Leucine/glutamine and v-ATPase/lysosomal acidification via mTORC1 activation are required for position-dependent regeneration

n animal regeneration, control of position-dependent cell proliferation is crucial for the complete restoration of patterned appendages in terms of both, shape and size. However, detailed mechanisms of this process are largely unknown. In this study, we identified leucine/glutamine and v-ATPase/lysosomal acidification, via mechanistic target of rapamycin complex 1 (mTORC1) activation, as effectors of amputation plane-dependent zebrafish caudal fin regeneration. mTORC1 activation, which functions in cell proliferation, was regulated by lysosomal acidification possibly via v-ATPase activity at 3 h post amputation (hpa). Inhibition of lysosomal acidification resulted in reduced growth factor-related gene expression and suppression of blastema formation at 24 and 48 hpa, respectively. Along the proximal-distal axis, position-dependent lysosomal acidification and mTORC1 activation were observed from 3 hpa. We also report that Slc7a5 (L-type amino acid transporter), whose gene expression is position-dependent, is necessary for mTORC1 activation upstream of lysosomal acidification during fin regeneration. Furthermore, treatment with leucine and glutamine, for both proximal and distal fin stumps, led to an up-regulation in cell proliferation via mTORC1 activation, indicating that leucine/glutamine signaling possesses the ability to change the position-dependent regeneration. Our findings reveal that leucine/glutamine and v-ATPase/lysosomal acidification via mTORC1 activation are required for position-dependent zebrafish fin regeneration.This study was supported by grants from Grant-in-Aid for Scientific Research from the JSPS (KAKENHI JP15J05983) to K.T

Metabolic engineering of S. pombe via CRISPR-Cas9 genome editing for lactic acid production from glucose and cellobiose

We constructed D-lactic acid (D-LA) producing Schizosaccharomyces pombe using CRISPR-Cas9 system. Two PDC genes, intact L-LDH, a minor gene of alcohol dehydrogenase (SPBC337.11) were disrupted to attenuate ethanol production pathway. To increase the cellular supply of acetyl-CoA, an important metabolite for growth, we introduced bacterial acetylating acetaldehyde dehydrogenase enzyme genes. Two kinds of acetaldehyde dehydrogenase genes from Escherichia coli, mhpF and eutE, were expressed. D-LA production was achieved by expressing D-lactate dehydrogenase gene from Lactobacillus plantarum. The engineered strains efficiently consumed glucose and produced 25.2 g/liter of D-LA from 35.5 g/liter of consumed glucose with the yield of 0.71 g-D-LA / g-glucose. Finally, we expressed beta-glucosidase by cell surface display techniques, and the resultant strain produced 24.4 g/L of D-LA from 30 g/L of cellobiose in minimal medium with the yield of 0.68 g-D-LA / g-glucose. This is the first report to generate metabolically engineered S. pombe strain using CRISPR-Cas9 system and we showed the possibility of S. pombe for the production host cell of value-added chemicals

Unusual endoscopic findings of gastric neuroendocrine tumor

Gastric neuroendocrine tumor (NET) is sometimes found as a submucosal tumor on upper gastrointestinal endoscopy. Gastric NET with malignant profile and neuroendocrine carcinoma (NEC) show various forms which are difficult to distinguish from gastric cancer and other disease. We report a case of a cauliflower-shaped NET of the stomach. A 61-year-old man was referred to our hospital with a complaint of abdominal fullness. Upper gastrointestinal endoscopic examination revealed an unusual, whitish cauliflower-shaped tumor that belongs to Borrmann type I on the lesser curvature of the gastric antrum. Histological examination of the biopsy specimen revealed NET G2, because the tumor cells were CD56- and synaptophysin-positive by immunohistochemical analysis. A distal gastrectomy with D2 lymphadenectomy was performed. A recurrence in the liver was revealed by follow up computed tomography after 11 months from operation. Combined chemotherapy with irinotecan (CPT-11) plus cisplatin (CDDP) was treated. The patient achieved a partial response, but he died after 31 months from gastrectomy. There is no independent, large-scaled prospective study and no standard treatment for gastric NETs with distant metastases. Our case is reported with a literature review of the treatment of metastatic gastric NET G2

Transient IGF-1R inhibition combined with osimertinib eradicates AXL-low expressing EGFR mutated lung cancer

Drug tolerance is the basis for acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) including osimertinib, through mechanisms that still remain unclear. Here, we show that while AXL-low expressing EGFR mutated lung cancer (EGFRmut-LC) cells are more sensitive to osimertinib than AXL-high expressing EGFRmut-LC cells, a small population emerge osimertinib tolerance. The tolerance is mediated by the increased expression and phosphorylation of insulin-like growth factor-1 receptor (IGF-1R), caused by the induction of its transcription factor FOXA1. IGF-1R maintains association with EGFR and adaptor proteins, including Gab1 and IRS1, in the presence of osimertinib and restores the survival signal. In AXL-low-expressing EGFRmut-LC cell-derived xenograft and patient-derived xenograft models, transient IGF-1R inhibition combined with continuous osimertinib treatment could eradicate tumors and prevent regrowth even after the cessation of osimertinib. These results indicate that optimal inhibition of tolerant signals combined with osimertinib may dramatically improve the outcome of EGFRmut-LC

A new approach to evaluate irradiation hardening of ion-irradiated ferritic alloys by nano-indentation techniques

The present work investigates the irradiation hardening of Fe-based model ferritic alloys after Fe-ion irradiation experiments in order to deduce mechanistically based nominal hardness from the nano-indentation tests on the ion-irradiated surface. Ion-irradiation experiments were carried out at 290 °C with 6.4 MeV Fe[3+] ions. The constant stiffness measurement (CSM) was used to obtain the depth-profile of hardness. The results has been analyzed and discussed based on the Nix–Gao model and an extended film/substrate system hardness model. The depth-sensing nano-indentation techniques with CSM revealed that the hardness gradient of the unirradiated Fe-based model alloy can be explained through the indentation size effect (ISE). On the other hand, the gradient of ion-irradiated surface of these samples includes not only the ISE but also softer substrate effect (SSE). We propose a new approach to evaluate a nominal hardness, which may connect to the bulk hardness, from experimentally obtained nano-hardness depth profile data

Distribution of the Steller sea lion Eumetopias jubatus during winter in the northern Sea of Japan, along the west coast of Hokkaido, based on aerial and land sighting surveys

Aerial and land-based sighting surveys were conducted to clarify the distribution of Steller sea lions during winter in the northern Sea of Japan, along the west coast of Hokkaido, from 2001 to 2003. Aerial surveys revealed that sea lions gathered around the Rishiri-Rebun Islands in March 2002 and 2003, and between Iwanai and Cape Obana in February 2003. Higher numbers of sea lions were also confirmed at Cape Ofuyu and Cape Kamui on the central-west coast compared to that at the haul-out sites in the 1980s on the upper-west coast in March 2002 and 2003. Additionally, fisherman observed sea lions along the coast of the Tsugaru Strait from February to May 2003, where the presence of sea lions was not reported in the 1980s. These facts suggest that sea lions have recently expanded their distribution southward. Land-based surveys at the recent haul-out sites indicated that sea lions in the central-west coast were composed mainly of adult and subadult males (average: Cape Ofuyu 75.2%, Cape Kamui 69.5%), which stayed from November 2002 to May 2003. In this area, sea lions arrived earlier and stayed longer than in the 1980s