Fibrotic scar tissue limits central nervous system regeneration in adult mammals. The extent of fibrotic tissue generation and distribution of stromal cells across different lesions in the brain and ...spinal cord has not been systematically investigated in mice and humans. Furthermore, it is unknown whether scar-forming stromal cells have the same origin throughout the central nervous system and in different types of lesions. In the current study, we compared fibrotic scarring in human pathological tissue and corresponding mouse models of penetrating and non-penetrating spinal cord injury, traumatic brain injury, ischemic stroke, multiple sclerosis and glioblastoma. We show that the extent and distribution of stromal cells are specific to the type of lesion and, in most cases, similar between mice and humans. Employing in vivo lineage tracing, we report that in all mouse models that develop fibrotic tissue, the primary source of scar-forming fibroblasts is a discrete subset of perivascular cells, termed type A pericytes. Perivascular cells with a type A pericyte marker profile also exist in the human brain and spinal cord. We uncover type A pericyte-derived fibrosis as a conserved mechanism that may be explored as a therapeutic target to improve recovery after central nervous system lesions.
Maple syrup urine disease (MSUD) is a rare inborn error of metabolism resulting from a deficiency in the branched-chain alpha-ketoacid dehydrogenase complex. MSUD has been reported to be the most ...common inborn error of metabolism in the Philippines. We described all patients with maple syrup urine disease patients diagnosed through newborn screening during its first 2 years of implementation and the challenges encountered during their medical management. There were 24 patients diagnosed with maple syrup urine disease for the 2-year period. All patients needed hospital admission. The most common complication during hospital admission was infection, needing intravenous antibiotics which were given to 21 of the patients. Out of the 24 diagnosed, 16 patients are alive, while eight have died. Several neurologic and non-neurologic complications have been observed during the follow-up of the patients. The common challenges of MSUD management in a low-resource setting identified in this study were late diagnosis, lack of access to metabolic specialists and medical supplies, nosocomial septicemia, and protein deficiency. Aside from early properly timed collection, improvement in other logistical concerns will also help in earlier diagnosis. Mechanisms of transfer of critically ill patients must be improved. Hospitals in difficult-to-reach areas must be equipped to handle critical metabolic cases when transfers are not possible. Newborn screening has been proven to improve outcome in patients with MSUD but the success of the program in preventing disability is also dependent on improvements in other aspects of healthcare.
Chromosome organization is crucial for genome function. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional ...models of chromosome organization. Using the super-resolution microscopy methods of OligoSTORM and OligoDNA-PAINT, we trace 8 megabases of human chromosome 19, visualizing structures ranging in size from a few kilobases to over a megabase. Focusing on chromosomal regions that contribute to compartments, we discover distinct structures that, in spite of considerable variability, can predict whether such regions correspond to active (A-type) or inactive (B-type) compartments. Imaging through the depths of entire nuclei, we capture pairs of homologous regions in diploid cells, obtaining evidence that maternal and paternal homologous regions can be differentially organized. Finally, using restraint-based modeling to integrate imaging and Hi-C data, we implement a method-integrative modeling of genomic regions (IMGR)-to increase the genomic resolution of our traces to 10 kb.
Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at ...nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology.
Pig-to-primate renal xenotransplantation is plagued by early antibody-mediated graft loss which precludes clinical application of renal xenotransplantation. We evaluated whether temporary complement ...inhibition with anti-C5 antibody Tesidolumab could minimize the impact of early antibody-mediated rejection in rhesus monkeys receiving pig kidneys receiving costimulatory blockade-based immunosuppression.
Double (Gal and Sda) and triple xenoantigen (Gal, Sda, and SLA I) pigs were created using CRISPR/Cas. Kidneys from DKO and TKO pigs were transplanted into rhesus monkeys that had the least reactive crossmatches. Recipients received anti-C5 antibody weekly for 70 days, and T cell depletion, anti-CD154, mycophenolic acid, and steroids as baseline immunosuppression (n = 7). Control recipients did not receive anti-C5 therapy (n = 10).
Temporary anti-C5 therapy reduced early graft loss secondary to antibody-mediated rejection and improved graft survival (P < 0.01). Deleting class I MHC (SLA I) in donor pigs did not ameliorate early antibody-mediated rejection (table). Anti-C5 therapy did not allow for the use of tacrolimus instead of anti-CD154 (table), prolonging survival to a maximum of 62 days.
Inhibition of the C5 complement subunit prolongs renal xenotransplant survival in a pig to non-human primate model.
Degeneration of central nervous system tissue commonly occurs during neuroinflammatory conditions, such as multiple sclerosis and neurotrauma. During such conditions, neural stem/progenitor cell ...(NPC) populations have been suggested to provide new cells to degenerated areas. In the normal brain, NPCs from the subventricular zone generate neurons that settle in the olfactory bulb or striatum. However, during neuroinflammatory conditions NPCs migrate toward the site of injury to form oligodendrocytes and astrocytes, whereas newly formed neurons are less abundant. Thus, the specific NPC lineage fate decisions appear to respond to signals from the local environment. The instructive signals from inflammation have been suggested to rely on excessive levels of the free radical nitric oxide (NO), which is an essential component of the innate immune response, as NO promotes neuronal to glial cell fate conversion of differentiating rat NPCs in vitro. Here, we demonstrate that the NO‐induced neuronal to glial fate conversion is dependent on the transcription factor neuron‐restrictive silencing factor‐1 (NRSF)/repressor element‐1 silencing transcription (REST). Chromatin modification status of a number of neuronal and glial lineage restricted genes was altered upon NO‐exposure. These changes coincided with gene expression alterations, demonstrating a global shift toward glial potential. Interestingly, by blocking the function of NRSF/REST, alterations in chromatin modifications were lost and the NO‐induced neuronal to glial switch was suppressed. This implicates NRSF/REST as a key factor in the NPC‐specific response to innate immunity and suggests a novel mechanism by which signaling from inflamed tissue promotes the formation of glial cells. Stem Cells 2014;32:2539–2549
Abstract
Background
Hi-C experiments couple DNA-DNA proximity with next-generation sequencing to yield an unbiased description of genome-wide interactions. Previous methods describing Hi-C ...experiments have focused on the industry-standard Illumina sequencing. With new next-generation sequencing platforms such as BGISEQ-500 becoming more widely available, protocol adaptations to fit platform-specific requirements are useful to give increased choice to researchers who routinely generate sequencing data.
Results
We describe an in situ Hi-C protocol adapted to be compatible with the BGISEQ-500 high-throughput sequencing platform. Using zebra finch (Taeniopygia guttata) as a biological sample, we demonstrate how Hi-C libraries can be constructed to generate informative data using the BGISEQ-500 platform, following circularization and DNA nanoball generation. Our protocol is a modification of an Illumina-compatible method, based around blunt-end ligations in library construction, using un-barcoded, distally overhanging double-stranded adapters, followed by amplification using indexed primers. The resulting libraries are ready for circularization and subsequent sequencing on the BGISEQ series of platforms and yield data similar to what can be expected using Illumina-compatible approaches.
Conclusions
Our straightforward modification to an Illumina-compatible in situHi-C protocol enables data generation on the BGISEQ series of platforms, thus expanding the options available for researchers who wish to utilize the powerful Hi-C techniques in their research.
Hydrogen peroxide (H2O2) is a reactive oxygen species that is involved in immunity and neuroinflammation. Here, we investigated whether and how pathophysiological levels of H2O2 influenced the ...differentiation of neural progenitor cells (NPCs). H2O2 levels within the range measured at neuroinflammatory events were applied to rat primary NPC cultures during 24 h, and effects were assessed directly after exposure or in NPCs that were differentiated for 7 days after H2O2 removal. Exposed differentiated NPCs showed significantly increased numbers of neurons and oligodendrocytes compared with unexposed controls. To identify the possible origin of this differentiation result, we characterized the undifferentiated culture and found a significant increase in both OLIG2(+) cells and proliferative ASCL1(+) C cells that could contribute to both more neurons and oligodendrocytes. In addition, H2O2-induced neurogenesis was supported by western blot and paralleled by gene expression analyses, which revealed an increased expression of the proneural gene Ngn2 and the neuronally expressed gene β-III tubulin. To investigate potential mechanisms for the observed effects on NPC differentiation, we performed gene expression profile analyses for oxidative stress and antioxidant-related and chromatin modification genes where the expression of several important genes was affected by the exposure. Increased oligodendrocyte numbers correlated with increased expression of the chromatin modification enzyme Sirt2, suggesting the involvement of Sirt2 in oligodendrocyte differentiation. Our results suggest a modulatory effect on the differentiation potential of NPCs by H2O2. Our findings indicate that H2O2 exposure has significant effects on NPC proliferation, differentiation, and vulnerability. These results have implications for regeneration after any neuroinflammatory event.
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