Influence of incubation temperature and embryonic motility on the growth of members of Caiman yacare (Daudin, 1802)

This study aimed to evaluate whether skeletal development of the Pantanal Caiman (Caiman yacare) is similarly influenced by temperature variation and controlled increases in embryo motility. All eggs were incubated at 90% humidity and 29 °C for the first 45 days. Thereafter, the incubation temperature was either maintained at 29 °C and embryos were treated with 4-aminopyridine (4-AP) on days 46, 47, 48, and 49 (Group I, 29 °C 4-AP, n = 15); maintained at 29 °C (n = 14; Group II); or at 33 °C (n = 14, Group III). Embryonic movement was measured using an Egg Buddy® digital monitor on days 30, 35, 42, 49, 56, and 60, at which point embryos were euthanized and samples were collected for analysis. No differences were observed between groups with varying incubation temperatures. In contrast, embryonic motility was greater in embryos treated with 4-AP (P < 0.001) on day 49, and this was associated with higher proportions of snout-vent and hand lengths. This study demonstrates for the first time that pharmacologically induced increases in embryo motility result in phenotypic changes to the proportion of elements during prenatal ontogeny, thereby effectively altering the adaptation of the species to specific environments.

Behavioural and physiological plasticity in social hierarchies

Individuals occupying dominant and subordinate positions in social hierarchies exhibit divergent behaviours, physiology and neural functioning. Dominant animals express higher levels of dominance behaviours such as aggression, territorial defence and mate-guarding. Dominants also signal their status via auditory, visual or chemical cues. Moreover, dominant animals typically increase reproductive behaviours and show enhanced spatial and social cognition as well as elevated arousal. These biobehavioural changes increase energetic demands that are met via shifting both energy intake and metabolism and are supported by coordinated changes in physiological systems including the hypothalamic–pituitary–adrenal and hypothalamic–pituitary–gonadal axes as well as altered gene expression and sensitivity of neural circuits that regulate these behaviours. Conversely, subordinate animals inhibit dominance and often reproductive behaviours and exhibit physiological changes adapted to socially stressful contexts. Phenotypic changes in both dominant and subordinate individuals may be beneficial in the short-term but lead to long-term challenges to health. Further, rapid changes in social ranks occur as dominant animals socially ascend or descend and are associated with dynamic modulations in the brain and periphery. In this paper, we provide a broad overview of how behavioural and phenotypic changes associated with social dominance and subordination are expressed in neural and physiological plasticity. This article is part of the theme issue ‘The centennial of the pecking order: current state and future prospects for the study of dominance hierarchies’.

Unconventional metabolites in chromatin regulation

Chromatin, the complex of DNA and histone proteins, serves as a main integrator of cellular signals. Increasing evidence links cellular functional to chromatin state. Indeed, different metabolites are emerging as modulators of chromatin function and structure. Alterations in chromatin state are decisive for regulating all aspects of genome function and ultimately have the potential to produce phenotypic changes. Several metabolites such as acetyl-CoA, S-adenosyl methionine (SAM) or adenosine triphosphate (ATP) have now been well characterized as main substrates or cofactors of chromatin modifying enzymes. However, there are other metabolites that can directly interact with chromatin influencing its state or that modulate the properties of chromatin regulatory factors. Also, there is a growing list of atypical enzymatic and non-enzymatic chromatin modifications that originate from different cellular pathways that have not been in the limelight of chromatin research. Here, we summarize different properties and functions of uncommon regulatory molecules originating from intermediate metabolism of lipids, carbohydrates and amino acids. Based on the various modes of action on chromatin and the plethora of putative, so far not described chromatin regulating metabolites, we propose that there are more links between cellular functional state and chromatin regulation to be discovered. We hypothesize that these connections could provide interesting starting points for interfering with cellular epigenetic states at a molecular level.

Effects of Dithiothreitol on Fertilization and Early Development in Sea Urchin

The vitelline layer (VL) of a sea urchin egg is an intricate meshwork of glycoproteins that intimately ensheathes the plasma membrane. During fertilization, the VL plays important roles. Firstly, the receptors for sperm reside on the VL. Secondly, following cortical granule exocytosis, the VL is elevated and transformed into the fertilization envelope (FE), owing to the assembly and crosslinking of the extruded materials. As these two crucial stages involve the VL, its alteration was expected to affect the fertilization process. In the present study, we addressed this question by mildly treating the eggs with a reducing agent, dithiothreitol (DTT). A brief pretreatment with DTT resulted in partial disruption of the VL, as judged by electron microscopy and by a novel fluorescent polyamine probe that selectively labelled the VL. The DTT-pretreated eggs did not elevate the FE but were mostly monospermic at fertilization. These eggs also manifested certain anomalies at fertilization: (i) compromised Ca2+ signaling, (ii) blocked translocation of cortical actin filaments, and (iii) impaired cleavage. Some of these phenotypic changes were reversed by restoring the DTT-exposed eggs in normal seawater prior to fertilization. Our findings suggest that the FE is not the decisive factor preventing polyspermy and that the integrity of the VL is nonetheless crucial to the egg’s fertilization response.

AXL Knock-Out in SNU475 Hepatocellular Carcinoma Cells Provides Evidence for Lethal Effect Associated with G2 Arrest and Polyploidization

AXL, a member of the TAM family, is a promising therapeutic target due to its elevated expression in advanced hepatocellular carcinoma (HCC), particularly in association with acquired drug resistance. Previously, RNA interference was used to study its role in cancer, and several phenotypic changes, including attenuated cell proliferation and decreased migration and invasion, have been reported. The mechanism of action of AXL in HCC is elusive. We first studied the AXL expression in HCC cell lines by real-time PCR and western blot and showed its stringent association with a mesenchymal phenotype. We then explored the role of AXL in mesenchymal SNU475 cells by CRISPR-Cas9 mediated gene knock-out. AXL-depleted HCC cells displayed drastic phenotypic changes, including increased DNA damage response, prolongation of doubling time, G2 arrest, and polyploidization in vitro and loss of tumorigenicity in vivo. Pharmacological inhibition of AXL by R428 recapitulated G2 arrest and polyploidy phenotype. These observations strongly suggest that acute loss of AXL in some mesenchymal HCC cells is lethal and points out that its inhibition may represent a druggable vulnerability in AXL-high HCC patients.

A Core Omnigenic Non-coding Trait Governing Dex-Induced Osteoporotic Effects Identified Without DEXA

Iatrogenic glucocorticoid (GC)-induced osteoporosis (GIO) is an idiosyncratic form of secondary osteoporosis. Genetic predisposition among individuals may give rise to variant degree of phenotypic changes but there has yet been a documented unified pathway to explain the idiosyncrasy. In this study, we argue that the susceptibility to epigenetic changes governing molecular cross talks along the BMP and PI3K/Akt pathway may underline how genetic background dictate GC-induced bone loss. Concordantly, osteoblasts from BALB/c or C57BL/6 neonatal mice were treated with dexamethasone for transcriptome profiling. Furthermore, we also confirmed that GC-pre-conditioned mesenchymal stem cells (MSCs) would give rise to defective osteogenesis by instigating epigenetic changes which affected the accessibility of enhancer marks. In line with these epigenetic changes, we propose that GC modulates a key regulatory network involving the scavenger receptor Cd36 in osteoblasts pre-conditioning pharmacological idiosyncrasy in GIO.

The use of high-throughput microscopy in the characterisation of phenotypes associated with the Unfolded Protein Response in Saccharomyces cerevisiae

<p>Proteins traversing the secretory pathway begin their passage in the endoplasmic reticulum (ER) where they must be correctly folded and processed to pass quality control measures. Complications with this process can result in the accumulation of misfolded proteins, commonly referred to as ER-stress, which has been associated with a number of diseases. The unfolded protein response (UPR) is the cell’s mechanism of dealing with ER-stress and is activated via the IRE1-HAC1 pathway in yeast. Ire1p is the ER-stress sensor and upon recognising misfolded proteins Ire1 oligomerises and forms local clusters. Activated Ire1p then splices out an inhibitory intron from the UPR specific transcription factor Hac1p which goes on to initiate downstream responses to alleviate ER-stress. Here we utilise high-throughput microscopy and UPR-specific GFP reporter systems to characterise the UPR in the yeast Saccharomyces cerevisiae. High-throughput microscopy and automated image analysis is increasingly being used as a screening tool for investigating genome-wide collections of yeast strains, including the yeast deletion mutant array and the yeast GFP collection. We describe the use of GFP labelled Ire1p to visualise cluster formation as a reporter for early UPR recognition of misfolded proteins, as well as a GFP controlled by a Hac1p responsive promoter to measure downstream UPR activation. These UPR-specific GFP reporter systems were used to screen a collection of non-essential gene deletion strains, identifying gene deletions that induce UPR activation and thus are likely to function in the early secretory pathway. This included well known components such as the ALG members of the glycosylation pathway and various ER chaperones such as LHS1 and SCJ1. Additionally this analysis revealed 44 previously uncharacterised genes, suggesting there are still processes related to the secretory pathway that are yet to be described. Moreover, by inducing ER-stress in this screening system we revealed genes required for the normal activation of the UPR including ribosomal/translation and chromatin/transcriptionally related genes, as well as various genes from throughout the secretory pathway. Furthermore, we screened a collection of ~4000 strains, each expressing a different GFP fusion protein, under ER-stress conditions to identify protein expression and localisation changes induced by the UPR. Comparison to UPR deficient Δhac1 cells uncovered a set of UPR specific targets including 26 novel UPR targets that had not been identified in previous studies measuring changes at the transcript level. As part of this work, we developed a dual red fluorescent protein system to label cells for automated image segmentation to enable single cell phenotype measurements. Here we describe the use of texture analysis as a means of increasing automation in the identification of phenotypic changes across the proteome. These novel techniques may be more widely applied to screening GFP collections to increase automation of image analysis, particularly as manual annotation of phenotypic changes is a major bottleneck in high-throughput screening. The results presented here from microscopy based screening compare well with other techniques in the literature, but also provide new information highlighting the synergistic effects of integrating high-throughput imaging into traditional screening methodologies.</p>

The use of high-throughput microscopy in the characterisation of phenotypes associated with the Unfolded Protein Response in Saccharomyces cerevisiae

<p>Proteins traversing the secretory pathway begin their passage in the endoplasmic reticulum (ER) where they must be correctly folded and processed to pass quality control measures. Complications with this process can result in the accumulation of misfolded proteins, commonly referred to as ER-stress, which has been associated with a number of diseases. The unfolded protein response (UPR) is the cell’s mechanism of dealing with ER-stress and is activated via the IRE1-HAC1 pathway in yeast. Ire1p is the ER-stress sensor and upon recognising misfolded proteins Ire1 oligomerises and forms local clusters. Activated Ire1p then splices out an inhibitory intron from the UPR specific transcription factor Hac1p which goes on to initiate downstream responses to alleviate ER-stress. Here we utilise high-throughput microscopy and UPR-specific GFP reporter systems to characterise the UPR in the yeast Saccharomyces cerevisiae. High-throughput microscopy and automated image analysis is increasingly being used as a screening tool for investigating genome-wide collections of yeast strains, including the yeast deletion mutant array and the yeast GFP collection. We describe the use of GFP labelled Ire1p to visualise cluster formation as a reporter for early UPR recognition of misfolded proteins, as well as a GFP controlled by a Hac1p responsive promoter to measure downstream UPR activation. These UPR-specific GFP reporter systems were used to screen a collection of non-essential gene deletion strains, identifying gene deletions that induce UPR activation and thus are likely to function in the early secretory pathway. This included well known components such as the ALG members of the glycosylation pathway and various ER chaperones such as LHS1 and SCJ1. Additionally this analysis revealed 44 previously uncharacterised genes, suggesting there are still processes related to the secretory pathway that are yet to be described. Moreover, by inducing ER-stress in this screening system we revealed genes required for the normal activation of the UPR including ribosomal/translation and chromatin/transcriptionally related genes, as well as various genes from throughout the secretory pathway. Furthermore, we screened a collection of ~4000 strains, each expressing a different GFP fusion protein, under ER-stress conditions to identify protein expression and localisation changes induced by the UPR. Comparison to UPR deficient Δhac1 cells uncovered a set of UPR specific targets including 26 novel UPR targets that had not been identified in previous studies measuring changes at the transcript level. As part of this work, we developed a dual red fluorescent protein system to label cells for automated image segmentation to enable single cell phenotype measurements. Here we describe the use of texture analysis as a means of increasing automation in the identification of phenotypic changes across the proteome. These novel techniques may be more widely applied to screening GFP collections to increase automation of image analysis, particularly as manual annotation of phenotypic changes is a major bottleneck in high-throughput screening. The results presented here from microscopy based screening compare well with other techniques in the literature, but also provide new information highlighting the synergistic effects of integrating high-throughput imaging into traditional screening methodologies.</p>