Influence of DNA extraction kits on freshwater fungal DNA metabarcoding

[Background] Environmental DNA (eDNA) metabarcoding is a common technique for efficient biodiversity monitoring, especially of microbes. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes. [Methods] DNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extractmicrobial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results; i.e., operational taxonomic unit (OTU) number and composition of the extracted samples. [Results] No PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals

Degree of high phenotypic plasticity in wild populations of Daphnia in early spring

Inducible defenses have been empirically known as defensive phenotypes that are triggered by predator kairomones. We hypothesized that morphological defense of wild Daphnia varies not only with predator density, but also with the predator regime in the field. We observed how the morphological defenses of two Daphnia species (D. ambigua and D. pulex) changed according to the population density in the water column at daytime of predatory insect larvae Chaoborus flavicans in Lake Fukami-ike, Japan, from February to July. In both Daphnia species, the inducible defense morphology was highly expressed in March and April. Its degree of expression decreased, and did not change with increases in predator density from May to July. These seasons are generally considered as when the number of the larvae and fish increases, and predation becomes more active due to their growth and breeding. We suggest that the degree of inducible defense of Daphnia is higher at the end of the overwintering season, when Chaoborus larvae began to inhabit the water column during the daytime and is constant in other seasons regardless of predators regime changes. Field observation of wild populations is important for understanding the seasonal changes in the morphology, and to provide more realistic explanations of phenomena in inducible defense

Stimulating Effect of High Concentration of Calcium Ion on the Polymerization of the Tubulin-Colchicine Complex. −Relationship between Magnesium and Calcium−

The polymerization reaction was reversible by the addition of calcium and/or EGTA and cooling. The extent of inhibition was affected by the pH and magnesium ion concentration. Maximum inhibition of polymerization was observed at a concentration of around 100µM calcium ion. The presence of calcium in a polymerization buffer caused the critical concentration to increase. At above 250µM calcium ion, no inhibition was observed. The inhibitory effect is due to the high-affinity sites of calcium to the tubulin-colchicine complex and the stimulating effect is due to low-affinity sites. Abbreviations: EGTA, Ethlene-bis (oxyehylenenitrilo) tetraacetic acid; GTP, guanosine 5'-triphosphate

The neuroprotective effects of milk fat globule-EGF factor 8 against oligomeric amyloid β toxicity

BACKGROUND: Phosphatidylserine receptor is a key molecule that mediates the phagocytosis of apoptotic cells. Milk fat globule-EGF factor 8 (MFG-E8) is a phosphatidylserine receptor that is expressed on various macrophage lineage cells, including microglia in the central nervous system (CNS). Targeted clearance of degenerated neurons by microglia is essential to maintain healthy neural networks. We previously showed that the CX3C chemokine fractalkine is secreted from degenerated neurons and accelerates microglial clearance of neuronal debris via inducing the release of MFG-E8. However, the mechanisms by which microglia produce MFG-E8 and the precise functions of MFG-E8 are unknown. METHODS: The release of MFG-E8 from microglia treated with conditioned medium from neurons exposed to neurotoxic substances, glutamate or oligomeric amyloid β (oAβ) was measured by ELISA. The neuroprotective effects of MFG-E8 and MFG-E8 − induced microglial phagocytosis of oAβ were assessed by immunocytochemistry. The effects of MFG-E8 on the production of the anti-oxidative enzyme hemeoxygenase-1 (HO-1) were determined by ELISA and immunocytochemisty. RESULTS: MFG-E8 was induced in microglia treated with conditioned medium from neurons that had been exposed to neurotoxicants, glutamate or oAβ. MFG-E8 significantly attenuated oAβ-induced neuronal cell death in a primary neuron − microglia coculture system. Microglial phagocytosis of oAβ was accelerated by MFG-E8 treatment due to increased CD47 expression in the absence of neurotoxic molecule production, such as tumor necrosis factor-α, nitric oxide, and glutamate. MFG-E8 − treated microglia induced nuclear factor E(2) − related factor 2 (Nrf2) − mediated HO-1 production, which also contributed to neuroprotection. CONCLUSIONS: These results suggest that microglia release MFG-E8 in response to signals from degenerated neurons and that MFG-E8 protects oAβ-induced neuronal cell death by promoting microglial phagocytic activity and activating the Nrf2-HO-1 pathway. Thus, MFG-E8 may have novel roles as a neuroprotectant in neurodegenerative conditions

Video-assisted thoracic surgery attenuates perioperative oxidative stress response in lung cancer patients: a preliminary study

Objectives: Reactive oxygen species (ROS) function as key metabolites that can impair biological processes. The aim of this preliminary study was to assess the perioperative oxidative changes in lung cancer surgery.Methods: We measured the levels of blood hydroperoxides, a type of ROS, as an index of oxidative injury to cellular components, as well as the plasma ferric-reducing ability as an index of total antioxidant potential in 32 lung cancer patients. Hydroperoxides were measured by the levels of diacron reactive oxygen metabolites (d-ROMs). The antioxidant potential was determined by the biological antioxidant potential (BAP), which represents the levels of endogenous antioxidant enzymes.Results: Lung cancer patients had slightly affected oxidative stress before surgery. The d-ROM and BAP levels after surgery and were significantly decreased than before surgery (p< 0.001) and the levels recovered preoperatively at third postoperative day. The d-ROM level in video-assisted surgery group (n=17) was significantly decreased than those in thoracotomy group (n=15) at third and seventh post-operative day (p < 0.001, and < 0.02).Conclusions: Lung cancer patients had already exposed oxidative stress before surgery and surgical intervention also generates large amounts of ROS. Video-assisted thoracic surgery can reduce the ROS compared to the standard thoractomy

The Japanese Clinical Practice Guideline for acute kidney injury 2016

Acute kidney injury (AKI) is a syndrome which has a broad range of etiologic factors depending on different clinical settings. Because AKI has significant impacts on prognosis in any clinical settings, early detection and intervention are necessary to improve the outcomes of AKI patients. This clinical guideline for AKI was developed by a multidisciplinary approach with nephrology, intensive care medicine, blood purification, and pediatrics. Of note, clinical practice for AKI management which was widely performed in Japan was also evaluated with comprehensive literature search

Blockade of Gap Junction Hemichannel Suppresses Disease Progression in Mouse Models of Amyotrophic Lateral Sclerosis and Alzheimer's Disease

Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimer's disease mouse model.Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases

Production of Embryonic and Fetal-Like Red Blood Cells from Human Induced Pluripotent Stem Cells

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications