An exceptional muscle development commonly referred to as 'double-muscled' (Fig. 1) has been seen in several cattle breeds and has attracted considerable attention from beef producers. Double-muscled animals are characterized by an increase in muscle mass of about 20%, due to general skeletal-muscle hyperplasia-that is, an increase in the number of muscle fibers rather than in their individual diameter. Although the hereditary nature of the double-muscled condition was recognized early on, the precise mode of inheritance has remained controversial; monogenic (domainant and recessive), oligogenic and polygenic models have been proposed. In the Belgian Blue cattle breed (BBCB), segregation analysis performed both in experimental crosses and in the outbred population suggested an autosomal recessive inheritance. This was confirmed when the muscular hypertrophy (mh) locus was mapped 3.1 cM from microsatellite TGLA44 on the centromeric end of bovine chromosome 2 (ref. 5). We used a positional candidate approach to demonstrate that a mutation in bovine MSTN, which encodes myostatin, a member of the TGF beta superfamily, is responsible for the double-muscled phenotype. We report an 11-bp deletion in the coding sequence for the bioactive carboxy-terminal domain of the protein causing the muscular hypertrophy observed in Belgian Blue cattle.
BackgroundPreterm newborns are at high risk of developing neurodevelopmental deficits caused by neuroinflammation leading to perinatal brain injury. Human Wharton’s jelly mesenchymal stem cells (hWJ-MSC) derived from the umbilical cord have been suggested to reduce neuroinflammation, in part through the release of extracellular vesicle-like exosomes. Here, we studied whether exosomes derived from hWJ-MSC have anti-inflammatory effects on microglia-mediated neuroinflammation in perinatal brain injury.MethodsUsing ultracentrifugation, we isolated exosomes from hWJ-MSC culture supernatants. In an in vitro model of neuroinflammation, we stimulated immortalized BV-2 microglia and primary mixed glial cells with lipopolysaccharide (LPS) in the presence or absence of exosomes. In vivo, we introduced brain damage in 3-day-old rat pups and treated them intranasally with hWJ-MSC-derived exosomes.ResultshWJ-MSC-derived exosomes dampened the LPS-induced expression of inflammation-related genes by BV-2 microglia and primary mixed glial cells. The secretion of pro-inflammatory cytokines by LPS-stimulated primary mixed glial was inhibited by exosomes as well. Exosomes interfered within the Toll-like receptor 4 signaling of BV-2 microglia, as they prevented the degradation of the NFκB inhibitor IκBα and the phosphorylation of molecules of the mitogen-activated protein kinase family in response to LPS stimulation. Finally, intranasally administered exosomes reached the brain and reduced microglia-mediated neuroinflammation in rats with perinatal brain injury.ConclusionsOur data suggest that the administration of hWJ-MSC-derived exosomes represents a promising therapy to prevent and treat perinatal brain injury.Electronic supplementary materialThe online version of this article (10.1186/s13287-019-1207-z) contains supplementary material, which is available to authorized users.
Dysfunction and loss of neurons are the major characteristics of CNS disorders that include stroke, multiple sclerosis, and Alzheimer's disease. Activation of the Toll-like receptor 7 by extra-cellular micro-RNA let-7, a highly expressed microRNA in the CNS, induces neuronal cell death. Let-7 released from injured neurons and immune cells acts on neighboring cells, exacerbating CNS damage.Here we show that a synthetic peptide analogous to the mammalian PreImplantation factor (PIF) secreted by developing embryos and which is present in the maternal circulation during pregnancy inhibits the biogenesis of let-7 in both neuronal and immune cells of the mouse. The synthetic peptide, sPIF, destabilizes KH-type splicing regulatory protein (KSRP), a key microRNA-processing protein, in a Toll-like receptor 4 (TLR4)-dependent manner, leading to decreased production of let-7. Furthermore, s.c. administration of sPIF into neonatal rats following hypoxic-ischemic brain injury robustly rescued cortical volume and number of neurons and decreased the detrimental glial response, as is consistent with diminished levels of KSRP and let-7 in sPIF-treated brains. Our results reveal a previously unexpected mechanism of action of PIF and underscore the potential clinical utility of sPIF in treating hypoxic-ischemic brain damage. The newly identified PIF/TLR4/KSRP/ let-7 regulatory axis also may operate during embryo implantation and development.
Perinatal brain injury (PBI) in preterm birth is associated with substantial injury and dysmaturation of white and gray matter, and can lead to severe neurodevelopmental deficits. Mesenchymal stromal cells (MSC) have been suggested to have neuroprotective effects in perinatal brain injury, in part through the release of extracellular vesicles like exosomes. We aimed to evaluate the neuroprotective effects of intranasally administered MSC-derived exosomes and their potential to improve neurodevelopmental outcome after PBI. Exosomes were isolated from human Wharton’s jelly MSC supernatant using ultracentrifugation. Two days old Wistar rat pups were subjected to PBI by a combination of inflammation and hypoxia-ischemia. Exosomes were intranasally administered after the induction of inflammation and prior to ischemia, which was followed by hypoxia. Infrared-labeled exosomes were intranasally administered to track their distribution with a LI-COR scanner. Acute oligodendrocyte- and neuron-specific cell death was analyzed 24 h after injury in animals with or without MSC exosome application using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemical counterstaining. Myelination, mature oligodendroglial and neuronal cell counts were assessed on postnatal day 11 using immunohistochemistry, Western blot or RT-PCR. Morris water maze assay was used to evaluate the effect of MSC exosomes on long-term neurodevelopmental outcome 4 weeks after injury. We found that intranasally administered exosomes reached the frontal part of the brain within 30 min after administration and distributed throughout the whole brain after 3 h. While PBI was not associated with oligodendrocyte-specific cell death, it induced significant neuron-specific cell death which was substantially reduced upon MSC exosome application prior to ischemia. MSC exosomes rescued normal myelination, mature oligodendroglial and neuronal cell counts which were impaired after PBI. Finally, the application of MSC exosomes significantly improved learning ability in animals with PBI. In conclusion, MSC exosomes represent a novel prevention strategy with substantial clinical potential as they can be administered intranasally, prevent gray and white matter alterations and improve long-term neurodevelopmental outcome after PBI.
A synthetic peptide (sPIF) analogous to the mammalian embryo-derived PreImplantation Factor (PIF) enables neuroprotection in rodent models of experimental autoimmune encephalomyelitis and perinatal brain injury. The protective effects have been attributed, in part, to sPIF's ability to inhibit the biogenesis of microRNA let-7, which is released from injured cells during central nervous system (CNS) damage and induces neuronal death. Here, we uncover another novel mechanism of sPIF-mediated neuroprotection. Using a clinically relevant rat newborn brain injury model, we demonstrate that sPIF, when subcutaneously administrated, is able to reduce cell death, reverse neuronal loss and restore proper cortical architecture. We show, both in vivo and in vitro, that sPIF activates cyclic AMP dependent protein kinase (PKA) and calcium-dependent protein kinase (PKC) signaling, leading to increased phosphorylation of major neuroprotective substrates GAP-43, BAD and CREB. Phosphorylated CREB in turn facilitates expression of Gap43, Bdnf and Bcl2 known to have important roles in regulating neuronal growth, survival and remodeling. As is the case in sPIF-mediated let-7 repression, we provide evidence that sPIF-mediated PKA/PKC activation is dependent on TLR4 expression. Thus, we propose that sPIF imparts neuroprotection via multiple mechanisms at multiple levels downstream of TLR4. Given the recent FDA fast-track approval of sPIF for clinical trials, its potential clinical application for treating other CNS diseases can be envisioned.
AM and cytokines are significantly elevated after hypothermic CPB compared to normothermic CPB. MUF led to a significant reduction in cytokine and AM levels after hypothermic CPB, except for IL-2R. MUF showed minimal effect in normothermia. We conclude that MUF is an efficient way to remove cytokines and AM. However, we were unable to demonstrate any significant impact of MUF in outcome of adults after elective CABG.
Hypoxic-ischemic (HI) insult in the perinatal phase harbors a high risk of encephalopathy in the neonate. Brain cells undergo apoptosis, initiating neurodegeneration. So far, therapeutic approaches such as cooling remain limited. Transplantation of mesenchymal stem cells (MSCs) exhibits therapeutic success despite the short-time survival in the host brain, providing strong evidence that their beneficial effects are largely based on secreted factors, including extracellular vesicles (EVs). The aim of this study was to investigate the effects of human Wharton's jelly MSC (hWJ-MSC)-derived EVs on neuroprotection and neuroregeneration, using an in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R) mimicking HI injury in the mouse neuroblastoma cell line neuro2a (N2a). hWJ-MSC-derived EVs were isolated from cell culture supernatants by multistep centrifugation and identified by endosomal marker expression and electron microscopy. OGD/R significantly increased DNA fragmentation and caspase 3 ( Casp3) transcription in N2a cells relative to undamaged cells. OGD/R-mediated DNA fragmentation and Casp3 expression could be prevented as well as resolved by the addition of hWJ-MSC-derived EV before and after OGD, respectively. hWJ-MSC-derived EV also tended to increase the phosphorylation of the B cell lymphoma 2 (Bcl2) family member Bcl-2-antagonist of cell death (BAD) in N2a cells, when added prior or post OGD, thereby inactivating the proapoptotic function of BAD. Fluorescence confocal microscopy revealed the close localization of hWJ-MSC-derived EVs to the nuclei of N2a cells. Furthermore, EVs released their RNA content into the cells. The expression levels of the microRNAs (miRs) let-7a and let-7e, known regulators of Casp3, were inversely correlated to Casp3. Our data suggest that hWJ-MSC-derived EVs have the potential to prevent and resolve HI-induced apoptosis in neuronal cells in the immature neonatal brain. Their antiapoptotic effect seems to be mediated by the transfer of EV-derived let-7-5p miR.
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