Adoptive immunotherapy with functional T cells is potentially an effective therapeutic strategy for combating many types of cancer and viral infection. However, exhaustion of antigen-specific T cells represents a major challenge to this type of approach. In an effort to overcome this problem, we reprogrammed clonally expanded antigen-specific CD8(+) T cells from an HIV-1-infected patient to pluripotency. The T cell-derived induced pluripotent stem cells were then redifferentiated into CD8(+) T cells that had a high proliferative capacity and elongated telomeres. These "rejuvenated" cells possessed antigen-specific killing activity and exhibited T cell receptor gene-rearrangement patterns identical to those of the original T cell clone from the patient. We also found that this method can be effective for generating specific T cells for other pathology-associated antigens. Thus, this type of approach may have broad applications in the field of adoptive immunotherapy.

We address the challenge of detecting the contribution of noncoding mutations to disease with a deep-learning-based framework that predicts specific regulatory effects and the deleterious impact of genetic variants. Applying this framework to 1,790 Autism Spectrum Disorder (ASD) simplex families reveals disease causality of noncoding mutations: ASD probands harbor both transcriptional and post-transcriptional regulation-disrupting de novo mutations of significantly higher functional impact than unaffected siblings. Further analysis suggests involvement of noncoding mutations in synaptic transmission and neuronal development, and taken together with prior studies reveal a convergent genetic landscape of coding and noncoding mutations in ASD. We demonstrate that sequences carrying prioritized proband mutations possess allele-specific regulatory activity, and highlight a link between noncoding mutations and IQ heterogeneity in ASD probands. Our predictive genomics framework illuminates the role of noncoding mutations in ASD, prioritizes high impact mutations for further study, and is broadly applicable to complex human diseases.

p2y5 is an orphan G protein-coupled receptor that is closely related to the fourth lysophosphatidic acid (LPA) receptor, LPA 4 . Here we report that p2y5 is a novel LPA receptor coupling to the G 13 -Rho signaling pathway. "LPA receptor-null" RH7777 and B103 cells exogenously expressing p2y5 showed [ 3 H]LPA binding, LPA-induced [35 S]guanosine 5-3-O-(thio)triphosphate binding, Rho-dependent alternation of cellular morphology, and G s/13 chimeric protein-mediated cAMP accumulation. LPA-induced contraction of human umbilical vein endothelial cells was suppressed by small interfering RNA knockdown of endogenously expressed p2y5. We also found that 2-acyl-LPA had higher activity to p2y5 than 1-acyl-LPA. A recent study has suggested that p2y5 is an LPA receptor essential for human hair growth. We confirmed that p2y5 is a functional LPA receptor and propose to designate this receptor LPA 6 .Lysophosphatidic acid (LPA 3 ; 1-or 2-acyl-sn-glycero-3-phosphate) is a naturally occurring lipid mediator with diverse biological activities (1, 2). LPA plays important roles in many biological processes, such as the nervous system (3), tumor metastasis (4), wound healing (5), cardiovascular functions (6), and reproduction (7), through its specific G protein-coupled receptors (GPCRs). At least five subtypes of LPA receptors have been identified. Three receptors (LPA 1 (8), LPA 2 (9), and LPA 3 (10, 11)) share about 50% amino acid sequence identities, and form the Edg (endothelial differentiation gene) family together with the GPCRs for sphingosine 1-phosphate. Two additional LPA receptors, p2y9/LPA 4 (12) and GPR92/LPA 5 (13, 14), which show small similarities with the Edg family GPCRs, were recently identified. These LPA receptors, by coupling with different sets of G proteins, transduce various responses in many cell types. Depending on the functional coupling of a given LPA receptor to the G proteins, LPA activates diverse signaling cascades involving phosphoinositide 3-kinase, phospholipase C, mitogen-activated protein kinase, Rho-family GTPase, and adenylyl cyclase (2).The fact that p2y5 shares the highest sequence homology with p2y9/LPA 4 among all GPCRs (12) strongly suggested that LPA is a ligand for p2y5. However, we could not detect LPAinduced Ca 2ϩ mobilization or cAMP level changes in p2y5-overexpressing cells at the time of the identification of p2y9/ LPA 4 as the fourth LPA receptor in our laboratory (12). In the course of the further analysis of p2y5-overexpressing cells, we found that p2y5 actually responded to LPA with activation of the G 13 -Rho signaling pathway. Our results confirm the identification of p2y5 as an LPA receptor and extend the knowledge of the functional roles of LPA. EXPERIMENTAL PROCEDURESLipids-1-Oleoyl-LPA, 1-palmitoyl-LPA, 1-stearoyl-LPA, 1-myristoyl-LPA, and 1-arachidonoyl-LPA were purchased from Avanti Polar Lipids (Alabaster, AL). 1-Linoleoyl-LPA was from Echelon Biosciences (Salt Lake City, UT). These lipids were stored at Ϫ30°C (10 mM stock in 50% ethanol). Alkyl-OMPT (10 mM stock in d...

Blood is a commonly used biofluid for biomarker discovery. Although blood lipid metabolites are considered to be potential biomarker candidates, their fundamental properties are not well characterized. We aimed to (1) investigate the matrix type (serum vs. plasma) that may be preferable for lipid biomarker exploration, (2) elucidate age- and gender-associated differences in lipid metabolite levels, and (3) examine the stability of lipid metabolites in matrix samples subjected to repeated freeze-thaw cycles. Using liquid chromatography-mass spectrometry, we performed lipidomic analyses for fasting plasma and serum samples for four groups (15 subjects/group) of young and elderly (25–34 and 55–64 years old, respectively) males and females and for an additional aliquot of samples from young males, which were subjected to repeated freeze-thaw cycles. Lysophosphatidylcholine and diacylglycerol levels were higher in serum than in plasma samples, suggesting that the clotting process influences serum lipid metabolite levels. Gender-associated differences highlighted that the levels of many sphingomyelin species were significantly higher in females than in males, irrespective of age and matrix (plasma and serum). Age-associated differences were more prominent in females than in males, and in both matrices, levels of many triacylglycerols were significantly higher in elderly females than in young females. Plasma and serum levels of most lipid metabolites were reduced by freeze-thawing. Our results indicate that plasma is an optimal matrix for exploring lipid biomarkers because it represents the original properties of an individual’s blood sample. In addition, the levels of some blood lipid species of healthy adults showed gender- and age-associated differences; thus, this should be considered during biomarker exploration and its application in diagnostics. Our fundamental findings on sample selection and handling procedures for measuring blood lipid metabolites is important for ensuring the quality of biomarkers identified and its qualification process.

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. GPx4 is strongly expressed in the mitochondria of testis and spermatozoa. We previously found a significant decrease in the expression of GPx4 in spermatozoa from 30% of infertile human males diagnosed with oligoasthenozoospermia (Imai, H., Suzuki, K., Ishizaka, K., Ichinose, S., Oshima, H., Okayasu, I., Emoto, K., Umeda, M., and Nakagawa, Y. (2001) Biol. Reprod. 64, 674 -683). To clarify whether defective GPx4 in spermatocytes causes male infertility, we established spermatocyte-specific GPx4 knock-out mice using a CreloxP system. All the spermatocyte-specific GPx4 knock-out male mice were found to be infertile despite normal plug formation after mating and displayed a significant decrease in the number of spermatozoa. Isolated epididymal GPx4-null spermatozoa could not fertilize oocytes in vitro. These spermatozoa showed significant reductions of forward motility and the mitochondrial membrane potential. These impairments were accompanied by the structural abnormality, such as a hairpin-like flagella bend at the midpiece and swelling of mitochondria in the spermatozoa. These results demonstrate that the depletion of GPx4 in spermatocytes causes severe abnormalities in spermatozoa. This may be one of the causes of male infertility in mice and humans.A frequent cause of male infertility is defective sperm function, which is the main problem for close to a quarter of couples who attend infertility clinics (1-4). Considerable efforts are now focused on the identifying ultrastructural and/or molecular defects in the spermatozoa or seminal plasma to develop solutions to various types of male infertility.Phospholipid hydroperoxide glutathione peroxidase (GPx4) 2 is an intracellular selenoprotein that directly reduces peroxidized phospholipids produced in cell membranes (5). The GPx4 gene has a complex intron/exon structure (6, 7). Three different transcripts of GPx4 exist, differing in their 5Ј extension and coding for a cytosolic protein (non-mitochondrial GPx4), a mitochondrial protein (mitochondrial GPx4), and a nuclear protein (nucleolar GPx4), respectively (6, 7). After cleavage of the N-terminal mitochondrial import sequence of mitochondrial GPx4, the mature protein becomes identical to the 20-kDa non-mitochondrial GPx4 (8, 9). Nuclear GPx4 was recently identified as a sperm nucleus-specific 34-kDa selenoprotein (called snGPx, for sperm nucleus-specific glutathione peroxidase) (10). It is formed by use of an alternative promoter and start codon localized in the first intron of the GPx4 gene (7, 10, 11). We previously reported that 34-kDa GPx4 localized in nucleoli in several cell lines by using an N-terminal nucleolar import signal (11). We call hereafter nuclear GPx4 nucleolar GPx4, because non-mitochondrial 20-kDa GPx4 exists both in cytosol and in the nucleus (12). Expression of three types of GPx4 is induced significantly in testis during spermatogenesis, especiall...

BackgroundAlzheimer’s disease (AD), the most common cause of dementia among neurodegenerative diseases, afflicts millions of elderly people worldwide. In addition to amyloid-beta (Aβ) peptide and phosphorylated tau, lipid dysregulation is suggested to participate in AD pathogenesis. However, alterations in individual lipid species and their role in AD disease progression remain unclear.MethodsWe performed a lipidomic analysis using brain tissues and plasma obtained from mice expressing mutated human amyloid precursor protein (APP) and tau protein (Tg2576×JNPL3) (APP/tau mice) at 4 (pre-symptomatic phase), 10 (early symptomatic) and 15 months (late symptomatic).ResultsLevels of docosahexaenoyl (22:6) cholesterol ester (ChE) were markedly increased in APP/tau mice compared to controls at all stages examined. Several species of ethanolamine plasmalogens (pPEs) and sphingomyelins (SMs) showed different levels between brains from APP/tau and control mice at various stages of AD. Increased levels of 12-hydroxyeicosatetraenoic acid (12-HETE) during the early symptomatic phase were consistent with previous reports using human AD brain tissue. In addition, 19,20-dihydroxy-docosapentaenoic acid (19,20-diHDoPE) and 17,18-dihydroxy-eicosatetraenoic acid (17,18-diHETE), which are produced from docosahexaenoic acid and eicosapentaenoic acid via 19,20-epoxy-docosapentaenoic acid (19,20-EpDPE) and 17,18-epoxy-eicosatetraenoic acid (17,18-EpETE), respectively, were significantly increased in APP/tau brains during the pre-symptomatic phase, and concomitant increases occurred in plasma. Several arachidonic acid metabolites such as prostaglandin D2 (PGD2) and 15-hydroxyeicosatetraenoic acid (15-HETE), which have potential deteriorating and protective actions, respectively, were decreased in the early symptomatic phase of APP/tau mice. Significant decreases in phosphatidylcholines and PEs with polyunsaturated fatty acids were also detected in the late symptomatic phase, indicating a perturbation of membrane properties.ConclusionOur results provide fundamental information on lipid dysregulation during various stages of human AD.

Alternative polyadenylation (APA) is increasingly recognized to regulate gene expression across different cell-types, but obtaining APA maps from individual cell-types typically requires prior purification, a stressful procedure that can itself alter cellular states. Here, we describe a new platform, cTag-PAPERCLIP, that generates APA profiles from single cell populations in intact tissues; cTag-PAPERCLIP requires no tissue dissociation and preserves transcripts in native states. Applying cTag-PAPERCLIP to profile four major cell-types in the mouse brain revealed common APA preferences between excitatory and inhibitory neurons distinct from astrocytes and microglia, regulated in part by neuron-specific RNA-binding proteins NOVA2 and PTBP2. We further identified a role of APA in switching Araf protein isoforms during microglia activation, impacting production of downstream inflammatory cytokines. Our results demonstrate the broad applicability of cTag-PAPERCLIP and a previously undiscovered role of APA in contributing to protein diversity between different cell-types and cellular states within the brain.

Highlights d cTag-CLIP provides a strategy to study cell-specific RNA regulation in vivo d NOVA2 controls unique RNA splicing programs in inhibitory and excitatory neurons d NOVA2 cTag-CLIP reveals a new mechanism of cell-specific AS regulation d NOVA2 regulates intron retention as a cis-acting scaffold for AS factor PTBP2