Ethanol production from hydrolyzed kraft pulp by mono- and co-cultures of yeasts: the challenge of C6 and C5 sugars consumption

Second-generation bioethanol production’s main bottleneck is the need for a costly and technically di cult pretreatment due to the recalcitrance of lignocellulosic biomass (LCB). Chemical pulping can be considered as a LCB pretreatment since it removes lignin and targets hemicelluloses to some extent. Chemical pulps could be used to produce ethanol. The present study aimed to investigate the batch ethanol production from unbleached Kraft pulp of Eucalyptus globulus by separate hydrolysis and fermentation (SHF). Enzymatic hydrolysis of the pulp resulted in a glucose yield of 96.1 3.6% and a xylose yield of 94.0 7.1%. In an Erlenmeyer flask, fermentation of the hydrolysate using Saccharomyces cerevisiae showed better results than Sche ersomyces stipitis. At both the Erlenmeyer flask and bioreactor scale, co-cultures of S. cerevisiae and S. stipitis did not show significant improvements in the fermentation performance. The best result was provided by S. cerevisiae alone in a bioreactor, which fermented the Kraft pulp hydrolysate with an ethanol yield of 0.433 g g1 and a volumetric ethanol productivity of 0.733 g L1 h1, and a maximum ethanol concentration of 19.24 g L1 was attained. Bioethanol production using the SHF of unbleached Kraft pulp of E. globulus provides a high yield and productivity.publishe

Dynamic 3D Cell Rearrangements Guided by a Fibronectin Matrix Underlie Somitogenesis

Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis

Analysis of long-range gene regulation at the HoxD locus

Hoxd genes are essential for the development of the various body axes in vertebrates and hence the underlying regulatory mechanisms are of paramount importance. Among these various mechanisms are long-range acting enhancers, which are located in the two adjacent regulatory landscapes. Analyses of chromatin architecture at this gene cluster has revealed the existence of two topologically associating domains (TADs) flanking the cluster and encompassing these regulatory landscapes. However, the dynamics of such regulatory regions as well as the stability and functional contribution of specific enhancer-promoter interactions during development remains to be established. In this work, we analysed the 3D chromatin organization and transcription profile at the HoxD locus, at different time points during genital tubercle (GT) development, and observe that the 3D conformation of this regulatory region predates the embryonic emergence of the GT. Along with this tissue development, we observe a reduction in transcript levels correlating with a decrease in enhancer-promoter chromatin loops within the adjacent gene desert. This decrease occurs while maintaining a subset of CTCF/Cohesin associated contacts, which are preserved independently from the transcriptional status of the gene cluster. To further explore the functional contribution of this regulatory landscape, we used CRISPR-Cas9 technology to generate mice carrying partial deletions of this region, as well as targeted deletions of both transient (enhancer associated) and constitutive (CTCF/Cohesin associated) contacts. We observe that single deletions of both transient and constitutive contacts displayed little if any effect on Hoxd genes expression in the GT. On the contrary, the single deletion of a previous characterized Hoxd enhancer, the Prox element, or deletions comprising several enhancers, result in the reduction of Hoxd genes expression levels. Overall our results suggest that not all enhancer elements within a complex regulatory landscape have the same functional strength, and highlight the existence of a dynamic yet robust system to tightly regulate Hoxd genes expression

Subunits of the Drosophila actin-capping protein heterodimer regulate each other at multiple levels.

The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth

Expressing <i>HA-cpa</i> or <i>HA-cpa^ΔABD</i> suppresses apoptosis and restores Cpb levels of wing discs knocked-down for <i>cpa</i>.

<p>(A–A′ to F–F′) standard confocal sections of third instar wing imaginal discs with dorsal sides up. (A–A′ to C–C′) <i>sd</i>-Gal4 driving (A–A′) UAS-<i>cpa-IR^C10</i> and two copies of UAS-<i>mCD8-GFP</i> (green in A) or (B–B′) UAS-<i>cpa-IR^C10</i>, UAS-<i>HA-cpa^89E</i> and one copy of UAS-<i>mCD8-GFP</i> (green in B) or (C–C′) UAS-<i>cpa-IR^C10</i>, UAS-<i>HA-cpa^ΔABD</i> and one copy of UAS-<i>mCD8-GFP</i> (green in C). (D–D″ to F–F″) <i>T155</i>-Gal4; UAS-<i>flp</i> induced <i>cpa^107E</i> mutant clones marked by the absence of GFP (green) and expressing (E–E″) UAS-<i>HA-cpa^89E</i> or (F–F″) UAS-<i>HA-cpa^ΔABD</i> in the whole wing disc epithelium. Discs are stained with anti-activated-Caspase 3 (magenta), which monitors DRONC activation and (D–D″ to F–F″) anti-DE-Cad (cyan blue). The scale bars represent 30 µm. (G) quantification of total C3 area per disc area for the three genotypes shown in A–A′ to C–C′. The mean for <i>sd>cpa-IR^C10, 2XGFP</i> is 92.4 (n = 23); for <i>sd>cpa-IR^C10, HA-cpa^89E</i>, <i>1XGFP</i> is 0.7 (n = 10); for <i>sd>cpa-IR^C10, HA-cpa^ΔABD</i>, <i>1XGFP</i> is 51.4 (n = 20). Error bars indicate s.e.m. <i>P<0.0001</i> for comparison of lane 1 and 2. <i>P<0.0005</i> for comparison of lane 1 and 3. (H) quantification of total C3 area per disc area for the three genotypes shown in D–D″ to F–F″. The means for <i>T155>flp; cpa^107E</i> is 9.228 (n = 18); for <i>T155>flp; cpa^107E</i>; UAS-<i>HA-cpa^89E</i> is 0.608 (n = 12); for <i>T155>flp; cpa^107E</i>; UAS-<i>HA-cpa^ΔABD</i> is 4.329 (n = 17). Error bars indicate s.e.m. <i>P<0.0001</i> for comparison of <i>T155>flp; cpa^107E</i> and <i>T155>flp; cpa^107E</i>; UAS-<i>HA-cpa^89E</i> and <i>P<0.0048</i> for comparison of <i>T155>flp; cpa^107E</i> and <i>T155>flp; cpa^107E</i>; UAS-<i>HA-cpa^ΔABD</i>. (I and J) western blots on protein extracts from wing discs expressing two copies of UAS-<i>mCD8-GFP</i> (lane 1) or UAS<i>-cpa-IR^C10</i> and two copies of UAS-<i>mCD8-GFP</i> (lane 2) or UAS-<i>cpa-IR^C10</i> and UAS-<i>HA-cpa^89E</i> and one copy of UAS-<i>mCD8-GFP</i> (lane 3) or UAS<i>-cpa-IR^C10</i> and UAS-<i>HA-cpa^ΔABD</i> and one copy of UAS-<i>mCD8-GFP</i> (lane 4) under <i>sd</i>-Gal4 control, blotted with (I) anti-Cpa (upper panel) and anti-H3 (lower panel) or (J) anti-Cpb (upper panel) and anti-H3 (lower panel). (K) mean intensity of the ratio of Cpb intensity signals between posterior and anterior wing compartments of <i>hh</i>-Gal4 driving two copies of UAS-<i>mCD8-GFP</i> (lane 1) or UAS-<i>cpa-IR^C10</i> and two copies of UAS-<i>mCD8-GFP</i> (lane 2) or UAS-<i>cpa-IR^C10</i> and UAS-<i>HA-cpa^ΔABD</i> and one copy of UAS-<i>mCD8-GFP</i> (lane 3). The mean for lane 1 is 1.064 (n = 20), for lane 2 is 0.822 (n = 17), for lane 3 is 0.883 (n = 24). Error bars indicate s.e.m.. <i>P<0.0001</i> for comparison of lanes 1 and 2 or 3 or <i>P<0.0085</i> for comparison of lanes 2 and 3.</p

Sequential in <i>cis</i> mutagenesis in vivo reveals various functions for CTCF sites at the mouse <i>HoxD</i> cluster

Mammalian Hox gene clusters contain a range of CTCF binding sites. In addition to their importance in organizing a TAD border, which isolates the most posterior genes from the rest of the cluster, the positions and orientations of these sites suggest that CTCF may be instrumental in the selection of various subsets of contiguous genes, which are targets of distinct remote enhancers located in the flanking regulatory landscapes. We examined this possibility by producing an allelic series of cumulative in cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border. In the most impactful alleles, the global chromatin architecture of the locus was modified, yet not drastically, illustrating that CTCF sites located on one side of a strong TAD border are sufficient to organize at least part of this insulation. Spatial colinearity in the expression of these genes along the major body axis was nevertheless maintained, despite abnormal expression boundaries. In contrast, strong effects were scored in the selection of target genes responding to particular enhancers, leading to the misregulation of Hoxd genes in specific structures. Altogether, while most enhancer–promoter interactions can occur in the absence of this series of CTCF sites, the binding of CTCF in the Hox cluster is required to properly transform a rather unprecise process into a highly discriminative mechanism of interactions, which is translated into various patterns of transcription accompanied by the distinctive chromatin topology found at this locus. Our allelic series also allowed us to reveal the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer–promoter interactions, and some doing both, whereas they all together contribute to the formation of a TAD border. This variety of tasks may explain the amazing evolutionary conservation in the distribution of these sites among paralogous Hox clusters or between various vertebrates. </p

Overexpressing full length <i>HA-cpa</i> and <i>cpb</i> prevents wing growth, while ectopic expression of <i>HA-cpa^ΔABD</i> and/or <i>cpb^L262R</i> has the opposite effect.

<p>(A, B, C, D and E) merge between adult wings expressing in green UAS<i>-mCD8GFP</i> under <i>nub</i>-Gal4 control and in magenta (A) UAS-<i>HA-cpa^89E</i> and UAS-<i>cpb⁷</i> or (B) UAS-<i>HA-cpa^ΔABD</i> and UAS-<i>cpb⁷</i> or (C) UAS-<i>cpb^L262R</i> and one copy of UAS-<i>mCD8-GFP</i> or (D) UAS-<i>cpb^L262R</i> and UAS<i>-HA-cpa^89E</i> or (E) UAS-<i>cpb^L262R</i> and UAS-<i>HA-cpa^ΔABD</i> under <i>nub</i>-Gal4 control. (A′, B′, C′ D′ and E′) magnification of hairs on adult wings for the genotypes shown in A, B, C, D and E. (F) quantification of relative wing size normalized to <i>nub>GFP</i> control for <i>nub</i>-Gal4 driving UAS-<i>mCD8-GFP</i> (lane 1) or UAS-<i>HA-cpa^89E</i> and UAS-<i>cpb⁷</i> (lane 2) or UAS-<i>HA-cpa^ΔABD</i> and UAS-<i>cpb⁷</i> (lane 3) or UAS-<i>cpb^L262R</i> and one copy of UAS-<i>mCD8-GFP</i> (lane 4) or UAS-<i>cpb^L262R</i> and UAS-<i>HA-cpa^89E</i> (lane 5) or UAS-<i>cpb^L262R</i> and UAS-<i>HA-cpa^ΔABD</i> (lane 6). The mean for lane 1 is 1(n = 32), for lane 2 is 0.9702 (n = 12), for lane 3 is 1.119 (n = 13), for lane 4 is 1.061 (n = 24), for lane 5 is 1.015 (n = 13), for lane 6 is 1.051 (n = 13). Error bars indicate s.e.m.. <i>P<0.015</i> for comparison of lanes 1 and 2. <i>P<0.0001</i> for comparison of lanes 1 and 3 or 4 or 6 and for comparison of lane 4 and 5.</p

Loss of <i>cpa</i> or <i>cpb</i> reduces both Cpa and Cpb protein levels.

<p>(A) western blot on protein extracts from embryos expressing UAS-<i>mCD8GFP</i> (lane 1) or <i>UAS-HA-cpa^89E</i> (lane 2) under <i>da</i>-Gal4 control or homozygote mutant for the <i>cpa^69E</i> allele (lane 3), blotted with anti-Cpa (upper panel) and anti-H3 (lower panel). (B) western blot on protein extracts from embryos expressing UAS-<i>mCD8GFP</i> (lane 1) or UAS-<i>cpb⁷</i> (lane 2) under <i>da</i>-Gal4 control or homozygote mutant for the <i>cpb^M143</i> allele (lane 3), blotted with anti-Cpb (upper panel) and anti-H3 (lower panel). (C and D) western blots on protein extracts from wing imaginal discs expressing UAS-<i>mCD8GFP</i> (lane 1) or UAS-<i>HA-cpa^89E</i> (lane 2) or UAS-<i>cpa-IR^C10</i> (lane 3) or UAS-<i>cpb⁷</i> (lane 4) or UAS-<i>cpb-IR⁴⁵⁶⁶⁸</i> (lane 5) under <i>sd</i>-Gal4 control, blotted with (C) anti-Cpa (upper panel) and anti-H3 (lower panel) or (D) anti-Cpb (upper panel) and anti-H3 (lower panel). (E–E″ to J–J′) optical cross sections through distal wing disc epithelium of third instar larvae with apical side up in which <i>hh</i>-Gal4 drives (E–E″ and F–F″) UAS-<i>mCD8-GFP</i> (green in E and F) and (G–G′ and H–H′) UAS-<i>cpa-IR^C10</i> or (I–I′ and J–J′) UAS-<i>cpb-IR⁴⁵⁶⁶⁸</i>. Discs are stained with (E–E″, G–G′ and I–I′) anti-Cpa (magenta) or (F–F″, H–H′ and J–J′) anti-Cpb (magenta) and (E–E″ and F–F″) anti-Arm. The arrows in G′, H′, I′ and J′ mark the limits of the posterior compartment boundary. The scale bars represent 15 µm. (K and L) graphs of (K) <i>cpa</i> or (L) <i>cpb</i> mRNA levels measured by five independent qRT-PCR in wing imaginal discs expressing UAS-<i>mCD8GFP</i> (lane 1) or UAS-<i>cpa-IR^C10</i> (lane 2) or UAS-<i>cpb-IR⁴⁵⁶⁶⁸</i> (lane 3) under <i>sd</i>-Gal4 control. (K) the mean for <i>sd>GFP</i> is 1.084; for <i>sd>cpa-IR^C10</i> is 0.4328; for <i>sd>cpb-IR⁴⁵⁶⁶⁸</i> is 1.155. <i>P<0.0027</i> for comparison of lane 1 and 2. (L) the mean for <i>sd>GFP</i> is 0.6210; for <i>sd>cpa-IR^C10</i> is 0.5037; for <i>sd>cpb-IR⁴⁵⁶⁶⁸</i> is 0.2375. <i>P<0.0049</i> for comparison of lane 1 and 3. n.s. indicates a non-significant <i>P</i>. Error bars indicate s.e.m.</p