Exploring DNA methylation patterns in copper exposed Folsomia candida and Enchytraeus crypticus

Accumulating evidence shows that epigenetics-mediated phenotypic plasticity plays a role in an organism’s ability to deal with environmental stress. However, to date, the role of epigenetic modifications in response to stress is hardly investigated in soil invertebrates. The main objective of this proof of principle study was to explore whether total cytosine and locus-specific CpG methylation are present in two important ecotoxicological model organisms, the springtail Folsomia candida and the potworm Enchytraeus crypticus, and if so, whether methylation patterns might change with increased toxicant exposure. LC-MS/MS analyses and bisulfite sequencing were performed to identify the CpG methylation state of the organisms. We show here, for the first time, a total level of 1.4% 5-methyl cytosine methylation in the genome of E. crypticus, and an absence of both total cytosine and locus-specific CpG methylation in F. candida. In E. crypticus, methylation of CpG sites was observed in the coding sequence (CDS) of the housekeeping gene Elongation Factor 1α, while the CDS of the stress inducible Heat Shock Protein 70 gene almost lacked methylation. This confirms previous observations that DNA methylation differs between housekeeping and stress-inducible genes in invertebrates. DNA methylation patterns in E. crypticus were not affected by exposure to copper (II) sulfate pentahydrate (CuSO4·5H2O) mixed in with LUFA 2.2 soil at sublethal effect concentrations that decreased reproduction by 10%, 20% and 50%. Although, differences in CpG methylation patterns between specific loci suggest a functional role for DNA methylation in E. crypticus, genome-wide bisulfite sequencing is needed to verify whether environmental stress affects this epigenetic hallmark

P-Glycoprotein Acts as an Immunomodulator during Neuroinflammation

Background: Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system in which autoreactive myelin-specific T cells cause extensive tissue damage, resulting in neurological deficits. In the disease process, T cells are primed in the periphery by antigen presenting dendritic cells (DCs). DCs are considered to be crucial regulators of specific immune responses and molecules or proteins that regulate DC function are therefore under extensive investigation. We here investigated the potential immunomodulatory capacity of the ATP binding cassette transporter P-glycoprotein (Pgp). P-gp generally drives cellular efflux of a variety of compounds and is thought to be involved in excretion of inflammatory agents from immune cells, like DCs. So far, the immunomodulatory role of these ABC transporters is unknown. Methods and Findings: Here we demonstrate that P-gp acts as a key modulator of adaptive immunity during an in vivo model for neuroinflammation. The function of the DC is severely impaired in P-gp knockout mice (Mdr1a/1b-/-), since both DC maturation and T cell stimulatory capacity is significantly decreased. Consequently, Mdr1a/1b-/- mice develop decreased clinical signs of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Reduced clinical signs coincided with impaired T cell responses and T cell-specific brain inflammation. We here describe the underlying molecular mechanism and demonstrate that P-gp is crucial for the secretion of pro-inflammatory cytokines such as TNF-alpha and IFN-gamma. Importantly, the defect in DC function can be restored by exogenous addition of these cytokines. Conclusions: Our data demonstrate that P-gp downmodulates DC function through the regulation of pro-inflammatory cytokine secretion, resulting in an impaired immune response. Taken together, our work highlights a new physiological role for P-gp as an immunomodulatory molecule and reveals a possible new target for immunotherap

A molecular map of murine lymph node blood vascular endothelium at single cell resolution

Blood vascular endothelial cells (BECs) control the immune response by regulating blood flow and immune cell recruitment in lymphoid tissues. However, the diversity of BEC and their origins during immune angiogenesis remain unclear. Here we profile transcriptomes of BEC from peripheral lymph nodes and map phenotypes to the vasculature. We identify multiple subsets, including a medullary venous population whose gene signature predicts a selective role in myeloid cell (vs lymphocyte) recruitment to the medulla, confirmed by videomicroscopy. We define five capillary subsets, including a capillary resident precursor (CRP) that displays stem cell and migratory gene signatures, and contributes to homeostatic BEC turnover and to neogenesis of high endothelium after immunization. Cell alignments show retention of developmental programs along trajectories from CRP to mature venous and arterial populations. Our single cell atlas provides a molecular roadmap of the lymph node blood vasculature and defines subset specialization for leukocyte recruitment and vascular homeostasis

Next-generation sequencing-based genome diagnostics across clinical genetics centers: Implementation choices and their effects

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care

Antenatal treatment in two Dutch families with pyridoxine-dependent seizures

Contains fulltext : 88199.pdf (publisher's version ) (Closed access)Incidental reports suggest that antenatal treatment of pyridoxine dependent seizures (PDS) may improve neurodevelopmental outcome of affected patients. Two families with PDS are reported, both with two affected siblings. Antenatal treatment with pyridoxine was instituted during the second pregnancy in each family (50 and 60 mg daily from 3 and 10 weeks of gestation, respectively). Perinatal characteristics and neurodevelopmental outcome at 4 (Family A) and 12 (Family B) years of age were compared between the untreated and treated child within each family. Meconium-stained amniotic fluid was present in both first pregnancies and abnormal foetal movements were noticed in one. In the treated infants, pregnancy and birth were uncomplicated. In family A, postnatal pyridoxine supplementation prevented neonatal seizures. Both children in family A were hypotonic and started walking after 2 years of age; both had white matter changes on MRI, and the first child was treated for squint. IQ was 73 and 98 in the antenatally untreated and treated child, respectively. The second child in family B developed seizures on the seventh day, because pyridoxine maintenance therapy had not been instituted after birth. Seizures responded rapidly to pyridoxine supplementation. MRI showed large ventricles and a mega cisterna magna. IQ was 80 and 106 in the antenatally untreated and treated child respectively. Both children had normal motor development. These results suggest that antenatal pyridoxine supplementation may be effective in preventing intrauterine seizures, decreasing the risk of complicated birth and improving neurodevelopmental outcome in PDS.1 maart 201

Stromal cells and immune cells involved in formation of lymph nodes and their niches

Secondary lymphoid organs are critical for efficient interaction between innate antigen presenting cells and adaptive lymphocytes in order to start adaptive immune responses. The efficiency by which these cellular subsets meet is highly increased by the orchestrating role of stromal cells within the secondary lymphoid organs. These cells provide cytokines, chemokines and cell surface receptors necessary for survival and guided migration. This increases the likelihood that antigen specific adaptive immune responses occur. Already from initial formation of secondary lymphoid organs, the interaction of immune cells with stromal cells is crucial and this interaction continues during immune activation. With the recent discovery of many stromal cell subsets new immune micro-niches with specific functions that are orchestrated by stromal cells will be discovered. Here, we will discuss how the development of lymph nodes as well as their specific niches is guided by the interaction of immune cells and stromal cells

The influence of Added Mass on Optimal Step Length in Running

Purpose: To examine the influence of induced changes in the morphology of the leg by adding mass on the optimal step length (OSL) in experienced runners to get more insight into parameters that influence preferred step length (PSL) and OSL. Methods: Thirteen experienced male runners (mean age 26.9 ± 6.1 y, height 183.7 ± 7.1 cm, mass 71.8 ± 5.9 kg) ran on a treadmill in 3 different conditions: unloaded (UL), loaded with 2 kg mass at the ankles (MA), and loaded with 2 kg mass at the hips (MH) at 7 different step lengths (SLs). SL deviations were expressed as deviations in relative leg length (%LL) from the individual PSL: 0%LL, ±5%LL, ±10%LL, and ±15%LL. Trials lasted 8 min, and 8 min of rest was given between trials. Oxygen uptake (V̇O2) was expressed as a fraction of V̇O2 at PSL + 0%LL in the unloaded condition (%V̇O2). The %SL with the lowest value of %V̇O2 was considered the OSL for this group of participants. Results: OSL at the UL condition was 6% shorter than PSL. The MA condition resulted in a 7%LL larger OSL than at UL and MH (P < .05). Conlusions: The mass distribution of the leg is a determinant of the OSL. As a consequence of the added mass to the ankles, OSL was 7%LL longer. Morphological characteristics of the leg might therefore play an important role in determining the runner’s individual optimal SL

The Role of Endothelial Cells and TNF-Receptor Superfamily Members in Lymphoid Organogenesis and Function During Health and Inflammation

Lymph nodes (LNs) are crucial for the orchestration of immune responses. LN reactions depend on interactions between incoming and local immune cells, and stromal cells. To mediate these cellular interactions an organized vascular network within the LN exists. In general, the LN vasculature can be divided into two components: blood vessels, which include the specialized high endothelial venules that recruit lymphocytes from the bloodstream, and lymphatic vessels. Signaling via TNF receptor (R) superfamily (SF) members has been implicated as crucial for the development and function of LNs and the LN vasculature. In recent years the role of cell-specific signaling of TNFRSF members in different endothelial cell (EC) subsets and their roles in development and maintenance of lymphoid organs has been elucidated. Here, we discuss recent insights into EC-specific TNFRSF member signaling and highlight its importance in different EC subsets in LN organogenesis and function during health, and in lymphocyte activation and tertiary lymphoid structure formation during inflammation

The Role of Endothelial Cells and TNF-Receptor Superfamily Members in Lymphoid Organogenesis and Function During Health and Inflammation

Lymph nodes (LNs) are crucial for the orchestration of immune responses. LN reactions depend on interactions between incoming and local immune cells, and stromal cells. To mediate these cellular interactions an organized vascular network within the LN exists. In general, the LN vasculature can be divided into two components: blood vessels, which include the specialized high endothelial venules that recruit lymphocytes from the bloodstream, and lymphatic vessels. Signaling via TNF receptor (R) superfamily (SF) members has been implicated as crucial for the development and function of LNs and the LN vasculature. In recent years the role of cell-specific signaling of TNFRSF members in different endothelial cell (EC) subsets and their roles in development and maintenance of lymphoid organs has been elucidated. Here, we discuss recent insights into EC-specific TNFRSF member signaling and highlight its importance in different EC subsets in LN organogenesis and function during health, and in lymphocyte activation and tertiary lymphoid structure formation during inflammation