (2), console (2)
- Gunnel mounted stainless steel rod holders (6)
- Heavy-duty rub rail
- Integral swim platform with covered telescoping swim ladder and stainless steel grab rail
- Motorwell drains
- Self-bailing cockpit sole
- Stainless steel thru hull anchor roller davit
- Stainless steel toe rails
- Transom mounted stainless steel rod holders (3)
- Under gunnel rod racks
- Welded stainless steel low profile grab rails
Console / Head
- 12-V receptacle
- Access panels to electronics
- Acrylic entry door with lock
- Bi-level molded console footrests
- Cast stainless steel steering wheel with knob
- Compass
- Drink holders (2)
- Dry storage areas (2)
- Easy access storage space for personal items
- Electronics surface that accommodates two 12" displays
- Enclosed head / storage area in console with easy clean liner
- Fold-down step inside console
- Instrumentation and electrical switch panel with circuit breaker protection and illuminated text
- Interior courtesy light
- Magazine rack storage
- Portable head with pump-out
- Remote ignition key switches with pushbutton start at helm
- Stainless steel port light with screen
- Urethane console visor
- Custom fiberglass cooler with grab handles (cooler slides forward to create sun pad filler)
- Fold-away stern seat
- Forward custom console cooler seat with backrest
- T-Top
- Bow area floodlight
- Cockpit floodlights (2)
- Dome light (red and white)
- Life jacket storage
- Rod holders (5)
Deluxe Leaning Post With Livewell
- Bait prep area
- Drink holders (2)
- Fold-down footrest
- Netted storage area on starboard side of leaning post
- Pressurized aerated livewell with clear top window and blue interior (40 gal)
- Rod holders (2)
- Sink with freshwater and pullout sprayer
- Stainless steel drink holders (2)
- Tackle storage drawers (3)
- Tip-out trash can
- Upholsterd leaning post with flip-up bolster and backrest
- Welded stainless steel grab rail
- White and red livewell lights
- Aft tackle storage center (port side)
- Bow anchor locker with drain
- Dry storage areas in console (3)
- Forward deck storage / seats (port and starboard) with drains, gaskets and lockable rod storage (3 per side)
- Forward in-floor storage / fishbox with pump-out and two 5 gal buckets
- Leaning post tackle storage
- Lockable console storage
- Netted storage area on side of leaning post
- Port and starboard aft fishboxes with pump-out and drains
- Port and starboard under gunnel net storage
- Under gunnel rod racks (2 per side)
- 186 gal fuel tank
- Automatic bilge pumps (750 GPH forward, 1,100 GPH aft)
- Battery trays (3) with switches
- Electric horn
- Electrical distribution / breaker panel with key switches (inside console)
- Fishbox pump-out
- Freshwater system with 28 gal tank, sink at leaning post and transom shower
- LED navigation lights
- Raw water washdown
- Recessed electric trim tabs with indicators
- Stainless steel propellers
The Company offers the details of this vessel in good faith but cannot guarantee or warrant the accuracy of this information nor warrant the condition of the vessel. A buyer should instruct his agents, or his surveyors, to investigate such details as the buyer desires validated. This vessel is offered subject to prior sale, price change, or withdrawal without notice.
Vessels listed are at various locations in the USA.


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Ho getting Aviation Training Academy soon
Ghana Airports Company Limited (GACL) and Africa World Airlines (AWA) officials, led by Yvonne Nana Afriyie Opare, Managing Director of GACL, recently conducted a thorough inspection of potential locations at Ho Airport with the vision of establishing an Aviation Training Academy.
Togbe Afede XIV, Chairman of AWA, and Agbogbomefia of Asogli, have set their sights on transforming the Ho Airport into a prominent aviation training hub.
The team was in the Volta Regional capital to explore various sites for a hangar and accompanying facilities, engaging in discussions with stakeholders, including representatives from the Ghana Civil Aviation Authority.
This collaborative effort aims to give rise to a comprehensive pilot training academy and a maintenance, repair, and overhaul facility.
The initiative holds the promise of generating employment opportunities and catalyzing economic growth not only in Ho but also in the surrounding regions.
Togbe Afede highlighted the airport's unique suitability for training and maintenance activities, emphasizing its serene environment as a conducive factor for effective aviation education.
The anticipated project is poised to have a positive impact on both tourism and the aviation sector, contributing to the overall development of the region.
Construction is expected to commence within the year, pending necessary approvals, and the collaboration is projected to yield mutual benefits for institutions like Ho Technical University.
This ambitious venture signals a significant step towards enhancing Ghana's aviation capabilities and fostering educational and economic opportunities in the Ho area.
The post Ho getting Aviation Training Academy soon appeared first on The Herald ghana.
The Herald ghana