Organismal complexity, cell differentiation and gene expression: human over mouse

We present a molecular and cellular phenomenon underlying the intriguing increase in phenotypic organizational complexity. For the same set of human–mouse orthologous genes (11 534 gene pairs) and homologous tissues (32 tissue pairs), human shows a greater fraction of tissue-specific genes and a greater ratio of the total expression of tissue-specific genes to housekeeping genes in each studied tissue, which suggests a generally higher level of evolutionary cell differentiation (specialization). This phenomenon is spectacularly more pronounced in those human tissues that are more directly involved in the increase of complexity, longevity and body size (i.e. it is reflected on the organismal level as well). Genes with a change in expression breadth show a greater human–mouse divergence of promoter regions and encoded proteins (i.e. the functional genomics data are supported by the structural analysis). Human also shows the higher expression of translation machinery. The upstream untranslated regions (5′UTRs) of human mRNAs are longer than mouse 5′UTRs (even after correction for the difference in genome sizes) and contain more uAUG codons, which suggest a more complex regulation at the translational level in human cells (and agrees well with the augmented cell specialization).     

Multiplexed Component Analysis to Identify Genes Contributing to the Immune Response during Acute SIV Infection

Immune response genes play an important role during acute HIV and SIV infection. Using an SIV macaque model of AIDS and CNS disease, our overall goal was to assess how the expression of genes associated with immune and inflammatory responses are longitudinally changed in different organs or cells during SIV infection. To compare RNA expression of a panel of 88 immune-related genes across time points and among three tissues – spleen, mesenteric lymph nodes (MLN) and peripheral blood mononuclear cells (PBMC) – we designed a set of Nanostring probes. To identify significant genes during acute SIV infection and to investigate whether these genes are tissue-specific or have global roles, we introduce a novel multiplexed component analysis (MCA) method. This combines multivariate analysis methods with multiple preprocessing methods to create a set of 12 “judges”; each judge emphasizes particular types of change in gene expression to which cells could respond, for example, the absolute or relative size of expression change from baseline. Compared to bivariate analysis methods, our MCA method improved classification rates. This analysis allows us to identify three categories of genes: (a) consensus genes likely to contribute highly to the immune response; (b) genes that would contribute highly to the immune response only if certain assumptions are met – e.g. that the cell responds to relative expression change rather than absolute expression change; and (c) genes whose contribution to immune response appears to be modest. We then compared the results across the three tissues of interest; some genes are consistently highly-contributing in all tissues, while others are specific for certain tissues. Our analysis identified CCL8, CXCL10, CXCL11, MxA, OAS2, and OAS1 as top contributing genes, all of which are stimulated by type I interferon. This suggests that the cytokine storm during acute SIV infection is a systemic innate immune response against viral replication. Furthermore, these genes have approximately equal contributions to all tissues, making them possible candidates to be used as non-invasive biomarkers in studying PBMCs instead of MLN and spleen during acute SIV infection experiments. We identified clusters of genes that co-vary together and studied their correlation with regard to other gene clusters. We also developed novel methods to faithfully visualize multi-gene correlations on two-dimensional polar plots, and to visualize tissue specificity of gene expression responses.     

Transcriptional Profiling of Adult Neural Stem-Like Cells from the Human Brain

There is a great potential for the development of new cell replacement strategies based on adult human neural stem-like cells. However, little is known about the hierarchy of cells and the unique molecular properties of stem- and progenitor cells of the nervous system. Stem cells from the adult human brain can be propagated and expanded in vitro as free floating neurospheres that are capable of self-renewal and differentiation into all three cell types of the central nervous system. Here we report the first global gene expression study of adult human neural stem-like cells originating from five human subventricular zone biopsies (mean age 42, range 33–60). Compared to adult human brain tissue, we identified 1,189 genes that were significantly up- and down-regulated in adult human neural stem-like cells (1% false discovery rate). We found that adult human neural stem-like cells express stem cell markers and have reduced levels of markers that are typical of the mature cells in the nervous system. We report that the genes being highly expressed in adult human neural stem-like cells are associated with developmental processes and the extracellular region of the cell. The calcium signaling pathway and neuroactive ligand-receptor interactions are enriched among the most differentially regulated genes between adult human neural stem-like cells and adult human brain tissue. We confirmed the expression of 10 of the most up-regulated genes in adult human neural stem-like cells in an additional sample set that included adult human neural stem-like cells (n = 6), foetal human neural stem cells (n = 1) and human brain tissues (n = 12). The NGFR, SLITRK6 and KCNS3 receptors were further investigated by immunofluorescence and shown to be heterogeneously expressed in spheres. These receptors could potentially serve as new markers for the identification and characterisation of neural stem- and progenitor cells or as targets for manipulation of cellular fate.     

Variable Combinations of Specific Ephrin Ligand/Eph Receptor Pairs Control Embryonic Tissue Separation

Ephrins and Eph receptors are involved in the establishment of vertebrate tissue boundaries. The complexity of the system is puzzling, however in many instances, tissues express multiple ephrins and Ephs on both sides of the boundary, a situation that should in principle cause repulsion between cells within each tissue. Although co-expression of ephrins and Eph receptors is widespread in embryonic tissues, neurons, and cancer cells, it is still unresolved how the respective signals are integrated into a coherent output. We present a simple explanation for the confinement of repulsion to the tissue interface: Using the dorsal ectoderm–mesoderm boundary of the Xenopus embryo as a model, we identify selective functional interactions between ephrin–Eph pairs that are expressed in partial complementary patterns. The combined repulsive signals add up to be strongest across the boundary, where they reach sufficient intensity to trigger cell detachments. The process can be largely explained using a simple model based exclusively on relative ephrin and Eph concentrations and binding affinities. We generalize these findings for the ventral ectoderm–mesoderm boundary and the notochord boundary, both of which appear to function on the same principles. These results provide a paradigm for how developmental systems may integrate multiple cues to generate discrete local outcomes.     

Discovery of novel splice forms and functional analysis of cancer-specific alternative splicing in human expressed sequences

We report here a genome-wide analysis of alternative splicing in 2 million human expressed sequence tags (ESTs), to identify splice forms that are up-regulated in tumors relative to normal tissues. We found strong evidence (P < 0.01) of cancer-specific splice variants in 316 human genes. In total, 78% of the cancer-specific splice forms we detected are confirmed by human-curated mRNA sequences, indicating that our results are not due to random mis-splicing in tumors; 73% of the genes showed the same cancer-specific splicing changes in tissue-matched tumor versus normal datasets, indicating that the vast majority of these changes are associated with tumorigenesis, not tissue specificity. We have confirmed our EST results in an independent set of experimental data provided by human-curated mRNAs (P-value 10^–5.7). Moreover, the majority of the genes we detected have functions associated with cancer (P-value 0.0007), suggesting that their altered splicing may play a functional role in cancer. Analysis of the types of cancer-specific splicing shifts suggests that many of these shifts act by disrupting a tumor suppressor function. Sur prisingly, our data show that for a large number (190 in this study) of cancer-associated genes cloned originally from tumors, there exists a previously uncharacterized splice form of the gene that appears to be predominant in normal tissue.     

Dally Proteoglycan Mediates the Autonomous and Nonautonomous Effects on Tissue Growth Caused by Activation of the PI3K and TOR Pathways

How cells acquiring mutations in tumor suppressor genes outcompete neighboring wild-type cells is poorly understood. The phosphatidylinositol 3-kinase (PI3K)–phosphatase with tensin homology (PTEN) and tuberous sclerosis complex (TSC)-target of rapamycin (TOR) pathways are frequently activated in human cancer, and this activation is often causative of tumorigenesis. We utilized the Gal4-UAS system in Drosophila imaginal primordia, highly proliferative and growing tissues, to analyze the impact of restricted activation of these pathways on neighboring wild-type cell populations. Activation of these pathways leads to an autonomous induction of tissue overgrowth and to a remarkable nonautonomous reduction in growth and proliferation rates of adjacent cell populations. This nonautonomous response occurs independently of where these pathways are activated, is functional all throughout development, takes place across compartments, and is distinct from cell competition. The observed autonomous and nonautonomous effects on tissue growth rely on the up-regulation of the proteoglycan Dally, a major element involved in modulating the spreading, stability, and activity of the growth promoting Decapentaplegic (Dpp)/transforming growth factor β(TGF-β) signaling molecule. Our findings indicate that a reduction in the amount of available growth factors contributes to the outcompetition of wild-type cells by overgrowing cell populations. During normal development, the PI3K/PTEN and TSC/TOR pathways play a major role in sensing nutrient availability and modulating the final size of any developing organ. We present evidence that Dally also contributes to integrating nutrient sensing and organ scaling, the fitting of pattern to size.     

Exposure to non-ionizing radiation provokes changes in rat thyroid morphology and expression of HSP-90

Non-ionizing radiation at 2.45 GHz may modify the morphology and expression of genes that codify heat shock proteins (HSP) in the thyroid gland. Diathermy is the therapeutic application of non-ionizing radiation to humans for its beneficial effects in rheumatological and musculo-skeletal pain processes. We used a diathermy model on laboratory rats subjected to maximum exposure in the left front leg, in order to study the effects of radiation on the nearby thyroid tissue. Fifty-six rats were individually exposed once or repeatedly (10 times in two weeks) for 30 min to 2.45 GHz radiation in a commercial chamber at different non-thermal specific absorption rates (SARs), which were calculated using the finite difference time domain technique. We used immunohistochemistry methods to study the expression of HSP-90 and morphological changes in thyroid gland tissues. Ninety minutes after radiation with the highest SAR, the central and peripheral follicles presented increased size and the thickness of the peripheral septa had decreased. Twenty-four hours after radiation, only peripheral follicles radiated at 12 W were found to be smaller. Peripheral follicles increased in size with repeated exposure at 3 W power. Morphological changes in the thyroid tissue may indicate a glandular response to acute or repeated stress from radiation in the hypothalamic–pituitary–thyroid axis. Further research is needed to determine if the effect of this physical agent over time may cause disease in the human thyroid gland.     

Biological and molecular profile of fracture non‐union tissue: A systematic review and an update on current insights

Fracture non‐union represents a common complication, seen in 5%–10% of all acute fractures. Despite the enhancement in scientific understanding and treatment methods, rates of fracture non‐union remain largely unchanged over the years. This systematic review investigates the biological, molecular and genetic profiles of both (i) non‐union tissue and (ii) non–union‐related tissues, and the genetic predisposition to fracture non‐union. This is crucially important as it could facilitate earlier identification and targeted treatment of high‐risk patients, along with improving our understanding on pathophysiology of fracture non‐union. Since this is an update on our previous systematic review, we searched the literature indexed in PubMed Medline; Ovid Medline; Embase; Scopus; Google Scholar; and the Cochrane Library using Medical Subject Heading (MeSH) or Title/Abstract words (non‐union(s), non‐union(s), human, tissue, bone morphogenic protein(s) (BMPs) and MSCs) from August 2014 (date of our previous publication) to 2 October 2021 for non‐union tissue studies, whereas no date restrictions imposed on non–union‐related tissue studies. Inclusion criteria of this systematic review are human studies investigating the characteristics and properties of non‐union tissue and non–union‐related tissues, available in full‐text English language. Limitations of this systematic review are exclusion of animal studies, the heterogeneity in the definition of non‐union and timing of tissue harvest seen in the included studies, and the search term MSC which may result in the exclusion of studies using historical terms such as ‘osteoprogenitors’ and ‘skeletal stem cells’. A total of 24 studies (non‐union tissue: n = 10; non–union‐related tissues: n = 14) met the inclusion criteria. Soft tissue interposition, bony sclerosis of fracture ends and complete obliteration of medullary canal are commonest macroscopic appearances of non‐unions. Non‐union tissue colour and surrounding fluid are two important characteristics that could be used clinically to distinguish between septic and aseptic non‐unions. Atrophic non‐unions had a predominance of endochondral bone formation and lower cellular density, when compared against hypertrophic non‐unions. Vascular tissues were present in both atrophic and hypertrophic non‐unions, with no difference in vessel density between the two. Studies have found non‐union tissue to contain biologically active MSCs with potential for osteoblastic, chondrogenic and adipogenic differentiation. Proliferative capacity of non‐union tissue MSCs was comparable to that of bone marrow MSCs. Rates of cell senescence of non‐union tissue remain inconclusive and require further investigation. There was a lower BMP expression in non‐union site and absent in the extracellular matrix, with no difference observed between atrophic and hypertrophic non‐unions. The reduced BMP‐7 gene expression and elevated levels of its inhibitors (Chordin, Noggin and Gremlin) could potentially explain impaired bone healing observed in non‐union MSCs. Expression of Dkk‐1 in osteogenic medium was higher in non‐union MSCs. Numerous genetic polymorphisms associated with fracture non‐union have been identified, with some involving the BMP and MMP pathways. Further research is required on determining the sensitivity and specificity of molecular and genetic profiling of relevant tissues as a potential screening biomarker for fracture non‐unions.     

Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.