Adult monozygotic twins discordant for intra-uterine growth have indistinguishable genome-wide DNA methylation profiles

Background

Low birth weight is associated with an increased adult metabolic disease risk. It is widely discussed that poor intra-uterine conditions could induce long-lasting epigenetic modifications, leading to systemic changes in regulation of metabolic genes. To address this, we acquire genome-wide DNA methylation profiles from saliva DNA in a unique cohort of 17 monozygotic monochorionic female twins very discordant for birth weight. We examine if adverse prenatal growth conditions experienced by the smaller co-twins lead to long-lasting DNA methylation changes.

Results

Overall, co-twins show very similar genome-wide DNA methylation profiles. Since observed differences are almost exclusively caused by variable cellular composition, an original marker-based adjustment strategy was developed to eliminate such variation at affected CpGs. Among adjusted and unchanged CpGs 3,153 are differentially methylated between the heavy and light co-twins at nominal significance, of which 45 show sensible absolute mean β-value differences. Deep bisulfite sequencing of eight such loci reveals that differences remain in the range of technical variation, arguing against a reproducible biological effect. Analysis of methylation in repetitive elements using methylation-dependent primer extension assays also indicates no significant intra-pair differences.

Conclusions

Severe intra-uterine growth differences observed within these monozygotic twins are not associated with long-lasting DNA methylation differences in cells composing saliva, detectable with up-to-date technologies. Additionally, our results indicate that uneven cell type composition can lead to spurious results and should be addressed in epigenomic studies.     

Two closely related human members of chitinase-like family, CHI3L1 and CHI3L2, activate ERK1/2 in 293 and U373 cells but have the different influence on cell proliferation

The activation of extracellular signal-regulated kinases (ERK1/2) has been associated with specific outcomes. Sustained activation of ERK1/2 by nerve growth factor (NGF) is associated with translocation of ERKs to the nucleus of PC12 cells and precedes their differentiation into sympathetic-like neurons whereas transient activation by epidermal growth factor (EGF) leads to cell proliferation. It was demonstrated that different growth factors initiating the same cellular signaling pathways may lead to the different cell destiny, either to proliferation or to the inhibition of mitogenesis and apoptosis. Thus, further investigation on kinetic differences in activation of certain signal cascades in different cell types by biologically different agents are necessary for understanding the mechanisms as to how cells make a choice between proliferation and differentiation.It was reported that chitinase 3-like 1 (CHI3L1) protein promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts similarly to insulin-like growth factor 1 (IGF1). Both are involved in mediating the mitogenic response through the signal-regulated kinases ERK1/2. In addition, CHI3L1 which is highly expressed in different tumors including glioblastomas possesses oncogenic properties. As we found earlier, chitinase 3-like 2 (CHI3L2) most closely related to human CHI3L1 also showed increased expression in glial tumors at both the RNA and protein levels and stimulated the activation of the MAPK pathway through phosphorylation of ERK1/2 in 293 and U87 MG cells. The work described here demonstrates the influence of CHI3L2 and CHI3L1 on the duration of MAPK cellular signaling and phosphorylated ERK1/2 translocation to the nucleus. In contrast to the activation of ERK1/2 phosphorylation by CHI3L1 that leads to a proliferative signal (similar to the EGF effect in PC12 cells), activation of ERK1/2 phosphorylation by CHI3L2 (similar to NGF) inhibits cell mitogenesis and proliferation.     

Border Patrol Gone Awry: Lung NKT Cell Activation by Francisella tularensis Exacerbates Tularemia-Like Disease

The respiratory mucosa is a major site for pathogen invasion and, hence, a site requiring constant immune surveillance. The type I, semi-invariant natural killer T (NKT) cells are enriched within the lung vasculature. Despite optimal positioning, the role of NKT cells in respiratory infectious diseases remains poorly understood. Hence, we assessed their function in a murine model of pulmonary tularemia—because tularemia is a sepsis-like proinflammatory disease and NKT cells are known to control the cellular and humoral responses underlying sepsis. Here we show for the first time that respiratory infection with Francisella tularensis live vaccine strain resulted in rapid accumulation of NKT cells within the lung interstitium. Activated NKT cells produced interferon-γ and promoted both local and systemic proinflammatory responses. Consistent with these results, NKT cell-deficient mice showed reduced inflammatory cytokine and chemokine response yet they survived the infection better than their wild type counterparts. Strikingly, NKT cell-deficient mice had increased lymphocytic infiltration in the lungs that organized into tertiary lymphoid structures resembling induced bronchus-associated lymphoid tissue (iBALT) at the peak of infection. Thus, NKT cell activation by F. tularensis infection hampers iBALT formation and promotes a systemic proinflammatory response, which exacerbates severe pulmonary tularemia-like disease in mice.     

Serotyping of Toxoplasma gondii in Cats (Felis domesticus) Reveals Predominance of Type II Infections in Germany

Background

Cats are definitive hosts of Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany.

Methodology

To establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7).

Findings

Positive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III.

Conclusions

Cats are able to mount a clonal type-specific antibody response against T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany.     

Computing thermomechanical properties of dry homopolymers used as raw materials for formulation of biomedical hydrogels

Different static properties have been calculated with COMPASS force field for polyacrylamide, poly(2-hydroxyethylacrylate) (HEA), poly(2-hydroxyethylmethacrylate) (HEMA), poly(glycidylmethacrylate) (GMA), polyethylene glycol (PEG), and poly(2,2,2-trifluoroethylmethacrylate) (TFEM). For each polymers, the calculated values were averaged on five equilibrated configurations of amorphous cell composed of one atactic chain containing 100 repeat units. The ranking obtained from the densities calculated at 300 K is TFEM?>?HEA?≈?xpolycrylamide?>?HEMA?≈?GMA?>?PEG. Concerning the glass transition temperature we have obtained polyacrylamide?>?HEMA?≈?GMA?≈?HEA?>?PEG, and polyacrylamide?>?HEMA?≈?HEA?>?GMA?≈?PEG?>?TFEM for the bulk modulus. The calculated results, when available, have been compared with experimental data coming from literature.     

Bi-PROF

The use of next generation sequencing has expanded our view on whole mammalian methylome patterns. In particular, it provides a genome-wide insight of local DNA methylation diversity at single nucleotide level and enables the examination of single chromosome sequence sections at a sufficient statistical power. We describe a bisulfite-based sequence profiling pipeline, Bi-PROF, which is based on the 454 GS-FLX Titanium technology that allows to obtain up to one million sequence stretches at single base pair resolution without laborious subcloning. To illustrate the performance of the experimental workflow connected to a bioinformatics program pipeline (BiQ Analyzer HT) we present a test analysis set of 68 different epigenetic marker regions (amplicons) in five individual patient-derived xenograft tissue samples of colorectal cancer and one healthy colon epithelium sample as a control. After the 454 GS-FLX Titanium run, sequence read processing and sample decoding, the obtained alignments are quality controlled and statistically evaluated. Comprehensive methylation pattern interpretation (profiling) assessed by analyzing 10²-10⁴ sequence reads per amplicon allows an unprecedented deep view on pattern formation and methylation marker heterogeneity in tissues concerned by complex diseases like cancer.     

The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers

The pregnancy-associated glycoproteins (PAGs) are a large gene family expressed in trophoblast cells of ruminant ungulates. The detection of PAGs (more specifically, PAG-1) in maternal serum has served as the basis for pregnancy detection in cattle. Unfortunately, PAG-1 and/or antigenically-related PAGs exhibit a long half-life in maternal serum (>8 d) and can be detected 80-100 d post-partum, thereby producing false positives in animals bred within 60-d of calving. The goal of the present studies was to develop a monoclonal-based assay that targeted early-pregnancy PAGs whose persistence in maternal serum post-partum might be relatively short-lived. Three anti-PAG monoclonal antibodies that recognized distinct subsets of PAGs were selected and used as trapping reagents in a 'sandwich' type of enzyme-linked immunosorbant assay (ELISA). A polyclonal antiserum with broad specificity was used for detecting bound PAGs. A total of 42 cows and heifers were bled daily on day 15, days 22 to 28, and then weekly throughout pregnancy and for 10 weeks (approximately 70 d) into the post-partum period. The ELISA was able to detect PAG in maternal serum of all animals unambiguously by day 28 post-insemination (PAG concentration: 8.75 +/- 3.04 ng/mL). In maternal serum, PAG concentrations peaked during the week of parturition at 588.9 +/- 249.9 ng/mL, and after calving, PAG was completely cleared (half-life: 4.3 d) by eight-week post-partum in 38 of 40 of the animals tested and was at very low concentrations in the remaining two (1.4 and 4.9 ng/mL, respectively). In summary, a monoclonal-based assay has been established that is sensitive enough to detect PAG in maternal serum by the forth week of pregnancy, but does not suffer from carry-over of antigen from a previous pregnancy.