Refinement of a morphological scoring system for postimplantation rabbit conceptuses

BACKGROUND: The rabbit is used extensively in developmental toxicity testing, yet basic information on rabbit embryo development is lacking. The goals of this study were to refine a rabbit embryo morphology scoring system, and use it to evaluate rabbit whole embryo cultures (WEC). METHODS: A total of 265 conceptuses were harvested between GD 8.0 and 12.0 (coitus = GD 0) at 6-hr intervals and examined in detail. Discreet developmental landmarks were then established for 18 morphological features and assigned scores ranging from 0 up to 6. The scoring system was then validated on a subset of randomly selected in vivo conceptuses, and was used to evaluate conceptuses grown for 12, 24, 36, or 48 hr in WEC beginning from GD 9.0 or 10.0. A few embryos also were examined using microscopic computed tomography (microCT)-based virtual histologytrade mark to assess the utility of this technology. RESULTS: Morphology scores of in vivo developed conceptuses increased linearly (r2 = 0.98) with advancing gestational age, from means of 0.0 on GD 8.0 to 67.9 on GD 12.0. Application of the scoring system, supplemented with evidence from Virtual histologytrade mark, indicated that the WEC system supported normal morphological development of rabbit conceptuses. However, when explanted at GD 9, the rate of development was about 20% slower than in vivo, whereas the rate of development in WEC from GD 10 was indistinguishable from in vivo. CONCLUSIONS: This work enhances the evaluation tools available to study mechanisms of normal and abnormal development in this widely used animal testing species.     

Three-dimensional preparation and imaging reveal intrinsic microtubule properties

We present an experimental investigation of microtubule dynamic instability in three dimensions, based on laser light-sheet fluorescence microscopy. We introduce three-dimensional (3D) preparation of Xenopus laevis egg extracts in Teflon-based cylinders and provide algorithms for 3D image processing. Our approach gives experimental access to the intrinsic dynamic properties of microtubules and to microtubule population statistics in single asters. We obtain evidence for a stochastic nature of microtubule pausing.     

Effects of the plant steroidal hormone, 24-epibrassinolide, on the mitotic index and growth of onion (Allium cepa) root tips

The purpose of the present study was to determine the effects of the steroidal plant hormone, 24-epibrassinolide (BL), on the mitotic index and growth of onion (Allium cepa) root tips. The classical Allium test was used to gather and quantify data on the rate of root growth, the stages of mitosis, and the number of mitoses in control and BL-treated groups of onions. Low doses of BL (0.005 ppm) nearly doubled the mean root length and the number of mitoses over that of controls. Intermediate doses of BL (0.05 ppm) also produced mean root lengths and number of mitoses that were significantly greater than those of the controls. The highest dose of BL (0.5 ppm) produced mean root lengths and number of mitoses that were less than control values, but the differences were not statistically significant. Examination of longitudinally sectioned root tips produced relatively similar results. This study confirms the suppositions of previous authors who have claimed that exogenously applied BL can increase the number of mitoses in plants, but failed to show cytogenetic data. This is the first report detailing the effects of BL on chromosomes and the cell cycle.     

Amyotrophic lateral sclerosis: an emerging era of collaborative gene discovery

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND). It is currently incurable and treatment is largely limited to supportive care. Family history is associated with an increased risk of ALS, and many Mendelian causes have been discovered. However, most forms of the disease are not obviously familial. Recent advances in human genetics have enabled genome-wide analyses of single nucleotide polymorphisms (SNPs) that make it possible to study complex genetic contributions to human disease. Genome-wide SNP analyses require a large sample size and thus depend upon collaborative efforts to collect and manage the biological samples and corresponding data. Public availability of biological samples (such as DNA), phenotypic and genotypic data further enhances research endeavors. Here we discuss a large collaboration among academic investigators, government, and non-government organizations which has created a public repository of human DNA, immortalized cell lines, and clinical data to further gene discovery in ALS. This resource currently maintains samples and associated phenotypic data from 2332 MND subjects and 4692 controls. This resource should facilitate genetic discoveries which we anticipate will ultimately provide a better understanding of the biological mechanisms of neurodegeneration in ALS.     

Isometric Scaling in Developing Long Bones Is Achieved by an Optimal Epiphyseal Growth Balance

One of the major challenges that developing organs face is scaling, that is, the adjustment of physical proportions during the massive increase in size. Although organ scaling is fundamental for development and function, little is known about the mechanisms that regulate it. Bone superstructures are projections that typically serve for tendon and ligament insertion or articulation and, therefore, their position along the bone is crucial for musculoskeletal functionality. As bones are rigid structures that elongate only from their ends, it is unclear how superstructure positions are regulated during growth to end up in the right locations. Here, we document the process of longitudinal scaling in developing mouse long bones and uncover the mechanism that regulates it. To that end, we performed a computational analysis of hundreds of three-dimensional micro-CT images, using a newly developed method for recovering the morphogenetic sequence of developing bones. Strikingly, analysis revealed that the relative position of all superstructures along the bone is highly preserved during more than a 5-fold increase in length, indicating isometric scaling. It has been suggested that during development, bone superstructures are continuously reconstructed and relocated along the shaft, a process known as drift. Surprisingly, our results showed that most superstructures did not drift at all. Instead, we identified a novel mechanism for bone scaling, whereby each bone exhibits a specific and unique balance between proximal and distal growth rates, which accurately maintains the relative position of its superstructures. Moreover, we show mathematically that this mechanism minimizes the cumulative drift of all superstructures, thereby optimizing the scaling process. Our study reveals a general mechanism for the scaling of developing bones. More broadly, these findings suggest an evolutionary mechanism that facilitates variability in bone morphology by controlling the activity of individual epiphyseal plates.     

Matrix Metalloproteinase 10 Degradomics in Keratinocytes and Epidermal Tissue Identifies Bioactive Substrates With Pleiotropic Functions*

Matrix metalloproteinases (MMPs) are important players in skin homeostasis, wound repair, and in the pathogenesis of skin cancer. It is now well established that most of their functions are related to processing of bioactive proteins rather than components of the extracellular matrix (ECM). MMP10 is highly expressed in keratinocytes at the wound edge and at the invasive front of tumors, but hardly any non-ECM substrates have been identified and its function in tissue repair and carcinogenesis is unclear. To better understand the role of MMP10 in the epidermis, we employed multiplexed iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) and monitored MMP10-dependent proteolysis over time in secretomes from keratinocytes. Time-resolved abundance clustering of neo-N termini classified MMP10-dependent cleavage events by efficiency and refined the MMP10 cleavage site specificity by revealing a so far unknown preference for glutamate in the P1 position. Moreover, we identified and validated the integrin alpha 6 subunit, cysteine-rich angiogenic inducer 61 and dermokine as novel direct MMP10 substrates and provide evidence for MMP10-dependent but indirect processing of phosphatidylethanolamine-binding protein 1. Finally, we sampled the epidermal proteome and degradome in unprecedented depth and confirmed MMP10-dependent processing of dermokine in vivo by TAILS analysis of epidermis from transgenic mice that overexpress a constitutively active mutant of MMP10 in basal keratinocytes. The newly identified substrates are involved in cell adhesion, migration, proliferation, and/or differentiation, indicating a contribution of MMP10 to local modulation of these processes during wound healing and cancer development. Data are available via ProteomeXchange with identifier PXD002474.Matrix metalloproteinases (MMPs)¹ are extracellular zinc-dependent endoproteinases that are highly expressed in tissues undergoing remodeling processes during development, in response to injury, or as a result of neoplastic transformation (1–3). MMP10, also known as stromelysin-2, gained particular interest in the skin, because of its specific and strong expression in wound edge keratinocytes as well as at the invasive front of epithelial tumors (4–6). Overexpression of a constitutively active MMP10 mutant in wound keratinocytes in mice led to scattering of these cells at the tip of the migrating wound epithelium, altered β1-integrin expression, reduced AKT phosphorylation and increased apoptosis (7). Lack of MMP10 in a lung infection model affected genes that are involved in the regulation of apoptosis, cell proliferation, immune response and signal transduction (8). In the gut, bone marrow-derived MMP10 had a protective role in experimental colitis with implications in macrophage polarization (9). MMP10 released from hepatocytes and macrophages positively contributed to liver regeneration (10), whereby it promoted hepatocarcinogenesis in a complicated crosstalk with chemokine signaling (11). Most recently, Rohani et al. demonstrated a role for macrophage-derived MMP10 in moderating scar formation by controlling collagenase activity of dermal macrophages (12).Similar complex phenotypes have been associated with activities of most MMPs that, however, were not related to processing of extracellular matrix (ECM) proteins, the classical MMP substrates (13), but of bioactive mediators, including cell surface receptors, growth factor binding proteins, proteases, inhibitors, cytokines, and chemokines (14, 15). This changed the view on MMPs as simple tissue degraders to precise modulators of diverse processes, such as cell proliferation, migration, differentiation, angiogenesis, apoptosis and immune response (2, 16). As an example, functions of MMP3, the closest homolog of MMP10, in keratinocyte differentiation (17), tumor cell invasion (18), and immune cell recruitment (19) could be explained by processing of non-ECM proteins that have been identified as direct substrates of this protease (2, 20) in addition to ECM components (21). However, because MMP10 has been mostly neglected in the quest for new MMP substrates, it remains to be elucidated, if it also exerts its functions in part by processing of bioactive proteins whose identification is instrumental in understanding the mechanisms of action of MMP10 in tissue repair and carcinogenesis.Recently, we applied iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS), a multiplexed quantitative proteomics workflow for identification of protease substrates in complex proteomes (22–24), to reveal new targets of MMP10 in secretomes from mouse embryonic fibroblasts (25). Moreover, to mimic MMP10 activity at the epidermal–dermal interface, we devised a new workflow that allowed monitoring both cellular origins and cleavages of substrates in mixed secretomes from keratinocytes and fibroblasts (26). However, this study focused on basement membrane components and missed additional information on cleavage kinetics. Thus, in this work, we employed time-resolved TAILS to identify novel MMP10 substrates in keratinocyte secretomes and mouse epidermal tissue, aiming at further characterizing the MMP10 substrate degradome in epidermal keratinocytes for a better understanding of its biological roles in the skin. Here, we identified novel bioactive substrates of MMP 10 in vitro and in the skin in vivo, which provide insight into its functions in wound repair and carcinogenesis. In addition, we revealed an unexpected preference of MMP10 for substrates that harbor a glutamate residue in P1 position, which might be exploited for the development of specific activity-based probes or inhibitors for this important wound- and tumor-related protease.     

The Role of Scale and Technology Maturity in Life Cycle Assessment of Emerging Technologies: A Case Study on Carbon Nanotubes

Life cycle assessment (LCA) has been applied for assessing emerging technologies, where large‐scale production data are generally lacking. This study introduces a standardized scheme for technology and manufacturing readiness levels to contextualize a technology's development stage. We applied the scheme to a carbon nanotube (CNT) LCA and found that, regardless of synthesis technique, CNT manufacturing will become less energy intensive with increased levels of readiness. We examined the influence of production volume on LCA results using primary data from a commercial CNT manufacturer with approximately 100 grams per day production volume and engineering design of a scaled‐up process with 1 tonne per day production capacity. The results show that scaling up could reduce 84% to 94% of its cradle‐to‐gate impacts, mainly as a result of the recycling of feedstock that becomes economically viable only beyond certain minimum production volume. This study shows that LCAs on emerging technologies based on immature data should be interpreted in conjunction with their technology and manufacturing readiness levels and reinforces the need of standardizing and communicating information on these readiness levels and scale of production in life cycle inventory practices.     

Regulatory MicroRNA Networks:Complex Patterns of Target Pathways for Disease-related and Housekeeping MicroRNAs

Blood-based micro RNA(mi RNA) signatures as biomarkers have been reported for various pathologies, including cancer, neurological disorders, cardiovascular diseases, and also infections. The regulatory mechanism behind respective mi RNA patterns is only partially understood. Moreover, ‘‘preserved' mi RNAs, i.e., mi RNAs that are not dysregulated in any disease,and their biological impact have been explored to a very limited extent. We set out to systematically determine their role in regulatory networks by defining groups of highly-dysregulated mi RNAs that contribute to a disease signature as opposed to preserved housekeeping mi RNAs. We further determined preferential targets and pathways of both dysregulated and preserved mi RNAs by computing multi-layer networks, which were compared between housekeeping and dysregulated mi RNAs. Of 848 mi RNAs examined across 1049 blood samples, 8 potential housekeepers showed very limited expression variations, while 20 mi RNAs showed highly-dysregulated expression throughout the investigated blood samples. Our approach provides important insights into mi RNAs and their role in regulatory networks. The methodology can be applied to systematically investigate the differences in target genes and pathways of arbitrary mi RNA sets.     

Interactions between evolutionary processes at high mutation rates

Evolution at high mutation rates is minimally affected by six processes: mutation-selection balance, error catastrophes, Muller's Ratchet, robustness and compensatory evolution, and clonal interference. Including all of these processes in a tractable, analytical model is difficult, but they can be captured in simulations that utilize realistic genotype-phenotype-fitness maps, as done here by modeling RNA folding. Subjecting finite, asexual populations to a range of mutation rates revealed simple criteria that predict when particular evolutionary processes are important. Populations were initiated with a genotype encoding the most fit phenotype. When purifying selection was strong relative to mutation, the initial genotype was replaced by one more mutationally robust, and the maximally fit phenotype was maintained in a mutation-selection balance where the deleterious mutation rate determined mean fitness. With weaker purifying selection, the most fit genotypes were lost. Although loss of the best genotype was ongoing and might have led to a progressive fitness decline, continual compensatory evolution led to an approximate fitness equilibration. Per total genomic mutation rate, mean fitness was similar for strong and weak purifying selection. These results represent a first step at separating interactions between evolutionary processes at high mutation rate, but additional theory is needed to interpret some outcomes.