Comparison and development of cross-study normalization methods for inter-species transcriptional analysis.

Performing joint analysis of gene expression datasets from different experiments can present challenges brought on by multiple factors-differences in equipment, protocols, climate etc. "Cross-study normalization" is a general term for transformations aimed at eliminating such effects, thus making datasets more comparable. However, joint analysis of datasets from different species is rarely done, and there are no dedicated normalization methods for such inter-species analysis. In order to test the usefulness of cross-studies normalization methods for inter-species analysis, we first applied three cross-study normalization methods, EB, DWD and XPN, to RNA sequencing datasets from different species. We then developed a new approach to evaluate the performance of cross-study normalization in eliminating experimental effects, while also maintaining the biologically significant differences between species and conditions. Our results indicate that all normalization methods performed relatively well in the cross-species setting. We found XPN to be better at reducing experimental differences, and found EB to be better at preserving biological differences. Still, according to our in-silico experiments, in all methods it is not possible to enforce the preservation of the biological differences in the normalization process. In addition to the study above, in this work we propose a new dedicated cross-studies and cross-species normalization method. Our aim is to address the shortcoming mentioned above: in the normalization process, we wish to reduce the experimental differences while preserving the biological differences. We term our method as CSN, and base it on the performance evaluation criteria mentioned above. Repeating the same experiments, the CSN method obtained a better and more balanced conservation of biological differences within the datasets compared to existing methods. To summarize, we demonstrate the usefulness of cross-study normalization methods in the inter-species settings, and suggest a dedicated cross-study cross-species normalization method that will hopefully open the way to the development of improved normalization methods for the inter-species settings.

Microheterogeneity in the Kinetics and Sex-Specific Response to Type I IFN.

The response to type I IFNs involves the rapid induction of prototypical IFN signature genes (ISGs). It is not known whether the tightly controlled ISG expression observed at the cell population level correctly represents the coherent responses of individual cells or whether it masks some heterogeneity in gene modules and/or responding cells. We performed a time-resolved single-cell analysis of the first 3 h after in vivo IFN stimulation in macrophages and CD4+ T and B lymphocytes from mice. All ISGs were generally induced in concert, with no clear cluster of faster- or slower-responding ISGs. Response kinetics differed between cell types: mostly homogeneous for macrophages, but with far more kinetic diversity among B and T lymphocytes, which included a distinct subset of nonresponsive cells. Velocity analysis confirmed the differences between macrophages in which the response progressed throughout the full 3 h, versus B and T lymphocytes in which it was rapidly curtailed by negative feedback and revealed differences in transcription rates between the lineages. In all cell types, female cells responded faster than their male counterparts. The ISG response thus seems to proceed as a homogeneous gene block, but with kinetics that vary between immune cell types and with sex differences that might underlie differential outcomes of viral infections.

A telencephalon cell type atlas for goldfish reveals diversity in the evolution of spatial structure and cell types.

Teleost fish form the largest group of vertebrates and show a tremendous variety of adaptive behaviors, making them critically important for the study of brain evolution and cognition. The neural basis mediating these behaviors remains elusive. We performed a systematic comparative survey of the goldfish telencephalon. We mapped cell types using single-cell RNA sequencing and spatial transcriptomics, resulting in de novo molecular neuroanatomy parcellation. Glial cells were highly conserved across 450 million years of evolution separating mouse and goldfish, while neurons showed diversity and modularity in gene expression. Specifically, somatostatin interneurons, famously interspersed in the mammalian isocortex for local inhibitory input, were curiously aggregated in a single goldfish telencephalon nucleus but molecularly conserved. Cerebral nuclei including the striatum, a hub for motivated behavior in amniotes, had molecularly conserved goldfish homologs. We suggest elements of a hippocampal formation across the goldfish pallium. Last, aiding study of the teleostan everted telencephalon, we describe substantial molecular similarities between goldfish and zebrafish neuronal taxonomies.

Genetics of Sex Differences in Immunity.

Women have a stronger immune response and a higher frequency of most autoimmune diseases than men. While much of the difference between men and women is due to the effect of gonadal hormones, genetic differences play a major role in the difference between the immune response and disease frequencies in women and men. Here, we focus on the immune differences between the sexes that are not downstream of the gonadal hormones. These differences include the gene content of the sex chromosomes, the inactivation of chromosome X in women, the consequences of non-random X inactivation and escape from inactivation, and the states that are uniquely met by the immune system of women-pregnancy, birth, and breast feeding. While these female-specific states are temporary and involve gonadal hormonal changes, they may leave a long-lasting footprint on the health of women, for example, by fetal cells that remain in the mother's body for decades. We also briefly discuss the immune phenotype of congenital sex chromosomal aberrations and experimental models that enable hormonal and the non-hormonal effects of the sex chromosomes to be disentangled. The increasing human life expectancy lengthens the period during which gonadal hormones levels are reduced in both sexes. A better understanding of the non-hormonal effects of sex chromosomes thus becomes more important for improving the life quality during that period.

Mapping the splicing landscape of the human immune system.

Most human genes code for more than one transcript. Different ratios of transcripts of the same gene can be found in different cell types or states, indicating differential use of transcription start sites or differential splicing. Such differential transcript use (DTUs) events provide an additional layer of regulation and protein diversity. With the exceptions of PTPRC and CIITA, there are very few reported cases of DTU events in the immune system. To rigorously map DTUs between different human immune cell types, we leveraged four publicly available RNA sequencing datasets. We identified 282 DTU events between five human healthy immune cell types that appear in at least two datasets. The patterns of the DTU events were mostly cell-type-specific or lineage-specific, in the context of the five cell types tested. DTUs correlated with the expression pattern of potential regulators, namely, splicing factors and transcription factors. Of the several immune related conditions studied, only sepsis affected the splicing of more than a few genes and only in innate immune cells. Taken together, we map the DTUs landscape in human peripheral blood immune cell types, and present hundreds of genes whose transcript use changes between cell types or upon activation.

Characterization of the Environmental Plasmidome of the Red Sea.

Plasmids contribute to microbial diversity and adaptation, providing microorganisms with the ability to thrive in a wide range of conditions in extreme environments. However, while the number of marine microbiome studies is constantly increasing, very little is known about marine plasmids, and they are very poorly represented in public databases. To extend the repertoire of environmental marine plasmids, we established a pipeline for the de novo assembly of plasmids in the marine environment by analyzing available microbiome metagenomic sequencing data. By applying the pipeline to data from the Red Sea, we identified 362 plasmid candidates. We showed that the distribution of plasmids corresponds to environmental conditions, particularly, depth, temperature, and physical location. At least 7 of the 362 candidates are most probably real plasmids, based on a functional analysis of their open reading frames (ORFs). Only one of the seven has been described previously. Three plasmids were identified in other public marine metagenomic data from different locations all over the world; these plasmids contained different cassettes of functional genes at each location. Analysis of antibiotic and metal resistance genes revealed that the same positions that were enriched with genes encoding resistance to antibiotics were also enriched with resistance to metals, suggesting that plasmids contribute site-dependent phenotypic modules to their ecological niches. Finally, half of the ORFs (50.8%) could not be assigned to a function, emphasizing the untapped potential of the unique marine plasmids to provide proteins with multiple novel functions. IMPORTANCE Marine plasmids are understudied and hence underrepresented in databases. Plasmid functional annotation and characterization is complicated but, if successful, may provide a pool of novel genes and unknown functions. Newly discovered plasmids and their functional repertoire are potentially valuable tools for predicting the dissemination of antimicrobial resistance, providing vectors for molecular cloning and an understanding of plasmid-bacterial interactions in various environments.

The interweaved signatures of common-gamma-chain cytokines across immunologic lineages.

"γc" cytokines are a family whose receptors share a "common-gamma-chain" signaling moiety, and play central roles in differentiation, homeostasis, and communications of all immunocyte lineages. As a resource to better understand their range and specificity of action, we profiled by RNAseq the immediate-early responses to the main γc cytokines across all immunocyte lineages. The results reveal an unprecedented landscape: broader, with extensive overlap between cytokines (one cytokine doing in one cell what another does elsewhere) and essentially no effects unique to any one cytokine. Responses include a major downregulation component and a broad Myc-controlled resetting of biosynthetic and metabolic pathways. Various mechanisms appear involved: fast transcriptional activation, chromatin remodeling, and mRNA destabilization. Other surprises were uncovered: IL2 effects in mast cells, shifts between follicular and marginal zone B cells, paradoxical and cell-specific cross-talk between interferon and γc signatures, or an NKT-like program induced by IL21 in CD8+ T cells.

The transcriptional landscape of the giant freshwater prawn: Embryonic development and early sexual differentiation mechanisms.

The giant freshwater prawn pjMacrobrachium rosenbergii is one of the best studied species in aquaculture. However, the transcriptional changes associated with embryonic development and the sexual differentiation mechanism of M. rosenbergii remain to be elucidated. To characterize the embryonic development of this prawn and to determine whether differential expression and differential splicing play roles in the early sexual differentiation of M. rosenbergii, we profiled five developmental days of male and female embryos by RNA sequencing. We identified modules of co-expressed genes representing waves of transcription that correspond to physiological processes in early embryonic development (such as the maternal-to-zygotic transition) up to preparation for life outside the egg (development of muscles, cuticle etc.). Additionally, we found that hundreds of genes are differentially expressed between sexes, most of them uncharacterized, suggesting that the sex differentiation mechanism of M. rosenbergii might contain clade-specific elements. The resulting first-of-a-kind transcriptional map of embryonic development of male and female M. rosenbergii will guide future studies to reveal the roles of specific genes and splicing isoforms in the embryonic development and sexual differentiation process of M. rosenbergii.

Immune Sexual Dimorphism: Connecting the Dots.

Despite numerous studies of immune sexual dimorphism, sexual differences are not rigorously mapped and dimorphic mechanisms are incompletely understood. Current immune research typically studies sex differences in specific cells, tissues, or diseases but without providing an integrated picture. To connect the dots, we suggest comprehensive research approaches to better our understanding of immune sexual dimorphism and its mechanisms.

DoChaP: the domain change presenter.

Alternative splicing results in multiple transcripts of the same gene, possibly encoding for different protein isoforms with different domains. Whereas it is possible to manually determine the effect of alternative splicing on the domain composition for a single event, the process requires the tedious integration of several data sources; it is error prone and not feasible for genome-wide characterization of domains affected by differential splicing. To fulfill the need for an automated solution, we developed the Domain Change Presenter (DoChaP, https://dochap.bgu.ac.il/), a web server for the visualization of exon-domain associations. DoChaP visualizes all transcripts of a given gene, the encoded proteins and their domains, and enables a comparison between the transcripts and between their protein products. The colors and organization make the structural effect of alternative splicing events on protein structures easily identified. To enable the study of the conservation of exons structure, alternative splicing, and the effect of alternative splicing on protein domains, DoChaP also provides a two-species comparison of exon-domain associations. DoChaP thus provides a unique and easy-to-use visualization of the exon-domain association and conservation, and will facilitate the study of the structural effects of alternative splicing in health and disease.