A quantitative image analysis pipeline for the characterization of filamentous fungal morphologies as a tool to uncover targets for morphology engineering: a case study using aplD in Aspergillus niger

Background Fungal fermentation is used to produce a diverse repertoire of enzymes, chemicals, and drugs for various industries. During submerged cultivation, filamentous fungi form a range of macromorphologies, including dispersed mycelia, clumped aggregates, or pellets, which have critical implications for rheological aspects during fermentation, gas/nutrient transfer, and, thus, product titres. An important component of strain engineering efforts is the ability to quantitatively assess fungal growth phenotypes, which will drive novel leads for morphologically optimized production strains. Results In this study, we developed an automated image analysis pipeline to quantify the morphology of pelleted and dispersed growth (MPD) which rapidly and reproducibly measures dispersed and pelleted macromorphologies from any submerged fungal culture. It (i) enables capture and analysis of several hundred images per user/day, (ii) is designed to quantitatively assess heterogeneous cultures consisting of dispersed and pelleted forms, (iii) gives a quantitative measurement of culture heterogeneity, (iv) automatically generates key Euclidian parameters for individual fungal structures including particle diameter, aspect ratio, area, and solidity, which are also assembled into a previously described dimensionless morphology number MN, (v) has an in-built quality control check which enables end-users to easily confirm the accuracy of the automated calls, and (vi) is easily adaptable to user-specified magnifications and macromorphological definitions. To concomitantly provide proof of principle for the utility of this image analysis pipeline, and provide new leads for morphologically optimized fungal strains, we generated a morphological mutant in the cell factory Aspergillus niger based on CRISPR-Cas technology. First, we interrogated a previously published co-expression networks for A. niger to identify a putative gamma-adaptin encoding gene (aplD) that was predicted to play a role in endosome cargo trafficking. Gene editing was used to generate a conditional aplD expression mutant under control of the titratable Tet-on system. Reduced aplD expression caused a hyperbranched growth phenotype and diverse defects in pellet formation with a putative increase in protein secretion. This possible protein hypersecretion phenotype could be correlated with increased dispersed mycelia, and both decreased pellet diameter and MN. Conclusion The MPD image analysis pipeline is a simple, rapid, and flexible approach to quantify diverse fungal morphologies. As an exemplar, we have demonstrated that the putative endosomal transport gene aplD plays a crucial role in A. niger filamentous growth and pellet formation during submerged culture. This suggests that endocytic components are underexplored targets for engineering fungal cell factories.DFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli

Constructing University Scientific and Technological Innovation Platform Based on the View of Industry-University-Research Cooperation

It is one of the most effective way of accelerating the transformation of scientific and technological achievements and improving the scientific and technological innovation ability (STIA) in university by Industry-university-research cooperation (IURC). It can provide the correct orientation and strong development power for constructing university scientific and technological innovation platform (USTIP). From the perspective of IURC, in order to construct USTIP, we must be close to enterprises. University is the technical support and the source of innovation in enterprise innovation, Technical requirements in enterprises is the driving force of university technological innovation. The cooperation between university and enterprise can integrate their high quality resources and enhance their ability in scientific and technological innovation. At the same time, constructing USTIP can be propitious to the formation of close IURC system

Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in Aspergillus niger submerged culture

Background: Filamentous fungal cell factories are used to produce numerous proteins, enzymes, and organic acids. Protein secretion and filamentous growth are tightly coupled at the hyphal tip. Additionally, both these processes require ATP and amino acid precursors derived from the citric acid cycle. Despite this interconnection of organic acid production and protein secretion/filamentous growth, few studies in fungi have identified genes which may concomitantly impact all three processes. Results: We applied a novel screen of a global co-expression network in the cell factory Aspergillus niger to identify candidate genes which may concomitantly impact macromorphology, and protein/organic acid fermentation. This identified genes predicted to encode the Golgi localized ArfA GTPase activating protein (GAP, AgeB), and ArfA guanine nucleotide exchange factors (GEFs SecG and GeaB) to be co-expressed with citric acid cycle genes. Consequently, we used CRISPR-based genome editing to place the titratable Tet-on expression system upstream of ageB, secG, and geaB in A. niger. Functional analysis revealed that ageB and geaB are essential whereas secG was dispensable for early filamentous growth. Next, gene expression was titrated during submerged cultivations under conditions for either protein or organic acid production. ArfA regulators played varied and culture-dependent roles on pellet formation. Notably, ageB or geaB expression levels had major impacts on protein secretion, whereas secG was dispensable. In contrast, reduced expression of each predicted ArfA regulator resulted in an absence of citric acid in growth media. Finally, titrated expression of either GEFs resulted in an increase in oxaloacetic acid concentrations in supernatants. Conclusion: Our data suggest that the Golgi may play an underappreciated role in modulating organic acid titres during industrial applications, and that this is SecG, GeaB and AgeB dependent in A. niger. These data may lead to novel avenues for strain optimization in filamentous fungi for improved protein and organic acid titres.TU Berlin, Open-Access-Mittel - 201

Green procurement in the private sector: a state of the art review between 1996 and 2013

NOTICE: this is the author’s version of a work that was accepted for publication in Journal of Cleaner Production. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Cleaner Production, Volume 85, 15 December 2014, Pages 122–133. DOI 10.1016/j.jclepro.2014.08.106The purpose of this paper is to identify the main themes of Green Procurement (GP) in the private sector through a literature review of papers published between 1996 and 2013 and to develop some future research directions. GP research has garnered interest from academics and industry alike. This is demonstrated by the increasing number of academic papers published in recent years. This literature review builds on the three themes in the adoption of GP identified from the literature: 1) the motivation and drivers for the implementation of GP; 2) barriers to the implementation of GP; 3) the performance impacts of the adoption of GP. Given that there are distinctive features of the private sector, compared to the public sector, this analysis focuses on GP in the private sector. The approach to GP holds important implications for managers, by directing limited resources towards projects which intersect both environmental performance and economic performance. The article discusses interesting findings, develops a conceptual framework of GP and suggests a number of directions for future research

Student engagement and voice in higher education: students’ perceptions

There has been intense debate about student engagement and student voice in institutions of higher education in the past decade or so. Most of the discussion has been theoretical or based on a cause-and-effect research design. With the aim of gathering student perspectives on student voice and its related mechanisms, this study collected the voice of 13 students, the majority of whom were international students, from one UK university. Participants include undergraduate (n=1), postgraduate taught (n=7), and PhD (n=5) students who voluntarily agreed to have an online interview with the researcher, a PhD intern of the Student Voice team. Findings indicate that participants have an overall positive and supportive view of student voice mechanisms at this institution, although some understandings are not adequate or accurate. Participants’ attitudes towards some commonly used communication channels indicate that they prioritise an interactive and dynamic tool to initiate dialogue with the university. Suggestions are put forward for managerial strategies for a sustainable and inclusive student voice mechanism.

A Comparison of Hierarchical and Non-Hierarchical Bayesian Approaches for Fitting Allometric Larch (Larix.spp.) Biomass Equations

Accurate biomass estimations are important for assessing and monitoring forest carbon storage. Bayesian theory has been widely applied to tree biomass models. Recently, a hierarchical Bayesian approach has received increasing attention for improving biomass models. In this study, tree biomass data were obtained by sampling 310 trees from 209 permanent sample plots from larch plantations in six regions across China. Non-hierarchical and hierarchical Bayesian approaches were used to model allometric biomass equations. We found that the total, root, stem wood, stem bark, branch and foliage biomass model relationships were statistically significant (p-values \u3c 0.001) for both the non-hierarchical and hierarchical Bayesian approaches, but the hierarchical Bayesian approach increased the goodness-of-fit statistics over the non-hierarchical Bayesian approach. The R2 values of the hierarchical approach were higher than those of the non-hierarchical approach by 0.008, 0.018, 0.020, 0.003, 0.088 and 0.116 for the total tree, root, stem wood, stem bark, branch and foliage models, respectively. The hierarchical Bayesian approach significantly improved the accuracy of the biomass model (except for the stem bark) and can reflect regional differences by using random parameters to improve the regional scale model accuracy

Physiological Responses and Tolerance Evaluation of Five Poplar Varieties to Waterlogging

Waterlogging resistance of five poplar varieties, ‘Danhongyang’ (DHY), ‘Juba-261’ (JB-261), ‘Zongqiansanhao’ (ZQ-3), ‘Zhonglin-2025’ (ZL-2025), and ‘Nanlin-895’ (NL-895), was evaluated under the simulated waterlogging conditions. Data on changes in leaf color and morphology as well as in biochemical indices, such as chlorophyll, malonaldehyde, soluble protein, soluble sugar content, superoxide dismutase (SOD), peroxidases (POD), ascorbate peroxidase (APX), glutathione reductase (GR), and glutathione peroxidase (GSH-PX) activities, relevant to submergence stress, were analyzed. The principal component analysis of the data identified the waterlogging resistance coefficient of the indices, which showed that waterflooding brought about different degrees of damage in the five poplar varieties, with DHY having the lowest waterlogging index. The leaf pigment content of the poplar was remarkably decreased by waterlogging, whereas malondialdehyde (MDA) and proline contents were enhanced, but in different extents among the poplar varieties. Nearly all other poplar varieties showed a tendency of decline in JB-261, ZQ-3, ZL-2025, NL-895, except for SOD activity in DHY, which increased under submergence stress. Poplar varieties had varying degree of changes in POD activity, and APX activity tended to increase upon waterlogging. GR also displayed increasing tendency in JB-261, ZL-2025 and NL-895, except for in ZQ-3, which declined under waterlogging stress. GSH-PX except for ZQ-3 displayed no significant change, which showed a tendency of decline in DHY, JB-261, ZL-2025, and NL895. Principal component analysis allowed us to reduce16 indices to four independent indices. The subordinate function analysis identified that the DHY variety had the highest waterlogging tolerance, whereas the NL-895 variety had the lowest waterlogging tolerance among tested varieties

Isolation and characterization of an Aux/IAA gene (LaIAA2) from Larix

The phytohormone auxin controls many aspects of plant development. Auxin/indole-3-acetic acid (Aux/IAA)  transcriptional factors are key regulators of auxin responses in plants. To investigate the effects of auxin on  gene expression during the rooting process of Larix cuttings, a subtractive cDNA library was constructed and  272 UniEST were obtained by using suppression subtractive hybridization (SSH). Based on a fragment of 272  UniEST, the full-length cDNA of LaIAA2, an Aux /IAA gene from Larix was isolated. Then, the response  expression of LaIAA2 to auxin was determined by treating with different sources and concentration of auxin and cycloheximide and the expression patterns of LaIAA2 were examined in different tissues. The results show  that LaIAA2 appears to be the first response gene of auxin and LaIAA2 gene was involved in the root  development and auxin signaling. The express pattern of LaIAA2 gene indicated that it might play a central role in root development, specially regulated lateral and adventitious root production.Key words: Aux/IAA gene family, auxin, LaIAA2, Lari