Structural Stabilization of Tissue for Embryo Phenotyping Using Micro-CT with Iodine Staining

The International Mouse Phenotyping Consortium has been established to conduct large-scale phenotyping of the approximately 23,000 single-gene knockout mice generated by the International Knockout Mouse Consortium to investigate the role of each gene in the mouse genome. Of the generated mouse lines, 30% are predicted to be embryonic lethal, requiring the implementation of imaging techniques and analysis tools specific to late gestation mouse embryo phenotyping. A well-adopted technique combines the use of iodinated contrast solutions and micro-computed tomography imaging. This simple iodine immersion technique provides superior soft-tissue contrast enhancement, however, the hypertonic nature of iodine promotes dehydration causing moderate to severe tissue deformation. Here, we combine the stabilizing properties of a hydrogel mesh with the enhanced contrast properties of iodine. The protocol promotes cross linking of tissue through formaldehyde fixation and the linking of hydrogel monomers to biomolecules. As a result, the hydrogel supports tissue structure and preserves its conformation taking advantage of iodine-enhanced soft tissue contrast to produce high quality mouse embryo images with minimal tissue distortion. Hydrogel stabilization substantially reduces intersample anatomical variation of mature mouse embryos subjected to iodine preparation protocols. A 20% and 50% reduction in intersample variation of normalized brain and lung volume is achieved through hydrogel stabilization, as well as a 20% reduction in variation in overall embryo anatomy as measured through image registration methods. This increases the sensitivity of computer automated analysis to reveal significant anatomical differences between mutant and wild-type mice.     

Phenotypic Switching Induced by Damaged Matrix Is Associated with DNA Methyltransferase 3A (DNMT3A) Activity and Nuclear Localization in Smooth Muscle Cells (SMC)

Extracellular matrix changes are often crucial inciting events for fibroproliferative disease. Epigenetic changes, specifically DNA methylation, are critical factors underlying differentiated phenotypes. We examined the dependency of matrix-induced fibroproliferation and SMC phenotype on DNA methyltransferases. The cooperativity of matrix with growth factors, cell density and hypoxia was also examined. Primary rat visceral SMC of early passage (0–2) were plated on native collagen or damaged/heat-denatured collagen. Hypoxia was induced with 3% O₂ (balanced 5% CO₂ and 95% N₂) over 48 hours. Inhibitors were applied 2–3 hours after cells were plated on matrix, or immediately before hypoxia. Cells were fixed and stained for DNMT3A and smooth muscle actin (SMA) or smooth muscle myosin heavy chain. Illumina 450 K array of CpG sites was performed on bisulfite-converted DNA from smooth muscle cells on damaged matrix vs native collagen. Matrix exquisitely regulates DNMT3A localization and expression, and influences differentiation in SMCs exposed to denatured matrix +/− hypoxia. Analysis of DNA methylation signatures showed that Matrix caused significant DNA methylation alterations in a discrete number of CpG sites proximal to genes related to SMC differentiation. Matrix has a profound effect on the regulation of SMC phenotype, which is associated with altered expression, localization of DNMTs and discrete changes DNA methylation.     

Mouriri morleyii sp. nov. (Melastomataceae) from Brazil,with notes on its foliar stomatal crypts

Mouriri morleyii R. Goldenb. & Meirelles sp. nov. can be distinguished from the other species in the genus by its large stomatal crypts (the largest ones in the genus), columnar sclereids, tetramerous flowers and calyx closed in bud. This new species was collected on an inselberg in the state of Espírito Santo, Brazil.     

The impact of prehistoric mining activities on the environment: a multidisciplinary study at the fen Schwarzenbergmoos (Brixlegg, Tyrol, Austria)

The exploitation of copper ore deposits of the northern Greywacke Zone was initiated by the implementation of metallurgic technologies in the Eastern Alps thousands of years ago. This multi-proxy study aimed to detect prehistoric mining phases in the vicinity of a prominent copper ore deposit in the Lower Inn Valley. Therefore we studied a peat core from a fen using pollen, micro charcoal and geochemical analyses. In the same fen, an archaeological investigation revealed an ore beneficiation site, well dated by dendrochronological analysis to the Late Bronze Age (9th century b.c.). First hints of mining activities reflected by the occurrence of anthropogenic indicators in the pollen diagram, associated with elevated metal values, at the beginning of the Bronze Age might result from early mineral prospecting and metallurgical experiments around the use of fahlore. The local ore deposit was then abandoned until during the Bronze Age mining activities started to increase. This is reflected by an expansion of the pioneer species Pinus and Larix on mine spoil heaps in the proximity. Concomitantly metal ratios and micro charcoal increase. From about 1000 to 850 b.c. a strong impact of mining activities is displayed in the multi-proxy data. The local forest was partly cleared on and in the vicinity of the fen. According to dendrochronological data the ore beneficiation plant was in use from about 900 to 870 b.c. Until about 700 b.c. another period with moderate impact by mining activities in the further vicinity of the fen shows up.     

Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C

Background

A number of neurodevelopmental syndromes are caused by mutations in genes encoding proteins that normally function in epigenetic regulation. Identification of epigenetic alterations occurring in these disorders could shed light on molecular pathways relevant to neurodevelopment.

Results

Using a genome-wide approach, we identified genes with significant loss of DNA methylation in blood of males with intellectual disability and mutations in the X-linked KDM5C gene, encoding a histone H3 lysine 4 demethylase, in comparison to age/sex matched controls. Loss of DNA methylation in such individuals is consistent with known interactions between DNA methylation and H3 lysine 4 methylation. Further, loss of DNA methylation at the promoters of the three top candidate genes FBXL5, SCMH1, CACYBP was not observed in more than 900 population controls. We also found that DNA methylation at these three genes in blood correlated with dosage of KDM5C and its Y-linked homologue KDM5D. In addition, parallel sex-specific DNA methylation profiles in brain samples from control males and females were observed at FBXL5 and CACYBP.

Conclusions

We have, for the first time, identified epigenetic alterations in patient samples carrying a mutation in a gene involved in the regulation of histone modifications. These data support the concept that DNA methylation and H3 lysine 4 methylation are functionally interdependent. The data provide new insights into the molecular pathogenesis of intellectual disability. Further, our data suggest that some DNA methylation marks identified in blood can serve as biomarkers of epigenetic status in the brain.     

Metagenomic Investigation of Plasma in Individuals with ME/CFS Highlights the Importance of Technical Controls to Elucidate Contamination and Batch Effects

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease causing indefinite fatigue. ME/CFS has long been hypothesised to have an infectious cause; however, no specific infectious agent has been identified. We used metagenomics to analyse the RNA from plasma samples from 25 individuals with ME/CFS and compare their microbial content to technical controls as well as three control groups: individuals with alternatively diagnosed chronic Lyme syndrome (N = 13), systemic lupus erythematosus (N = 11), and healthy controls (N = 25). We found that the majority of sequencing reads were removed during host subtraction, thus there was very low microbial RNA content in the plasma. The effects of sample batching and contamination during sample processing proved to outweigh the effects of study group on microbial RNA content, as the few differences in bacterial or viral RNA abundance we did observe between study groups were most likely caused by contamination and batch effects. Our results highlight the importance of including negative controls in all metagenomic analyses, since there was considerable overlap between bacterial content identified in study samples and control samples. For example, Proteobacteria, Firmicutes, Actinobacteria, and Bacteriodes were found in both study samples and plasma-free negative controls. Many of the taxonomic groups we saw in our plasma-free negative control samples have previously been associated with diseases, including ME/CFS, demonstrating how incorrect conclusions may arise if controls are not used and batch effects not accounted for.     

The language of everyday racism and microaggression in the workplace: Palestinian professionals in Israel

Based on interviews with Palestinian professionals in Jewish organizations in Israel, this article discloses a distinctive practice of ‘everyday racism’ and microaggression – a language of everyday racism. This ‘language of everyday racism’ refers to Hebrew words and expressions that are routinely used by Jews in their mundane conversations and that include the word ‘Arab’ when describing a deficiency or defect, some sort of unsightliness, filth, or general negativity (as in the expression ‘You're dressed like an Arab woman’). This article not only describes the language of everyday racism as a specific form of everyday racism and microaggression (national microaggression), it also illustrates how this language activates the Palestinian professionals in a reflexive manner. The discussion section describes how the internal dialectic between structure and agency is critical to understanding the language of everyday racism, which in turn acts as a mechanism of the inequality that underlies face-to-face interactions.     

Testing transgenic regulatory elements through live mouse imaging

To overcome positional and methylation effects on transgene expression, we developed a universal cloning cassette for in vivo assessment of regulatory elements using the luciferase reporter gene and the CCCD camera. Monitoring luciferase expression pattern in live mice enables screening of large numbers of transgenic founders quickly and inexpensively. We demonstrate that in the engineered transgenic mice, the chicken beta-globin 5'HS4 insulator did not always provide the desirable expression pattern, and the Island Element, responsible for the demethylation of the surrounding DNA region, was not beneficial. Both tested liver-specific and developmentally regulated promoters exhibited the expected expression pattern in most transgenic founders.