Historical and philosophical perspectives on the study of developmental bias

Throughout the recent history of research at the intersection of evolution and development, notions such as developmental constraint, evolutionary novelty, and evolvability have been prominent, but the term “developmental bias” has scarcely been used. And one may even doubt whether a unique and principled definition of bias is possible. I argue that the concept of developmental bias can still play a vital scientific role by means of setting an explanatory agenda that motivates investigation and guides the formulation of integrative explanatory frameworks. Less crucial is a definition that would classify patterns of phenotypic variation and unify variational patterns involving different traits and taxa as all being “bias.” Instead, what we should want is a concept that generates intellectual identity across various researchers, and that unites the diverse fields and approaches relevant to the study of developmental bias, from paleontology to behavioral biology. I point to some advantages of conducting research specifically under the label of “developmental bias,” compared with employing other, more common terms such as “evolvability.”     

Review of impact categories and environmental indicators for life cycle assessment of geotechnical systems

Life cycle assessment (LCA) has only had limited application in the geotechnical engineering discipline, though it has been widely applied to civil engineering systems such as pavements and roadways. A review of previous geotechnical LCAs showed that most studies have tracked a small set of impact categories, such as energy and global warming potential. Accordingly, currently reported environmental indicators may not effectively or fully capture important environmental impacts and tradeoffs associated with geotechnical systems, including those associated with land and soil resources. This research reviewed previous studies, methods, and models for assessment of land use and soil‐related impacts to understand their applicability to geotechnical LCA. The results of this review show that critical gaps remain in current knowledge and practice. In particular, further development or refinement of environmental indicators, impact categories, and cause–effect pathways is needed as they pertain to geotechnical applications—specifically those related to soil quality, soil functions, and the ecosystem services soils provide. In addition, many existing methods emerge from research on land use and land use change related to other disciplines (e.g., agriculture). For applicability to geotechnical projects, the resolution of many of these methods and resulting indicators need to be downscaled from the landscape/macro scale to the project scale. In the near term, practitioners of geotechnical LCA should begin tracking changes to soil properties and report impacts to land and soil resources qualitatively.     

Methanol-free biosynthesis of fatty acid methyl ester (FAME) in Synechocystis sp. PCC 6803

To meet the increasing global demand of biodiesel over the next decades, alternative methods for producing one of the key constituents of biodiesel (e.g. fatty acid methyl esters (FAMEs)) are needed. Algal biodiesel has been a long-term target compromised by excessive costs for harvesting and processing. In this work, we engineered cyanobacteria to convert carbon dioxide into excreted FAME, without requiring methanol as a methyl donor. To produce FAME, acyl-ACP, a product of the fatty acid biosynthesis pathway, was first converted into free fatty acid (FFA) by a thioesterase, namely ’UcFatB1 from Umbellularia californica. Next, by employing a juvenile hormone acid O-methyltransferase (DmJHAMT) from Drosophila melanogaster and S-adenosylmethionine (SAM) as a methyl donor, FFAs were converted into corresponding FAMEs. The esters were naturally secreted extracellularly, allowing simple product separation by solvent overlay as opposed to conventional algae biodiesel production where the algae biomass must first be harvested and processed for transesterification of extracted triacylglycerols (TAGs). By optimizing both the promoter and RBS elements, up to 120 mg/L of FAMEs were produced in 10 days. Quantification of key proteins and metabolites, together with constructs over-expressing SAM synthetase (MetK), indicated that ’UcFatB1, MetK, and DmJHAMT were the main factors limiting pathway flux. In order to solve the latter limitation, two reconstructed ancestral sequences of DmJHAMT were also tried, resulting in strains showing a broader methyl ester chain-length profile in comparison to the native DmJHAMT. Altogether, this work demonstrates a promising pathway for direct sunlight-driven conversion of CO₂ into excreted FAME.     

Engineering Saccharomyces cerevisiae for high yield production of α-amyrin via synergistic remodeling of α-amyrin synthase and expanding the storage pool

α-Amyrin is a plant-originated high-valued triterpene that is highly effective against several pathological ailments. α-Amyrin production by engineered Saccharomyces cerevisiae has been achieved by introducing α-amyrin synthase (αAS). However, the low yield of α-amyrin highly limits its industrial application; the low catalytic activity of αAS and the toxic effect of α-amyrin have been considered key elements. In this study, the highest yield of α-amyrin was obtained in engineered S. cerevisiae by remodeling α-amyrin synthase MdOSC1 and expanding the storage pool. The yield of α-amyrin was increased to 11-fold higher than that of the control by the triple mutant MdOSC1^{N11T/P250H/P373A} obtained based on the modeling analysis. Furthermore, key genes of MVA pathway were overexpressed to provide sufficient precursors, and DGA1 (Diacylglycerol acyltransferase) was overexpressed to expand the intracellular storage capacity. Finally, the as-constructed aAM12 strain produced 213.7 ± 12.4 mg/L α-amyrin in the shake flask and 1107.9 ± 76.8 mg/L in fed-batch fermentation; the fermentation yield was 106-fold higher than that of the original aAM1 strain under the same conditions, representing the highest α-amyrin yield in yeast reported to date. Microbial production of α-amyrin with over 1 g/L will be suitable for commercialization and can accelerate the industrial production of α-amyrin in yeast.     

Development of a 3D Tissue‐Engineered Skeletal Muscle and Bone Co‐culture System

In vitro 3D tissue‐engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co‐culture of TE skeletal muscle and bone are investigated. High‐glucose Dulbecco's modified Eagle medium (HG‐DMEM) supplemented with 20% fetal bovine serum followed by HG‐DMEM with 2% horse serum is found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an upregulation of RUNX2/CBFa1 in TE85s. Myotube formation is also evident within indirect contact monolayer cultures. Finally, in 3D co‐cultures, TE85 collagen/hydroxyapatite constructs have significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen‐based C2C12 skeletal muscle constructs; however, fusion within these constructs appears reduced. This work demonstrates the first report of the simultaneous co‐culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step toward a full in vitro 3D musculoskeletal junction model.     

Probing and Engineering the Fatty Acyl Substrate Selectivity of Starter Condensation Domains of Nonribosomal Peptide Synthetases in Lipopeptide Biosynthesis

Lipopeptides are produced by nonribosomal peptide synthetases (NRPSs) and contain diverse fatty acyl moieties that are major determinants of antibiotic potency. The lipid chains are incorporated into peptidyl backbones via lipoinitiation, a process comprising free fatty acid activation and the subsequent starter condensation domain (C1)‐catalyzed conjugation of fatty acyl moieties onto the aminoacyl substrates. Thus, a thorough understanding of lipoinitiation biocatalysts would significantly expand their potential to produce novel antibiotics. Here, biochemical assays, in silico analysis, and mutagenesis studies are used to ultimately identify the specific amino acid residues that control the fatty acyl substrate selectivity of C1 in lipopeptide A54145. In silico docking study has identified four candidate amino acids, and subsequent in vitro assays confirmed their functional contribution to the channel that controls substrate selectivity. Two engineered variants with single point mutations in C1 are found to alter the substrate selectivity toward nonnatural fatty acyl substrates. The detailed mechanistic insights into the catalytic contribution of C1 obtained from the present study will facilitate future NPRS biocatalyst efforts     

Overexpression of ER Protein Processing and Apoptosis Regulator Genes in Human Embryonic Kidney 293 Cells Improves Gene Therapy Vectors Production

Gammaretroviral and lentiviral vectors (γ‐RV and LV) are among the most used vectors in gene therapy. Currently, human embryonic kidney (HEK) 293 cells, the manufacture platform of choice for these vectors, provide low transducing particle yields, challenging their therapeutic applications and commercialization. This work studies metabolic pathways, focusing on endoplasmic reticulum (ER) protein processing and anti‐apoptotic mechanisms, influencing vector productivity in HEK 293 cell substrates. To that end, four candidate genes—protein disulfide isomerase family A member 2 gene, heat shock protein family A (Hsp70) member 5 gene, X‐box binding protein 1 gene (ER protein processing), and B‐cell lymphoma 2 protein gene (anti‐apoptotic)—are individually stably expressed in the cells. How their overexpression level influence vector yields is analyzed by establishing cell populations with incremental genomic copies of each. γ‐RV volumetric productivity increases up to 97% when overexpressing ER protein processing genes. LV volumetric production increases 53% when overexpressing the anti‐apoptotic gene. Improvements are associated with higher cell specific productivities and dependent on gene overexpression level, highlighting the importance of fine‐tuning gene expression. Overall, this work discloses gene engineering targets enabling efficient gene therapy product manufacture showing that ER protein processing and anti‐apoptotic pathways are pivotal to producer cell performance.     

The role of extracellular matrix in biomechanics and its impact on bioengineering of cells and 3D tissues

The cells and tissues of the human body are constantly exposed to exogenous and endogenous forces that are referred to as biomechanical cues. They guide and impact cellular processes and cell fate decisions on the nano-, micro- and macro-scale, and are therefore critical for normal tissue development and maintaining tissue homeostasis. Alterations in the extracellular matrix composition of a tissue combined with abnormal mechanosensing and mechanotransduction can aberrantly activate signaling pathways that promote disease development. Such processes are therefore highly relevant for disease modelling or when aiming for the development of novel therapies.In this mini review, we describe the main biomechanical cues that impact cellular fates. We highlight their role during development, homeostasis and in disease. We also discuss current techniques and tools that allow us to study the impact of biomechanical cues on cell and tissue development under physiological conditions, and we point out directions, in which in vitro biomechanics can be of use in the future.     

Model‐assisted identification of metabolic engineering strategies for Jatropha curcas lipid pathways

Efficient approaches to increase plant lipid production are necessary to meet current industrial demands for this important resource. While Jatropha curcas cell culture can be used for in vitro lipid production, scaling up the system for industrial applications requires an understanding of how growth conditions affect lipid metabolism and yield. Here we present a bottom‐up metabolic reconstruction of J. curcas supported with labeling experiments and biomass characterization under three growth conditions. We show that the metabolic model can accurately predict growth and distribution of fluxes in cell cultures and use these findings to pinpoint energy expenditures that affect lipid biosynthesis and metabolism. In addition, by using constraint‐based modeling approaches we identify network reactions whose joint manipulation optimizes lipid production. The proposed model and computational analyses provide a stepping stone for future rational optimization of other agronomically relevant traits in J. curcas.     

黄土区土质与土石质塿土堆积体水力侵蚀过程差异

利用室内模拟降雨试验,研究了不同雨强及坡度条件下黄土区土质(不含砾石)与土石质(砾石质量分数30%)塿土堆积体的水动力学特征、侵蚀特征及侵蚀动力机制的差异。结果表明: 砾石存在改变了堆积体坡面的水动力学特性,与土质坡面相比,土石质坡面的流速、弗汝德数、单位径流功率和过水断面单位能分别减少1.7%～49.7%、6.7%～60.6%、2.0%～44.6%和1.0%～26.7%;曼宁糙率系数、径流剪切力分别增加6.2%～169.4%、5.7%～79.3%。2.0、2.5 mm·min^-1雨强下,土石质坡面侵蚀速率较土质坡面降低26.2%～89.9%,砾石的减沙效益显著。2种堆积体的侵蚀速率与水动力学参数间均可用线性函数拟合,与土质坡面相比,土石质坡面的可蚀性参数均降低,降幅为56.1%～73.3%;而临界水动力学参数中径流剪切力增加11.1%,径流功率、单位径流功率和过水断面单位能分别减少25.4%、64.0%和5.0%。砾石的存在一定程度上控制了工程堆积体坡面降雨侵蚀过程。