Tribute to Dr. Takuo Aoyagi, inventor of pulse oximetry.

INTRODUCTION: Dr. Takuo Aoyagi invented pulse oximetry in 1974. Pulse oximeters are widely used worldwide, most recently making headlines during the COVID-19 pandemic. Dr. Aoyagi passed away on April 18, 2020, aware of the significance of his invention, but still actively searching for the theory that would take his invention to new heights. METHOD: Many people who knew Dr. Aoyagi, or knew of him and his invention, agreed to participate in this tribute to his work. The authors, from Japan and around the world, represent all aspects of the development of medical devices, including scientists and engineers, clinicians, academics, business people, and clinical practitioners. RESULTS: While the idea of pulse oximetry originated in Japan, device development lagged in Japan due to a lack of business, clinical, and academic interest. Awareness of the importance of anesthesia safety in the US, due to academic foresight and media attention, in combination with excellence in technological innovation, led to widespread use of pulse oximetry around the world. CONCLUSION: Dr. Aoyagi's final wish was to find a theory of pulse oximetry. We hope this tribute to him and his invention will inspire a new generation of scientists, clinicians, and related organizations to secure the foundation of the theory.

Ecological and genomic profiling of anaerobic methane-oxidizing archaea in a deep granitic environment.

Recent single-gene-based surveys of deep continental aquifers demonstrated the widespread occurrence of archaea related to Candidatus Methanoperedens nitroreducens (ANME-2d) known to mediate anaerobic oxidation of methane (AOM). However, it is unclear whether ANME-2d mediates AOM in the deep continental biosphere. In this study, we found the dominance of ANME-2d in groundwater enriched in sulfate and methane from a 300-m deep underground borehole in granitic rock. A near-complete genome of one representative species of the ANME-2d obtained from the underground borehole has most of functional genes required for AOM and assimilatory sulfate reduction. The genome of the subsurface ANME-2d is different from those of other members of ANME-2d by lacking functional genes encoding nitrate and nitrite reductases and multiheme cytochromes. In addition, the subsurface ANME-2d genome contains a membrane-bound NiFe hydrogenase gene putatively involved in respiratory H2 oxidation, which is different from those of other methanotrophic archaea. Short-term incubation of microbial cells collected from the granitic groundwater with 13C-labeled methane also demonstrates that AOM is linked to microbial sulfate reduction. Given the prominence of granitic continental crust and sulfate and methane in terrestrial subsurface fluids, we conclude that AOM may be widespread in the deep continental biosphere.

Deep microbial life in high-quality granitic groundwater from geochemically and geographically distinct underground boreholes.

Deep granitic aquifer is one of the largest, but least understood, microbial habitats. To avoid contamination from the surface biosphere, underground drilling was conducted for 300 m deep granitic rocks at the Mizunami underground research laboratory (URL), Japan. Slightly alkaline groundwater was characterized by low concentrations of dissolved organic matter and sulfate and the presence of > 100 nM H2 . The initial biomass was the highest (∼10(5) cells ml(-1) ) with the dominance of Hydrogenophaga spp., whereas the phylum Nitrospirae became predominant after 3 years with decreasing biomass (∼10(4) cells ml(-1) ). One week incubation of groundwater microbes after 3 years with (13) C-labelled bicarbonate and 1% H2 and subsequent single-cell imaging with nanometer-scale secondary ion mass spectrometry demonstrated that microbial cells were metabolically active. Pyrosequencing of microbial communities in groundwater retrieved at 3-4 years after drilling at the Mizunami URL and at 14 and 25 years after the drilling at the Grimsel Test Site, Switzerland, revealed the occurrence of common Nitrospirae lineages at the geographically distinct sites. As the close relatives of the Nitrospirae lineages were exclusively detected from deep groundwaters and terrestrial hot springs, it suggests that these bacteria are indigenous and potentially adapted to the deep terrestrial subsurface.

Biogeochemical signals from deep microbial life in terrestrial crust.

In contrast to the deep subseafloor biosphere, a volumetrically vast and stable habitat for microbial life in the terrestrial crust remains poorly explored. For the long-term sustainability of a crustal biome, high-energy fluxes derived from hydrothermal circulation and water radiolysis in uranium-enriched rocks are seemingly essential. However, the crustal habitability depending on a low supply of energy is unknown. We present multi-isotopic evidence of microbially mediated sulfate reduction in a granitic aquifer, a representative of the terrestrial crust habitat. Deep meteoric groundwater was collected from underground boreholes drilled into Cretaceous Toki granite (central Japan). A large sulfur isotopic fractionation of 20-60‰ diagnostic to microbial sulfate reduction is associated with the investigated groundwater containing sulfate below 0.2 mM. In contrast, a small carbon isotopic fractionation (<30‰) is not indicative of methanogenesis. Except for 2011, the concentrations of H2 ranged mostly from 1 to 5 nM, which is also consistent with an aquifer where a terminal electron accepting process is dominantly controlled by ongoing sulfate reduction. High isotopic ratios of mantle-derived 3He relative to radiogenic 4He in groundwater and the flux of H2 along adjacent faults suggest that, in addition to low concentrations of organic matter (<70 µM), H2 from deeper sources might partly fuel metabolic activities. Our results demonstrate that the deep biosphere in the terrestrial crust is metabolically active and playing a crucial role in the formation of reducing groundwater even under low-energy fluxes.

Novel microbial populations in deep granitic groundwater from Grimsel Test Site, Switzerland.

Freshwater aquifers in granitic rocks are widespread microbial habitats in the terrestrial subsurface. Microbial populations in deep granitic groundwater from two recently drilled (1 and 2 years) and two old boreholes (14 and 25 years) were compared. The 16S rRNA gene sequences related to "Candidatus Magnetobacterium bavaricum", Thermodesulfovibrio spp. of Nitrospirae (90.5-93.1 % similarity) and a novel candidate division with <90 % similarity to known cultivated species were dominant in all boreholes. Most of the environmental clones closely related to the novel lineages in Nitrospirae, which have been detected exclusively in deep groundwater samples. In contrast, betaproteobacterial sequences related to the family Rhodocyclaceae were obtained only from the recently drilled boreholes, which had higher total cell numbers. Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) analysis supported the result from clone library analysis; betaproteobacterial cells were dominantly detected in recently drilled boreholes. These results suggest that while indigenous microbial populations represented by the novel phylotypes persisted in the boreholes for 25 years, betaproteobacterial species disappeared after 2 years owing to the change of substrate availability.

A new DNA extraction method by controlled alkaline treatments from consolidated subsurface sediments.

Microbial communities that thrive in subterranean consolidated sediments are largely unknown owing to the difficulty of extracting DNA. As this difficulty is often attributed to DNA binding onto the silica-bearing sediment matrix, we developed a DNA extraction method for consolidated sediment from the deep subsurface in which silica minerals were dissolved by being heated under alkaline conditions. NaOH concentrations (0.07 and 0.33 N), incubation temperatures (65 and 94 °C) and incubation times (30-90 min) before neutralization were evaluated based on the copy number of extracted prokaryotic DNA. Prokaryotic DNA was detected by quantitative PCR analysis after heating the sediment sample at 94 °C in 0.33 N NaOH solution for 50-80 min. Results of 16S rRNA gene sequence analysis of the extracted DNA were all consistent with regard to the dominant occurrence of the metallophilic bacterium, Cupriavidus metallidurans, and Pseudomonas spp. Mineralogical analysis revealed that the dissolution of a silica mineral (opal-CT) during alkaline treatment was maximized at 94 °C in 0.33 N NaOH solution for 50 min, which may have resulted in the release of DNA into solution. Because the optimized protocol for DNA extraction is applicable to subterranean consolidated sediments from a different locality, the method developed here has the potential to expand our understanding of the microbial community structure of the deep biosphere.