For the advancement of methylated and unmethylated LCR HPV16 subjected to in vitro methylation with the SssI CpGmethylase

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Версія від 12:20, 20 серпня 2018, створена Trippimple9 (обговореннявнесок) (For the advancement of methylated and unmethylated LCR HPV16 subjected to in vitro methylation with the SssI CpGmethylase)

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The vulnerability of DKU80 strains to targeted double-strand breaks suggested we could perhaps rescue them through homologous recombination and, in the process, introduce desired point mutations. We observed that a 90 bp oligonucleotide homologous to the targeted region of PKG indeed improved the survival rate of DKU80 parasites transfected with pU6-PKG. Furthermore, a mutation included in the oligonucleotide was successfully incorporated into the parasite genome in a third of the surviving population. Similarly, targeting of the 39 end of the CDPK3 locus allowed us to incorporate an epitope tag into the open reading frame, which could be observed in 15% of the population three days post transfection, again in the absence of selection. As predicted the rate of endogenous tagging was significantly reduced in wild-type parasites, consistent with the NHEJ-mediated repair of targeted double-stranded breaks in this strain. These results highlight that although gene disruption is most readily achieved in wild-type strains, genome editing is most efficiently performed in the absence of NHEJ. However, deletion of KU80 should be easily generated in any genetic background through CRISPR/Cas9. Similar manipulations should be feasible throughout the parasite genome with just minimal investment in the construction of the targeting plasmid. The ease of CRISPR/Cas9 targeting, design and implementation, coupled with its efficiency, will greatly enhance our ability to manipulate the T. gondii genome. The methods described here should enable genetic manipulation of any transfection-competent strain and permit iterated modification of the parasite genome without concern for genetic markers or antibiotic selection. These tools have the potential to transform our analysis of the parasite genome by providing a truly multiplexable platform for genome manipulation. Improved neonatal and intensive care has enabled the survival of preterm infants with very low birth weights. These infants are at increased risk for nosocomial infection, and Staphylococcus epidermidis is the predominant pathogen isolated from blood cultures obtained in the neonatal intensive care unit. Increasing evidence suggests that neonatal brain injury is associated with infection/inflammation, but the underlying mechanisms are incompletely characterized. Preterm infants in particular have an increased risk of brain injury, which is predominantly located in the cerebral white matter, although recently a high frequency of grey matter injury has also been reported. Moreover, very low birth weight premature infants manifest cerebellar abnormalities. Infection/inflammation stimulates innate and subsequent adaptive immune responses via the Toll-like Receptor family of pattern-recognition receptors that can be stimulated with specific agonists. TLRs exist in a wide range of tissues outside the immune system, including the central nervous system. TLR2 forms heterodimers with TLR1 and TLR6, and these receptor complexes recognize molecules expressed on Gram-positive bacteria, such as peptidoglycan, lipopeptides, and lipoproteins, and they also mediate recognition of whole bacteria such as Staphylococcus epidermidis. Of note, TLR2 is selectively up-regulated in the peripheral blood mononuclear cells of human newborns infected with Gram-positive bacteria. With respect to the CNS, a role for TLR2 signaling in adult mouse brain injury has been suggested, as summarized by a most recent review but there are few reports that define the role of TLR2 signaling in neonatal brain injury. However, there are studies that suggest that TLR2 and TLR4 are the principal TLRs present on microglia which are involved in the innate immune response to infection/ hypoxia-ischemia; for a most recent review, please see. Of note, neonates demonstrate a distinct functional expression of the TLR system, and therefore studies of outcome in adult models cannot be directly extrapolated to newborns. In the present study, we hypothesized that stimulation of TLR2 during a critical period of neonatal brain development would have a detrimental effect on the immature brain, which may be measurable as changes in adult behavior. We used a synthetic lipopeptide, Pam3CysSerLys4, as a specific TLR2 agonist, that was administrated systemically to newborn wild-type and TLR2 deficient mice from postnatal day 3 to PND11 to evaluate short and long-term effects on the developing mouse brain. After repeated administration of 5 mg/kg Pam3CSK4 once a day from PND3 to PND11, brain weight was decreased compared with endotoxin-free saline-treated animals at PND12. In contrast, there was no difference between endotoxin-free saline-treated animals and LPS-treated animals. We found no infarctions, dilatation of the cerebral ventricles, or morphological signs of cell death in any of the brain regions examined after administration of Pam3CSK4 or LPS. There was a significant increase in both the absolute spleen and liver weights in animals treated with Pam3CSK4 and LPS compared with those treated with endotoxin-free saline at PND12 as well as the relative spleen and liver weight to body weight ratio. The whole body weight was not different between groups at PND12. No mortality or other signs of morbidity were found during the entire study period. To characterize the inflammatory response after Pam3CSK4 treatment, we first analyzed the cytokine/chemokine production by multiplex ELISA in brain homogenate samples at 6 hours after the first Pam3CSK4 treatment at PND3, in comparison with saline and LPS treated mice. It was found that 5 mg/kg Pam3CSK4 treatment induced elevated levels of IL-1ß, IL-6, KC, MCP-1, similar to those cytokines and chemokines induced by 0.3 mg/kg LPS. IL-1ß was an exception in that a significant increase was noted in Pam3CSK4-treated pups compared with LPS-treated pups. Of note, IL-6 was significantly increased by Pam3CSK4 but not by LPS. TNF-a levels did not change in either of the two treatment groups. IL-10 and IL-17 levels were below the limits of detection in all brain homogenate samples tested. To further examine the inflammatory response, we stained brain sections for the microglia marker Iba-1.