Injury-induced inflammatory signaling and hematopoiesis in Drosophila

Inflammatory response in Drosophila to sterile (axenic) injury in embryos and adults has received some attention in recent years, and most concentrate on the events at the injury site. Here we focus on the effect sterile injury has on the hematopoietic organ, the lymph gland, and the circulating blood cells in the larva, the developmental stage at which major events of hematopoiesis are evident. In mammals, injury activates Toll-like receptor (TLR)/NFκB signaling in macrophages, which then express and secrete secondary, pro-inflammatory cytokines. In Drosophila larvae, distal puncture injury of the body wall epidermis causes a rapid activation of Toll and Jun kinase (JNK) signaling throughout the hematopoietic system and the differentiation of a unique blood cell type, the lamellocyte. Furthermore, we find that Toll and JNK signaling are coupled in their activation. Secondary to this Toll/JNK response, a cytokine, Upd3, is induced as a Toll pathway transcriptional target, which then promotes JAK/STAT signaling within the blood cells. Toll and JAK/STAT signaling are required for the emergence of the injury-induced lamellocytes. This is akin to the derivation of specialized macrophages in mammalian systems. Upstream, at the injury site, a Duox- and peroxide-dependent signal causes the activation of the proteases Grass and SPE needed for the activation of the Toll-ligand Spz, but microbial sensors or the proteases most closely associated with them during septic injury are not involved in the axenic inflammatory response.

Assessing the co-variability of DNA methylation across peripheral cells and tissues: Implications for the interpretation of findings in epigenetic epidemiology

Most epigenome-wide association studies (EWAS) quantify DNA methylation (DNAm) in peripheral tissues such as whole blood to identify positions in the genome where variation is statistically associated with a trait or exposure. As whole blood comprises a mix of cell types, it is unclear whether trait-associated DNAm variation is specific to an individual cellular population. We collected three peripheral tissues (whole blood, buccal epithelial and nasal epithelial cells) from thirty individuals. Whole blood samples were subsequently processed using fluorescence-activated cell sorting (FACS) to purify five constituent cell-types (monocytes, granulocytes, CD4+ T cells, CD8+ T cells, and B cells). DNAm was profiled in all eight sample-types from each individual using the Illumina EPIC array. We identified significant differences in both the level and variability of DNAm between different sample types, and DNAm data-derived estimates of age and smoking were found to differ dramatically across sample types from the same individual. We found that for the majority of loci variation in DNAm in individual blood cell types was only weakly predictive of variance in DNAm measured in whole blood, although the proportion of variance explained was greater than that explained by either buccal or nasal epithelial samples. Covariation across sample types was much higher for DNAm sites influenced by genetic factors. Overall, we observe that DNAm variation in whole blood is additively influenced by a combination of the major blood cell types. For a subset of sites, however, variable DNAm detected in whole blood can be attributed to variation in a single blood cell type providing potential mechanistic insight about EWAS findings. Our results suggest that associations between whole blood DNAm and traits or exposures reflect differences in multiple cell types and our data will facilitate the interpretation of findings in epigenetic epidemiology.

Telomere length varies substantially between blood cell types in a reptile

Telomeres are repeat sequences of non-coding DNA-protein molecules that cap or intersperse metazoan chromosomes. Interest in telomeres has increased exponentially in recent years, to now include their ongoing dynamics and evolution within natural populations where individuals vary in telomere attributes. Phylogenetic analyses show profound differences in telomere length across non-model taxa. However, telomeres may also differ in length within individuals and between tissues. The latter becomes a potential source of error when researchers use different tissues for extracting DNA for telomere analysis and scientific inference. A commonly used tissue type for assessing telomere length is blood, a tissue that itself varies in terms of nuclear content among taxa, in particular to what degree their thrombocytes and red blood cells (RBCs) contain nuclei or not. Specifically, when RBCs lack nuclei, leucocytes become the main source of telomeric DNA. RBCs and leucocytes differ in lifespan and how long they have been exposed to ‘senescence' and erosion effects. We report on a study in which cells in whole blood from individual Australian painted dragon lizards ( Ctenophorus pictus ) were identified using flow cytometry and their telomere length simultaneously measured. Lymphocyte telomeres were on average 270% longer than RBC telomeres, and in azurophils (a reptilian monocyte), telomeres were more than 388% longer than those in RBCs. If this variation in telomere length among different blood cell types is a widespread phenomenon, and DNA for comparative telomere analyses are sourced from whole blood, evolutionary inference of telomere traits among taxa may be seriously complicated by the blood cell type comprising the main source of DNA.

Assessing the co-variability of DNA methylation across peripheral cells and tissues: implications for the interpretation of findings in epigenetic epidemiology

Summary/AbstractBackgroundMost epigenome-wide association studies (EWAS) quantify DNA methylation (DNAm) in peripheral tissues such as whole blood to identify positions in the genome where variation is statistically associated with a trait or exposure. As whole blood comprises a mix of cell types, it is unclear whether trait-associated variation is specific to an individual cellular population.MethodsWe collected three peripheral tissues (whole blood, buccal and nasal epithelial cells) from thirty individuals. Whole blood samples were subsequently processed using fluorescence-activated cell sorting (FACS) to purify five constituent cell-types (monocytes, granulocytes, CD4+ T cells, CD8+ T cells, and B cells). DNAm was profiled in all eight sample-types from each individual using the Illumina EPIC array.ResultsWe identified significant differences in both the level and variability of DNAm between different tissues and cell types, and DNAm data-derived estimates of age and smoking were found to differ dramatically across sample types from the same individual. We found that for the majority of loci variation in DNAm in individual blood cell types was only weakly predictive of variance in DNAm measured in whole blood, however, the proportion of variance explained was greater than that explained by either buccal or nasal tissues. Instead we observe that DNAm variation in whole blood is additively influenced by a combination of the major blood cell types. For a subset of sites variable DNAm detected in whole blood can be attributed to variation in a single blood cell type providing potential mechanistic insight.ConclusionsWe identified major differences in DNAm between blood cell types and peripheral tissues, with each sample type being characterized by a unique DNAm signature across multiple loci. Our results suggest that associations between whole blood DNAm and traits or exposures reflect differences in multiple cell types and provide important insights for the interpretation of EWAS performed in whole blood.Key MessagesWe identified major differences in DNA methylation between blood cell types and peripheral tissues, with each sample type being characterized by a unique DNA methylation signature across multiple loci.Estimates of DNAmAge and tobacco smoking from DNA methylation data can be highly variable across different sample types collected from the same individual at the same time.While individual blood cell types did predict more of the variation in whole blood compared to buccal epithelial and nasal epithelial cells, the percentage of variance explained was still small.Instead our data indicate that at the majority of sites, variation in multiple blood cell types additively combines to drive variation in DNA methylation in whole blood.There are subset of sites where variable DNA methylation detected in whole blood can be attributed to variation in a single blood cell type.

Robust Method for Semantic Segmentation of Whole-Slide Blood Cell Microscopic Images

Previous works on segmentation of SEM (scanning electron microscope) blood cell image ignore the semantic segmentation approach of whole-slide blood cell segmentation. In the proposed work, we address the problem of whole-slide blood cell segmentation using the semantic segmentation approach. We design a novel convolutional encoder-decoder framework along with VGG-16 as the pixel-level feature extraction model. The proposed framework comprises 3 main steps: First, all the original images along with manually generated ground truth masks of each blood cell type are passed through the preprocessing stage. In the preprocessing stage, pixel-level labeling, RGB to grayscale conversion of masked image and pixel fusing, and unity mask generation are performed. After that, VGG16 is loaded into the system, which acts as a pretrained pixel-level feature extraction model. In the third step, the training process is initiated on the proposed model. We have evaluated our network performance on three evaluation metrics. We obtained outstanding results with respect to classwise, as well as global and mean accuracies. Our system achieved classwise accuracies of 97.45%, 93.34%, and 85.11% for RBCs, WBCs, and platelets, respectively, while global and mean accuracies remain 97.18% and 91.96%, respectively.

Nascent RNA sequencing of peripheral blood leukocytes reveal gene expression diversity

Nuclear Run-On sequencing is a powerful method to measure transcription with high resolution, sensitivity, and directional information, which provides alternative perspective from existing methods such as chromatin immunoprecipitation or mRNA sequencing. Current form of Nuclear Run-On assays such as Precision Run-On sequencing (PRO-seq) involves multiple RNA chemistry steps including RNA end repairs and ligations. These have limited the widespread use of PRO-seq by requiring robust RNA handling skills and multiple days of effort. To solve this, we developed an ultrashort PRO-seq (uPRO) method that requires minimal steps. In uPRO, the requirement of only two reactions - RNA adaptor ligation and template switch reverse transcription - reduced the procedure into less than a single day. Using uPRO, we generated genome-wide transcription profiles of human haploid cell lines (HAP1) and peripheral blood samples combined with Chromatin Run-On sequencing (pChRO). Blood cell handling procedure is dramatically reduced using pChRO directly on crude chromatin preparations, and enables utilizing archived specimens. As a result, we identified individual differences in the transcriptional profiles of human whole blood from a small volume (~1 ml). We also generated blood cell type specific transcription data, and deconvoluted the nucleated blood cell compositions by modeling to the reference datasets. Overall, uPRO and pChRO provided a powerful platform to identify differentially expressed genes between individuals with minimal sample requirements.

Glycan and lectin biosensors

A short description about the importance of glycan biorecognition in physiological (blood cell type) and pathological processes (infections by human and avian influenza viruses) is provided in this review. Glycans are described as much better information storage media, compared to proteins or DNA, due to the extensive variability of glycan structures. Techniques able to detect an exact glycan structure are briefly discussed with the main focus on the application of lectins (glycan-recognising proteins) in the specific analysis of glycans still attached to proteins or cells/viruses. Optical, electrochemical, piezoelectric and micromechanical biosensors with immobilised lectins or glycans able to detect a wide range of analytes including whole cells/viruses are also discussed.