Biosynthesis of unnatural glycolipids possessing diyne moiety in the acyl chain in the green sulfur photosynthetic bacterium Chlorobaculum tepidum grown by supplementation of 10,12-heptadecadiynic acid

AbstractUnnatural glycolipids possessing the diyne moiety in their acyl groups were successfully biosynthesized in the green sulfur photosynthetic bacterium Chlorobaculum (Cba.) tepidum by cultivation with supplementation of 10,12-heptadecadiynic acid. Monogalactosyldiacylglycerol (MGDG) and rhamnosylgalactosyldiacylglycerol (RGDG) esterified with one 10,12-heptadecadiynic acid were primarily formed in the cells, and small amounts of glycolipids esterified with the two unnatural fatty acids can also be detected. The relative ratio of these unnatural glycolipids occupied in the total glycolipids was estimated to be 49% based on HPLC analysis using a evaporative light scattering detector. These results indicate that the acyl groups in glycolipids, which play important roles in the formation of extramembranous antenna complexes called chlorosomes, can be modified in vivo by cultivation of green sulfur photosynthetic bacteria with exogenous synthetic fatty acids. Visible absorption and circular dichroism spectra of Cba. tepidum containing the unnatural glycolipids demonstrated the formation of chlorosomes, indicating that the unnatural glycolipids in this study did not interfere with the biogenesis of chlorosomes

Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

Background: Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool forelucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patientperipheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimuminvasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitorcells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally requireHSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogrammingefficiencies, making the overall procedure costly, laborious, and time-consuming.Methods: We have established a highly efficient method for generating iPSCs from non-mobilized PB-derivedCD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and werekept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads,with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vectorSeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generationseries, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time wererecorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cellreceptor gene rearrangement, pluripotency markers, and differentiation capability were examined.Results：We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay.Conclusion：This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases

The action of BDNF on GABAA currents changes from potentiating to suppressing during maturation of rat hippocampal CA1 pyramidal neurons

During the development of the hippocampus, the action of GABA shifts from depolarizing to hyperpolarizing, and brain-derived neurotrophic factor (BDNF) has important roles in GABAergic transmission. We demonstrate that BDNF (20 ng ml−1) rapidly and reversibly potentiates postsynaptic GABAA receptor-mediated currents (by 80.5 ± 14.3 %, n = 10) in hippocampal CA1 pyramidal neurons isolated from postnatal day (P)6 rats, using nystatin-perforated patch-clamp recordings. This potentiation is caused by an elevation of intracellular Ca2+ that occurs in response to the activation of Trk B receptor tyrosine kinase and phospholipase C-γ. The modulation of the GABAA responses by BDNF in hippocampal CA1 pyramidal neurons isolated from P10 rats was more diverse (from potentiating to inhibitory), and at P14, BDNF induced a long-lasting inhibition. In addition, Ca2+/calmodulin-dependent protein kinase 2 plays important roles in the potentiating, but not in the inhibitory effect, of BDNF on the GABAA responses. These results suggest that changes in the intracellular signalling pathway could contribute to the developmental shift of the actions of BDNF on inhibitory systems