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.
Hwang et al. develop cTag-PAPERCLIP to profile mRNA 3′ ends in individual cell-types from intact tissues
in vivo
. They use cTag-PAPERCLIP to show that mRNA alternative polyadenylation contributes to protein diversity between different cell-types and cellular states within the brain.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Circadian rhythms are known to influence a variety of biological phenomena such as cell cycle, sleep-wake rhythm, hormone release and other important physiological functions. Given that cell cycle ...entry of hibernating hematopoietic stem cells (HSCs) plays a critical role in controlling hematopoiesis, we asked functional significance of the clock gene Bmal1, which plays a central role in regulating circadian rhythms as a transcription factor. Here we investigated the necessity of Bmal1 for HSC functions using Bmal1 deficient (Bmal1⁻/⁻) mice.
Using colony-forming assays in vitro, we found that the frequency of mixed colony formation between Bmal1⁺/⁺ and Bmal1⁻/⁻ CD34-KSL cells does not differ significantly. Competitive bone marrow assays also revealed that Bmal1⁻/⁻ bone marrow cells competed normally with wild-type cells and displayed long-term multi-hematopoietic lineage reconstitution. In addition, there were no significant differences in the frequencies and hibernation state of bone marrow HSCs between Bmal1⁺/⁺ and Bmal1⁻/⁻ mice, suggesting that they are independent of circadian rhythms.
This paper discusses the necessity of circadian rhythms for HSC functions. Our data clearly shows that a key circadian clock gene Bmal1 is dispensable for intrinsic functions of HSCs, such as differentiation, proliferation and repopulating ability.
Biomarkers will play important roles in disease diagnosis, drug development, and the proper use of drugs. Blood is considered the best biofluid for biomarker research because it is easy to access and ...a wealth of data are available. However, previous studies revealed that several ionic metabolites showed different levels (including presence or absence) in plasma and serum. Thus, attention should be paid to selecting the best biofluid for biomarker exploration. Many lipid molecules have biological significance and thus would be candidate biomarkers. However, no comprehensive study revealing differences in lipid metabolite levels between plasma and serum has been undertaken. Furthermore, gender differences have not been reported. To clarify the difference in the levels of lipid metabolites between human plasma and serum from both genders, we performed lipid metabolomic analysis using liquid chromatography-mass spectrometry-based systems for phospholipids (PLs), lysoPLs, sphingomyelins, ceramides and oxidative fatty acids. Our results revealed that most of the lipid metabolites were present at similar levels in plasma and serum and in males and females. However, several oxidative fatty acid metabolites showed differences. Of the metabolites related to clotting processes, three showed higher levels in serum than in plasma, and three were detected only in serum. Furthermore, four metabolites were present at different levels between males and females, and two were detected only in males. Thus, attention should be paid to the selection of plasma or serum when utilizing these lipid metabolites as biomarkers.
Abstract 2984
T lymphocytes play central roles in cellular immunity, exerting their proliferative and effector activities when they recognize antigens via T-cell receptors (TCRs) in HLA-restricted ...and antigen-specific manner. Adoptive cell transfer therapy (ACT), the administration of ex vivo-activated and -expanded autologous tumor-reactive T lymphocytes, is currently one of the effective methods for immunotherapy, especially for treatment of metastatic solid tumors including melanoma. However, the successful applications of this method are currently limited for tumor therapies. To broaden the range of the application of ACT, we endeavored to develop easier method to obtain cells that carry antigen-specific TCR genes. For the purpose, generation of induced pluripotent stem (iPS) cells from an antigen-reactive single T lymphocyte is attractive and rewarding way. iPS cells have a capacity for unlimited self-renewal while maintaining pluripotency. These features may enable us to induce an unlimited number of T lymphocytes, especially high proliferative naïve / central memory-type T lymphocytes, showing reactivity to specific antigens. If they retain properties of naïve T lymphocytes, they may proliferate for a longer period and achieve better therapeutic effects than their peripheral blood counterparts expanded in vitro.
Peripheral T lymphocytes were isolated from healthy volunteers. Then reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) were transduced into fresh or frozen / thawed T lymphocytes. T lymphocyte-derived iPS-like colonies were observed within 3 weeks and they were isolated and clonally expanded. They exhibited standard ES-like morphology, cell surface marker expression, alkaline phosphatase activity, as well as differentiation potential into various tissues related to all three germ layers. Human TCRs are encoded in four genes (TCRA, TCRB, TCRG, TCRD), which should be genetically assembled in an irreversible manner during T-lymphocyte development. This feature allowed us to retrospectively confirm that the iPS cells were generated from T lymphocyte. The TCR gene rearrangements encoded in an iPS colony were clonal for all iPS lines, indicating that each iPS colony was derived from a single T lymphocyte. Sequence analyses of TCR genes revealed whether the rearrangements were productive, and the productivity might promise the conservation of TCR genes rearrangement during the reprogramming process.
Next, we tried to re-differentiate T lymphocyte derived-iPS (T-iPS) cells into T cells by co-culturing them with murine stromal cell layers (OP9 and OP9-DL1). T-cell differentiation was evidenced by the expression of T-cell markers, such as CD5, CD7, CD27, CD4, CD8, TCR α β and CD3. We obtained 33.5 ± 17.9% CD4+ CD8+ double positive (DP) cells, 6.51 ± 5.40% CD4+ CD8− single positive (SP) cells and 3.80 ± 1.28% CD4− CD8+ SP cells. They could be activated via TCR stimulation, and produce cytokines as functionally matured T lymphocytes do. The re-differentiation efficiency of T-iPS cells was higher than those of other pluripotent stem cells, such as embryonic stem (ES) cells, fibroblasts derived-iPS cells, or cord blood derived-iPS cells. Transcribed TCR mRNA sequences in re-differentiated T cells were analyzed, and they were revealed to be identical to that engraved in the pre-differentiated T-iPS cells genome in CD4+ CD8+ DP phase. However, fully matured CD4+ CD8− or CD4− CD8+ SP phase cells had several TCRA gene rearrangement patterns distinct from the original T-iPS cell’s. On the other hand, TCRB gene maintained identity with the original. The variance of the sequences, especially antigen-recognition site sequences, indicated that the antigen-specificity in the original T lymphocyte might be converted during DP to SP transition process in vitro.
These data indicate that functionally matured T cells were generated by re-differentiating T-iPS cells in vitro, and that re-assemble of TCRA genes could take place during SP T cell maturation process. In order to fulfill the T-iPS-mediated immunotherapy, we need to overcome the obstacle of further TCRA gene rearrangements. We think the solution lies in refinement of the re-differentiation method for controlling the expression of RAG1 and RAG2 recombinases or for inhibiting their activities.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Adequate blood flow (Qb) is necessary for effective hemodialysis (HD). Aim of the study was to examine relationship between the actually delivered Qb (dQb) and reported Qb (rQb) with dialysis ...machine. One hundred HD patients with arteriovenous fistula were enrolled. Delivered Qb was measured at the beginning and end of each HD session. dQb/rQb < 1 indicated a discrepancy between actual dQb and rQb reported using a dialysis machine. In addition, dQb/rQb was examined in HD patients using needles of different gauges during treatment. The average levels of dQb/rQb at start and end of HD session were 1.01 ± 0.04 and 0.98 ± 0.05, respectively. In the 16 gauge and 17 gauge needle groups, the percentage of patients with dQb/rQb < 1 increased in accordance with the increase in rQb or as the HD session progressed. In the 15 gauge needle group, the percentage of patients with dQb/rQb < 1 was <50% at any level of rQb. Selection of needle gauge is important factors for determining actual dQb in HD patients.
Biomarkers are useful tools as indicators/predictors of disease severity and drug responsiveness, and thus, are expected to make drug development more efficient and to accelerate proper use of ...approved drugs. Many academic achievements on biomarkers have been reported, but only several biomarkers are used in drug development and clinical settings. We first show our results on the pharmacogenomic analysis of the anti-cancer drug irinotecan and of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). UGT1A1*6 and *28 were significantly associated with altered pharmacokinetics of an irinotecan metabolite, SN-38, and with increased frequency of severe neutropenia. HLA* 58:01 and HLA-B*15:11/HLA-A*31:01 were associated with SJS/TEN by allopurinol and carbamazepine, respectively. Our papers have been cited in the package inserts of irinotecan and allopurinol. In addition to these genomic biomarkers, metabolomic biomarkers, which can reflect the disease phenotype and drug responsiveness, have been exploring for 12 major diseases in Japan, as a part of a multi-omics team with multi-national centers. In animal models of dilated cardiomyopathy and Alzheimer's disease, we found several changes in lipid metabolite levels in the diseased tissues. Moreover, two oxidized fatty acids were correlatively changed in the brain and plasma from Alzheimer's model mice before its onset, and thus, could be candidates for predictive biomarkers. Finally, we propose/discuss several key issues for academic researches on biomarker discovery and development, especially for newly coming researchers in the field of pharmaceutical sciences. We hope that this review would help novel biomarker identification and qualification in Japan.
Abstract 490
T lymphocytes play central roles in cellular immunity, exerting their proliferative and effector activities when they recognize antigens, in HLA-restricted and antigen-specific manner, ...via T-cell receptors (TCRs). Successful treatment of leukemias/cancers with T-lymphocytes infusions is a direct proof that human immunity has the potential to eradicate cancers. However, continuous exposure to tumor/self antigens drives T lymphocytes into a highly exhausted state, with loss of potentials for long-term survival, proliferation, and effector functions that can end up with deletion of antigen-responding T-lymphocyte pools. Several workers have endeavored to develop clinical protocols for expanding antigen-responding T cells from the few naïve T-cell pools remaining in the patient. However, highly expanded T cells in such protocols have not proved fully effective so far, because functional losses like those in the patient occur during ex vivo manipulation. To overcome this obstacle to T-lymphocyte based immunotherapy, we endeavored to induce antigen-specific TCR-expressing T lymphocytes from induced pluripotent stem (iPS) cells, which were derived from antigen-reactive single T lymphocytes. iPS cells have a capacity for unlimited self-renewal while maintaining pluripotency. These features enabled us to induce an unlimited number of T lymphocytes, especially naïve T lymphocytes, showing reactivity to specific antigens. If they retain properties of naïve T lymphocytes, they may proliferate for a longer period and achieve better therapeutic effects than their peripheral blood counterparts expanded in vitro.
Peripheral T lymphocytes were isolated from healthy volunteers. Then three reprogramming factors (OCT4, SOX2, and KLF4) and additional factors (c-MYC and/or NANOG) were transduced into fresh or frozen/thawed T lymphocytes using a retrovirus. The virus-infected T lymphocytes were then transferred onto mouse embryonic fibroblasts (MEFs) in the presence of cytokines and chemicals favorable for T-lymphocyte survival/proliferation. iPS-like colonies were observed within 3 weeks after infections. Single T lymphocyte-derived colonies were isolated and clonally expanded. They exhibited standard ES-like morphology, cell surface markers and alkaline phosphatase activity, as well as differentiation potential into various tissues related to all three germ layers. Human TCRs are encoded in four genes (TCRA, TCRB, TCRG, TCRD), which should be genetically assembled in an irreversible manner during T-lymphocyte development. This feature allowed us to retrospectively confirm the iPS cells were generated from T lymphocyte. The TCR genes rearrangement encoded in an iPS colony was single in all iPS lines, indicating that the iPS colony was derived from single T lymphocyte. Sequence analyses of TCR genes revealed whether the rearrangements were productive, and the productivity might promise the conservation of TCR genes rearrangement during the reprogramming process. Next, we tried to re-differentiate T-lymphocyte derived-iPS (T-iPS) cells into T-lineage cells by co-culturing them with murine stromal cell layers (OP9 and OP9-DL1). These T-lineage committed cells were expressed TCRab heterodimer and T-cell surface markers such as CD3. They could activate via TCR stimulation, and produce IL-2 and IFN-g as maturing T lymphocytes. The re-differentiation efficiency of T-iPS cells was higher than those of embryonic stem cells, fibroblasts derived-iPS cells, or cord blood derived-iPS cells. mRNA sequence of TCRs transcribed in re-differentiated T-lineage cells was identical to that engraved in the pre-differentiated T-iPS cells genome. The invariance of the sequence, especially antigen-recognition site sequence, indicated that the antigen-specificity in original T lymphocyte was conserved during re-differentiation process.
Here we show that the conservation of the antigen-specificity encoded in TCR genes throughout induction of T-iPS cells and re-differentiation into T-lineage cells. These data suggest that further optimization of these processes for clinical application could open the door to the development of novel T-lymphocyte therapy, repeatedly supplying patient-compatible and disease-specific naïve T lymphocytes.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
To establish a fixed-time artificial insemination (AI) protocol in pigs after treatment for synchronization of ovulation with a combination of prostaglandin F2α (PGF2α), equine chorionic gonadotropin ...and human chorionic gonadotropin (hCG), we investigated the reproductive hormone profiles and ovulation in the synchronized sows and determined a satisfactory timing for a single AI to provide sufficient reproductive performance. Plasma progesterone concentrations significantly decreased on the day following PGF2α treatment. A preovulatory surge of luteinizing hormone was observed in all synchronized sows, peaking at 26.0±1.2 hours after hCG treatment, and ovulation was detected at 44.8±2.3 hours after treatment. When a timed AI was performed once at 24 or 36 hours after hCG treatment, normal blastcysts were collected from all of the synchronized pigs. Moreover, farrowing rate and average litter size did not differ between the synchronized pigs inseminated once at 24 or 36 hours after hCG treatment and cyclic pigs inseminated multiple times during natural estrus.
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