Ied by chemical phosphorus assay, as described elsewhere [42].Lipidomics AnalysesTotal lipids (0.five x 108 tachyzoites) have been fractionated on chloroformequilibrated silica 60 columns. Neutral lipids were eluted by acetone washing of your column. Phospholipids were subsequently eluted in 5 columnvolumes of chloroform/methanol/water (1:9:1). Each lipid fraction was collected, dried under nitrogen stream at 37 , and stored at 20 for downstream assays. Internal common PtdCho (44:two) was mixed with extracted lipids to calibrate the recovery yield of major lipids. 1020 l aliquots of phospholipid extract in chloroform/methanol (1:1) were introduced onto a HILIC column (Kinetex, 2.six m) at a flow rate of 1 ml/min to resolve distinctive phospholipid classes, essentially as described elsewhere [43]. Column effluent was introduced into either a four,000 QTRAP mass spectrometer (AB Sciex, Framingham, MA) or LTQXL (Thermo Scientific, Waltham, MA), and analyzed in the unfavorable ion mode employing electrospray ionization. Data have been processed applying the proprietary application with the respective instrument suppliers. Lipidomics information reported in this perform have been deposited inside the Dryad repository [44]: http://dx.doi.org/10.5061/dryad.564scIn Vivo Parasite Infection and Cerebral HistopathologyC57BL/6J mice had been infected with extracellular Aldose Reductase Inhibitors Related Products tachyzoites with the RHku80hxgprt (parental), tgpts or tgpts/TgPTSHA strains. Parasites for in vivo infections have been propagated in HFF cells. Fresh hostfree tachyzoites have been syringereleased immediately after 40 hr of infection, filtered (5 m), then injected by way of intraperitoneal (i.p.) route (50 parasites of the parental and tgpts/ TgPTSHA strains; five x 102 or five x 103 of tgpts strain). Animals were monitored for mortality and morbidity three instances a day over a period of four wk. An inoculum of 50 parental tachyzoites (form I) was utilised to challenge the tgptsimmunized animals, which had been monitored for more 4 wk. Cysts had been harvested in the brains of female NMRI mice infected with T. gondii on the ME49 strain five to six months earlier (i.p.), as described just before [45]. The tgptsvaccinated mice (500 parasites) were challenged together with the form II parasites (ME49, three cysts i.p. in 200 l) 4 wk after the major infection. A handle group of na e animals was also integrated. Parasite burden within the mouse brain was estimated by counting cysts and semiquantitative realtime PCR following yet another four wk of infection with all the ME49 strain. Brain tissue was mechanically homogenized in 1 ml sterile PBS and cysts have been counted making use of a light microscope. For qPCR,PLOS Biology | DOI:10.1371/journal.pbio.November 13,18 /Phosphatidylthreonine Is Essential for the Parasite Virulenceperfused brain tissue samples have been snapfrozen and stored at 80 [46]. 30 mg tissue was applied to purify nucleic acids (Nitrobenzylthioinosine web QIAgen kit). FastStart Important DNA Green Master (Roche, Germany) was mixed with genomic DNA (90 ng) in triplicate reactions, which had been created in a LightCycler 480 Instrument II (Roche, Germany). The parasite burden (target: TgB1 gene) was estimated relative to mouse (reference: argininosuccinate lyase, MmASL). Primers for the TgB1 and MmASL genes are listed in S1 Table. For cerebral histopathology, brain tissues isolated from infected animals have been immersed in four paraformaldehyde for numerous days. Samples have been embedded in paraffin, sliced into 4m thick sections, deparaffinized and then stained with hematoxylineosin stain, as described elsewhere [47]. Slides were developed utilizing the Bon.
