Single bacterial cell detection using a mutant luciferase

Previously, we constructed a genetically modified luciferase of Photinus pyralis that generates more than 10-fold higher luminescence intensity than the wild-type enzyme. In this study, we demonstrate that this modified luciferase enables us to detect ATP at 10⁻¹⁸ mol, which is almost equal to the quantity contained in a single bacterial cell. Consequently,we have been able to detect bacterial contamination in samples as low as one colony-forming unit (c.f.u.) for both Escherichia coli and Bacillus subtilis.     

Sensitive and specific method for the determination of josamycin in human plasma by liquid chromatography–mass spectrometry

A highly sensitive and specific method for the determination of josamycin in human plasma by LC–MS was developed and validated. Josamycin was extracted from human plasma by a single-step liquid–liquid extraction and analyzed by LC–MS via an electrospray ionization interface. Selected ion monitoring was used to detect josamycin and its internal standard. The intra-day precision and accuracy, expressed as C.V. and R.E., ranged from 2.8% to 13.5% and −10.3% to 7.6%, respectively. The lower limit of detection was 0.1 ng/ml and the lower limit of quantitation was set at 1 ng/ml when 0.5 ml of plasma was used. No endogenous interference was observed in human plasma obtained from drug-free volunteers.     

Cellular abnormalities of blood vessels as targets in cancer

Tumor blood vessels have multiple structural and functional abnormalities. They are unusually dynamic, and naturally undergo sprouting, proliferation, remodeling or regression. The vessels are irregularly shaped, tortuous, and lack the normal hierarchical arrangement of arterioles, capillaries and venules. Endothelial cells in tumors have abnormalities in gene expression, require growth factors for survival and have defective barrier function to plasma proteins. Pericytes on tumor vessels are also abnormal. Aberrant endothelial cells and pericytes generate defective basement membrane. Angiogenesis inhibitors can stop the growth of tumor vessels, prune existing vessels and normalize surviving vessels. Loss of endothelial cells is not necessarily accompanied by simultaneous loss of pericytes and surrounding basement membrane, which together can then provide a scaffold for regrowth of tumor vessels. Rapid vascular regrowth reflects the ongoing drive for angiogenesis and bizarre microenvironment in tumors that promote vascular abnormalities and thereby create therapeutic targets.     

Novel Mechanisms of Trafficking Defect Caused by KCNQ1 Mutations Found in Long QT Syndrome

Long QT syndrome (LQTS) is a hereditary arrhythmia caused by mutations in genes for cardiac ion channels, including a potassium channel, KvLQT1. Inheritance of LQTS is usually autosomal-dominant, but autosomal-recessive inheritance can be observed in patients with LQTS accompanied by hearing loss. In this study, we investigated the functional alterations caused by KCNQ1 mutations, a deletion (delV595) and a frameshift (P631fs/19), which were identified in compound heterozygous state in two patients with autosomal-recessive LQTS not accompanied by hearing loss. Functional analyses showed that both mutations impaired cell surface expression due to trafficking defects. The mutations severely affected outward potassium currents without apparent dominant negative effects. It was found that delV595 impaired subunit binding, whereas P631fs/19 was retained in endoplasmic reticulum due to the newly added 19-amino acid sequence containing two retention motifs (R⁶³³GR and R⁶⁴⁶LR). This is the first report of novel mechanisms for trafficking abnormality of cardiac ion channels, providing us new insights into the molecular mechanisms of LQTS.     

Improving the interpretability of climate landscape metrics: An ecological risk analysis of Japan's Marine Protected Areas

Conservation efforts strive to protect significant swaths of terrestrial, freshwater and marine ecosystems from a range of threats. As climate change becomes an increasing concern, these efforts must take into account how resilient‐protected spaces will be in the face of future drivers of change such as warming temperatures. Climate landscape metrics, which signal the spatial magnitude and direction of climate change, support a convenient initial assessment of potential threats to and opportunities within ecosystems to inform conservation and policy efforts where biological data are not available. However, inference of risk from purely physical climatic changes is difficult unless set in a meaningful ecological context. Here, we aim to establish this context using historical climatic variability, as a proxy for local adaptation by resident biota, to identify areas where current local climate conditions will remain extant and future regional climate analogues will emerge. This information is then related to the processes governing species’ climate‐driven range edge dynamics, differentiating changes in local climate conditions as promoters of species range contractions from those in neighbouring locations facilitating range expansions. We applied this approach to assess the future climatic stability and connectivity of Japanese waters and its network of marine protected areas (MPAs). We find 88% of Japanese waters transitioning to climates outside their historical variability bounds by 2035, resulting in large reductions in the amount of available climatic space potentially promoting widespread range contractions and expansions. Areas of high connectivity, where shifting climates converge, are present along sections of the coast facilitated by the strong latitudinal gradient of the Japanese archipelago and its ocean current system. While these areas overlap significantly with areas currently under significant anthropogenic pressures, they also include much of the MPA network that may provide stepping‐stone protection for species that must shift their distribution because of climate change.     

MCP-1 expressed by osteoclasts stimulates osteoclastogenesis in an autocrine/paracrine manner

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays a critical role in the recruitment and activation of leukocytes. Here, we describe that multinuclear osteoclast formation was significantly inhibited in cells derived from MCP-1-deficient mice. MCP-1 has been implicated in the regulation of osteoclast cell-cell fusion; however defects of multinuclear osteoclast formation in the cells from mice deficient in DC-STAMP, a seven transmembrane receptor essential for osteoclast cell-cell fusion, was not rescued by recombinant MCP-1. The lack of MCP-1 in osteoclasts resulted in a down-regulation of DC-STAMP, NFATc1, and cathepsin K, all of which were highly expressed in normal osteoclasts, suggesting that osteoclast differentiation was inhibited in MCP-1-deficient cells. MCP-1 alone did not induce osteoclastogenesis, however, the inhibition of osteoclastogenesis in MCP-1-deficient cells was restored by addition of recombinant MCP-1, indicating that osteoclastogenesis was regulated in an autocrine/paracrine manner by MCP-1 under the stimulation of RANKL in osteoclasts.     

Induction of Macrophage-Like Immunosuppressive Cells from Mouse ES Cells That Contribute to Prolong Allogeneic Graft Survival

Recent progress in regenerative medicine has enabled the utilization of pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) as a donor resource for transplantation. However, immune suppression is still needed when the donor-recipient combination is allogeneic. Protection of ESCs-derived grafts from host immune response might be achieved thought the utilization of immunosuppressive cells generated from ESCs. In the present study, we show that a certain fraction of immunosuppressive cells can be generated from ESCs and help to suppress immune response against allogeneic grafts. ESCs-derived suppressor cells (ES-SCs) resembled macrophages in terms of cell surface molecule and gene expressions. Furthermore, gene expression analysis including microarray showed that ES-SCs have M1/M2 hybrid phenotype with high expression of genes correlated to immunosuppression of T cell response. Indeed, ES-SCs were effective to block allogeneic T cell proliferation in a nitric oxide-dependent manner, and prolonged the survival of ESCs-derived embryoid bodies or cardiomyocytes grafts transplanted into mouse kidney capsule. Thus, we consider the potential use of these ESCs-derived macrophage-like immunosuppressive cells as cellular therapies to promote long-term graft survival in future therapies.