A Comparative Study of Methods for Detecting Extraterrestrial Life in Exploration Missions to Mars and the Solar System II: Targeted Characteristics, Detection Techniques, and Their Combination for Survey, Detection, and Analysis.

We present a comparative study of the methods used in the search for extraterrestrial microorganism life, including a summary table where different life-detection techniques can be easily compared as an aid to mission and instrument design aimed at life detection. This is an extension of previous study, where detection techniques for a series of target characteristics and molecules that could constitute a positive life detection were evaluated. This comparison has been extended with a particular consideration to sources of false positives, the causes of negative detection, the results of detection techniques when presented regarding terrestrial life, and additional science objectives that could be achieved outside the primary aim of detecting life. These additions address both the scientific and programmatic side of exploration mission design, where a successful proposal must demonstrate probable outcomes and be able to return valuable results even if no life is found. The applicability of the life detection techniques is considered for Earth life, Earth-independent life (life emerging independently from that on Earth,) and Earth-kin life (sharing a common ancestor with life on Earth), and techniques effective in detecting Earth life should also be useful in the detection of Earth-kin life. However, their applicability is not guaranteed for Earth-independent life. As found in our previous study, there exists no realistic single detection method that can conclusively determine the discovery of extraterrestrial life, and no method is superior to all others. In this study, we further consider combinations of detection techniques and identify imaging as a valuable addition to molecule detection methods, even in cases where there is insufficient resolution to observe the detailed morphology of a microbial cell. The search for extraterrestrial life is further divided into a survey-and-detection and analysis-and-conclusion step. These steps benefit from different detection techniques, but imaging is necessary for both parts.

Comparative study of methods for detecting extraterrestrial life in exploration mission of Mars and the solar system.

The detection and analysis of extraterrestrial life are important issues of space science. Mars is among the most important planets to explore for extraterrestrial life, owing both to its physical properties and to its ancient and present environments as revealed by previous exploration missions. In this paper, we present a comparative study of methods for detecting extraterrestrial life and life-related substances. To this end, we have classified and summarized the characteristics targeted for the detection of extraterrestrial life in solar system exploration mission and the methods used to evaluate them. A summary table is presented. We conclude that at this moment (i) there is no realistic single detection method capable of concluding the discovery of extraterrestrial life, (ii) no single method has an advantage over the others in all respects, and (iii) there is no single method capable of distinguishing extraterrestrial life from terrestrial life. Therefore, a combination of complementary methods is essential. We emphasize the importance of endeavoring to detect extraterrestrial life without overlooking possible alien life forms, even at the cost of tolerating false positives. Summaries of both the targets and the detection methods should be updated continuously, and comparative studies of both should be pursued. Although this study assumes Mars to be a model site for the primary environment for life searches, both the targets and detection methods described herein will also be useful for searching for extraterrestrial life in any celestial environment and for the initial inspection of returned samples.

A rare case of multiple brain abscesses caused by apical periodontitis of deciduous teeth in congenital heart disease: a case report.

BACKGROUND: A brain abscess is a focal infection in which abscesses form in the brain. A brain abscess is a rare but fatal disease when rupture occurs into the ventricles. We report a case of multiple brain abscesses caused by a hematogenous infection from the apical periodontitis of deciduous teeth. CASE PRESENTATION: The patient was a 7-years and 8-months-old male with congenital heart disease. The patient sought medical attention due to fever and headache, for which he was started on three antibiotics with a diagnosis of multiple brain abscesses. Given that apical periodontitis of deciduous teeth was strongly suspected as the source of the brain abscess, the deciduous teeth were extracted. Immediately after deciduous teeth extraction, the patient's headache and neurological symptoms disappeared. CONCLUSIONS: After teeth extraction, a clear shrinkage of the brain abscess was observed, and the patient was discharged from the hospital.

Anti-RANKL Inhibits Thymic Function and Causes DRONJ in Mice.

Background: Denosumab, a human monoclonal antibody against receptor activator of nuclear factor-kappa B ligand (RANKL), is a novel bone antiresorptive agent used in patients with osteoporosis or metastatic bone cancer. Denosumab-related osteonecrosis of the jaw (DRONJ) has been recently reported in patients using denosumab. However, the mechanisms of DRONJ are not fully understood. Appropriate pathogenic mechanisms of DRONJ have yet to be established. Therefore, we investigated the pathogenesis of DRONJ in mice. Methods: Anti-mouse RANKL monoclonal antibody and melphalan were performed to create a mouse model of DRONJ-like lesions in female C57BL/6J mice. We examined the development of DRONJ-like lesions and immune function. Results: We showed that administration of anti-mouse RANKL monoclonal antibody and melphalan caused DRONJ-like lesions that recapitulated major clinical manifestations of the human disease, including the characteristic features of an open alveolar socket and exposed necrotic bone. In the analysis using a mouse model of DRONJ-like lesion, it was revealed that anti-mouse RANKL monoclonal antibody and melphalan suppress autoimmune regulator (AIRE) expression in the thymus and imbalanced T cell populations. Conclusion: This study suggests evidence of an immunity-based mechanism of DRONJ-like disease. This work may contribute to a better understanding of the pathogenesis of human DRONJ.

Osteoclast formation from mouse bone marrow cells on micro/nano-scale patterned surfaces.

OBJECTIVES: Osteoclasts can sense the surface topography of materials. However, it is difficult to identify the structural factors that affect osteoclast formation and its function. Furthermore, we hypothesized that the type of osteoclast precursor cells also affects osteoclastogenesis in the materials. In this study, we investigated the effects of defined micro/nanoscale patterns on osteoclastogenesis from bone marrow cells (BMCs). METHODS: Various cyclo-olefin polymer (COP) patterns were prepared using nanoimprinting. The effects of shape, size, and height of the patterns, and the wettability of the patterned surfaces on osteoclastogenesis from BMCs were evaluated in vitro. RESULTS: Osteoclast formation was promoted on pillars (diameter, 1 µm or 500 nm; height, 500 nm). Notably, osteoclastogenesis from BMCs was better promoted on hydrophobic pillars than on hydrophilic pillars. In contrast, decreased osteoclast formation was observed on the nanopillars (diameter, 100 nm; height, 200 nm). CONCLUSIONS: We demonstrated the promotion of osteoclast formation from BMCs on hydrophobic pillars with diameters of 1 µm and 500 nm. Some cellular behaviors in the patterns were dependent on the type of osteoclast precursor cells. The designed patterns are useful for designing the surface of dental implants or bone replacement materials with a controllable balance between osteoblast and osteoclast activities.

Bone resorption improvement by conditioned medium of stem cells from human exfoliated deciduous teeth in ovariectomized mice.

Stem cells from human exfoliated deciduous teeth (SHED) are mesenchymal stem cells with multipotent differentiation potential present in the dental pulp tissue of the deciduous teeth. SHED produce secretions that have immunomodulatory and regenerative functions. In the present study, we investigated the effects of SHED-conditioned medium (SHED-CM) on osteopenia induced by the ovariectomy (OVX) phenotype and its corresponding immunological changes. Eleven-week-old female C3H/HeJ mice were subjected to OVX. SHED-CM was administered intraperitoneally in these mice for 4 weeks starting immediately after OVX. SHED-CM improved bone mass after OVX and elevated the polarization of M2 macrophages in the peritoneal cavity. SHED-CM also suppressed an OVX-induced increase in interferon-γ (INF-γ) and interleukin-17 (IL-17) concentrations in the peripheral blood. Inhibition of M2 macrophage polarization with neutralizing antibodies did not reduce the concentration of IFN-γ and IL-17 in peripheral blood, which were increased by OVX, and did not alleviate osteopenia induced by the OVX phenotype. Mechanistically, these findings suggest that SHED-CM alleviates bone resorption by suppressing the activation of IFN-γ and IL-17 cells by polarizing M2 macrophages. In conclusion, our data indicate that SHED-CM contains active secretions that may have promising efficacy to ameliorate OVX-induced osteopenia. We suggest that SHED-CM has the potential to be used as a novel therapeutic agent to inhibit osteoporosis.

In situ biochemical characterization of Venus cloud particles using a life-signature detection microscope.

Much of the information about the size and shape of aerosols forming haze and the cloud layer of Venus is obtained from indirect inferences from nephelometers on probes and from the analysis of the variation of polarization with the phase angle and the glory feature from images of Venus. The microscopic imaging of Venus' aerosols has recently been advocated. Direct measurements from a fluorescence microscope can provide information on the morphology, density, and biochemical characteristics of the particles; thus, fluorescence microscopy is attractive for in situ particle characterization of the Venus cloud layer. Fluorescence imaging of Venus cloud particles presents several challenges owing to the sulfuric acid composition and corrosive effects. In this article, we identify the challenges and describe our approach to overcoming them for a fluorescence microscope based on an in situ biochemical and physical characterization instrument for use in the clouds of Venus from a suitable aerial platform. We report that pH adjustment using alkali was effective for obtaining fluorescence images and that fluorescence attenuation was observed after the adjustment, even when the acidophile suspension in concentrated sulfuric acid was used as a sample.

N-acetylcysteine attenuates PGE2 and ROS production stimulated by 4-META/MMA-based resin in murine osteoblastic cells.

This study examined the effects of N-acetylcysteine (NAC) on the inflammatory reactions of murine osteoblastic cells cultured on the 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate (4-META/MMA)-based resin. Superbond C&B (SB) was used as the 4-META/MMA-based resin and placed in a 48-well cell culture plate. The cells were cultured in αMEM (control) as well as on SB and SB in αMEM with NAC (SB+NAC). They were examined using the WST-1 proliferation assay, real-time PCR, enzyme-linked immunosorbent assay (ELISA), intracellular reactive oxygen species (ROS) measurements, and cellular glutathione (GSH) detection. COX-2 and IL-6 gene expressions were upregulated in SB; however, they were suppressed by NAC. Furthermore, PGE2 production in the culture medium was increased in SB, whereas NAC decreased the PGE2 production. NAC lowered the ROS level in the culture medium and significantly increased the intracellular GSH level. The present in vitro study demonstrated that NAC might be effective for dental material detoxification.

Draft Genome Sequence of Clostridium tagluense Strain A121T, Isolated from a Permafrost Core in the Canadian High Arctic.

Clostridium tagluense strain A121T was isolated from a permafrost core in the Canadian High Arctic. This endospore-forming strain is a strictly anaerobic, Gram-positive, and psychrotolerant bacterium. This article describes the draft genome sequence of C. tagluense strain A121T.

Patients with SATB2-associated syndrome exhibiting multiple odontomas.

Special AT-rich sequence-binding protein 2 (SATB2)-associated syndrome (SAS) is characterized by alterations of SATB2. Its clinical features include intellectual disability and craniofacial abnormalities, such as cleft palate, dysmorphic features, and dental abnormalities. Here, we describe three previously undiagnosed, unrelated patients with SAS who exhibited dental abnormalities, including multiple odontomas. Although isolated odontomas are common, multiple odontomas are rare. Individuals in families 1 and 3 underwent whole-exome sequencing. Patient 2 and parents underwent targeted amplicon sequencing. On the basis of the hg19/GRCh37 reference and the RefSeq mRNA NM_001172517, respective heterozygous mutations were found and validated in Patients 1, 2, and 3: a splice-site mutation (chr2:g.200137396C > T, c.1741-1G > A), a nonsense mutation (chr2:g.200213750G > A, c.847C > T, p.R283*), and a frame-shift mutations (chr2:g.200188589_200188590del, c.1478_1479del, p.Q493Rfs*19). All mutations occurred de novo. The mutations in Patients 1 and 3 were novel; the mutation in Patient 2 has been described previously. Tooth mesenchymal cells derived from Patient 2 showed diminished SATB2 expression. Multiple odontomas were evident in the patients in this report; however, this has not been recognized previously as a SAS-associated phenotype. We propose that multiple odontomas be considered as an occasional manifestation of SAS.

Expression and Dynamics of Podoplanin in Cultured Osteoblasts with Mechanostress and Mineralization Stimulus.

This study investigates the significance of the expression and dynamics of podoplanin in mechanostress and mineralization in cultured murine osteoblasts. Podoplanin increased in osteoblasts subjected to straining in non-mineralization medium, suggesting that the mechanostress alone is a podoplanin induction factor. In osteoblasts subjected to vertical elongation straining in the mineralization medium, the mRNA amounts of podoplanin, osteopontin, and osteocalcin were significantly larger than those in cells not subjected to straining, suggesting that mechanostress is the cause of a synergistic effect in the expression of these proteins. In osteoblasts in the mineralization medium, significant increases in osteocalcin mRNA occurred earlier in cells subjected to straining than in the cells not subjected to straining, suggesting that the mechanostress is a critical factor to enhance the expression of osteocalcin. Western blot and ELISA analysis showed increased podoplanin production in osteoblasts with longer durations of straining. There was significantly less mineralization product in osteoblasts with antibodies for podoplanin, osteopontin, and osteocalcin. There was also less osteopontin and osteocalcin produced in osteoblasts with anti-podoplanin. These findings suggest that mechanostress induces the production of podoplanin in osteoblasts and that podoplanin may play a role in mineralization in cooperation with bone-associated proteins.

Indomethacin inhibits melanogenesis via down-regulation of Mitf mRNA transcription.

Non-steroidal anti-inflammatory drugs (NSAIDs) exhibit several divergent biological effects. In this study, we investigated the effect of indomethacin on melanin synthesis using B16F1 melanoma cells. Indomethacin inhibited α-melanocyte stimulating hormone (α-MSH)-enhanced melanin synthesis in a dose-dependent manner. Western blotting analysis revealed that indomethacin significantly suppressed tyrosinase and Mitf protein levels. In a luciferase reporter assay, we found that indomethacin reduced tyrosinase promoter activity. Moreover, real-time RT-PCR analysis showed that indomethacin lowered mRNA levels of melanogenic genes, including Mitf. Together, our findings indicate that indomethacin inhibits melanogenesis via the suppression of Mitf transcription.

How much medium do you use for cell culture? Medium volume influences mineralization and osteoclastogenesis in vitro.

Bone is maintained by a balance between bone formation and resorption. This remodeling is controlled by a wide variety of systemic and local factors including hormones, cytokines and mechanical stresses. The present in vitro study examined the impact of medium volume, using 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 ml/well in a 24­well plate, on the differentiation of osteoblasts and osteoclasts. There were no differences in the alkaline phosphatase activity of osteoblasts amongst the groups; however, the area of mineral deposition was decreased in a media volume­dependent manner. A co­culture of osteoblastic cells with bone marrow cells revealed a reduction in the total number of osteoclastic tartrate­resistant acid phosphatase (TRAP)­positive multinuclear cells (≥2 nuclei), whereas the formation of large osteoclastic TRAP­positive multinuclear cells (≥8 nuclei) was increased, in a media volume­dependent manner. There were also no differences in receptor activator of nuclear factor­κB ligand mRNA and total osteoprotegerin (OPG) protein expression levels amongst the groups, however the concentration of OPG decreased in a media volume­dependent manner. In conclusion, the present study demonstrated that the suppression of mineralization in osteoblastic cells and the stimulation of osteoclast fusion are dependent on the medium volume, indicating that media volume is an important factor in in vitro cell culture systems.

Release from optimal compressive force suppresses osteoclast differentiation.

Bone remodeling is an important factor in orthodontic tooth movement. During orthodontic treatment, osteoclasts are subjected to various mechanical stimuli, and this promotes or inhibits osteoclast differentiation and fusion. It has been previously reported that the release from tensile force induces osteoclast differentiation. However, little is known about how release from compressive force affects osteoclasts. The present study investigated the effects of release from compressive force on osteoclasts. The number of tartrate­resistant acid phosphatase (TRAP)­positive multinucleated osteoclasts derived from RAW264.7 cells was counted, and gene expression associated with osteoclast differentiation and fusion in response to release from compressive force was evaluated by reverse transcription­quantitative polymerase chain reaction. Osteoclast number was increased by optimal compressive force application. On release from this force, osteoclast differentiation and fusion were suppressed. mRNA expression of NFATc1 was inhibited for 6 h subsequent to release from compressive force. mRNA expression of the other osteoclast­specific genes, TRAP, RANK, matrix metalloproteinase­9, cathepsin­K, chloride channel 7, ATPase H+ transporting vacuolar proton pump member I, dendritic cell­specific transmembrane protein and osteoclast stimulatory transmembrane protein (OC­STAMP) was significantly inhibited at 3 h following release from compressive force compared with control cells. These findings suggest that release from optimal compressive force suppresses osteoclast differentiation and fusion, which may be important for developing orthodontic treatments.

Archaeal communities of Arctic methane-containing permafrost.

In the present study, we used culture-independent methods to investigate the diversity of methanogenic archaea and their distribution in five permafrost samples collected from a borehole in the Kolyma River Lowland (north-east of Russia). Total DNA was extracted from methane-containing permafrost samples of different age and amplified by PCR. The resulting DNA fragments were cloned. Phylogenetic analysis of the sequences showed the presence of archaea in all studied samples; 60%-95% of sequences belonged to the Euryarchaeota. Methanogenic archaea were novel representatives of Methanosarcinales, Methanomicrobiales, Methanobacteriales and Methanocellales orders. Bathyarchaeota (Miscellaneous Crenarchaeota Group) representatives were found among nonmethanogenic archaea in all the samples studied. The Thaumarchaeota representatives were not found in the upper sample, whereas Woesearchaeota (formerly DHVEG-6) were found in the three deepest samples. Unexpectedly, the greatest diversity of archaea was observed at a depth of 22.3 m, probably due to the availability of the labile organic carbon and/or due to the migration of the microbial cells during the freezing front towards the bottom.

Optimal compressive force accelerates osteoclastogenesis in RAW264.7 cells.

Mechanical stress produced by orthodontic forces is a factor in the remodeling of periodontal ligaments (PDLs) and alveolar bone. It has been reported that the expression of a number of cytokines associated with osteoclastogenesis is upregulated when compressive forces act on osteoblasts and PDL cells. The present study investigated the effects of compressive forces on the formation of osteoclasts from the macrophage cell line RAW264.7. Compressive forces on osteoclasts were exerted using layers of 3, 5, 7, 9 or 14 glass cover slips on the 4th day of culture for 24 h. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. Osteoclastogenesis advanced rapidly on days four and five. The number of osteoclasts with >8 nuclei peaked when the force of 7 slips was applied, which was therefore regarded as the optimal compressive force. Alterations in the expression of osteoclast-associated genes are associated with changes in the differentiation and fusion of macrophages in response to compressive forces; therefore, osteoclast-associated genes were assessed by reverse transcription quantitative polymerase chain reaction in the present study. The mRNA expression of osteoclast­associated genes increased significantly after 3 h of optimal compression, whereas mRNA expression increased after 24 h in the control group. These findings suggested that osteoclastogenesis of macrophages was accelerated when an optimal compressive force was applied.

Rosmarinic acid and arbutin suppress osteoclast differentiation by inhibiting superoxide and NFATc1 downregulation in RAW 264.7 cells.

The present study investigated the effect of the natural polyphenols, rosmarinic acid and arbutin, on osteoclast differentiation in RAW 264.7 cells. Rosmarinic acid and arbutin suppressed osteoclast differentiation and had no cytotoxic effect on osteoclast precursor cells. Rosmarinic acid and arbutin inhibited superoxide production in a dose-dependent manner. mRNA expression of the master regulator of osteoclastogenesis, nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and the osteoclast marker genes, matrix metalloproteinase-9, tartrate-resistant acid phosphatase and cathepsin-K, decreased following treatments with rosmarinic acid and arbutin. Furthermore, resorption activity decreased with the number of osteoclasts. These results suggest that rosmarinic acid and arbutin may be useful for the prevention and treatment of bone diseases, such as osteoporosis, through mechanisms involving inhibition of superoxide and downregulation of NFATc1.

Recruitment of mesenchymal stem cells by stromal cell-derived factor 1α in pulp cells from deciduous teeth.

Dental pulp cells (DPCs), including dental pulp (DP) stem cells, play a role in dentine repair under certain conditions caused by bacterial infections associated with caries, tooth fracture and injury. Mesenchymal stem cells (MSCs) have also been shown to be involved in this process of repair. However, the mechanisms through which MSCs are recruited to the DP have not yet been elucidated. Therefore, the aim of the present in vitro study was to investigate whether stromal cell-derived factor 1α (SDF1)-C-X-C chemokine receptor type 4 (CXCR4) signaling is involved in tissue repair in the DP of deciduous teeth. A single-cell clone from DPCs (SDP11) and UE7T-13 cells were used as pulp cells and MSCs, respectively. The MG-63 and HuO9 cells, two osteosarcoma cell lines, were used as positive control cells. Reverse transcription polymerase chain reaction (RT-PCR) revealed that all cell lines (SDP11, UE7T-13 MG-63 and HuO9) were positive for both SDF1 and CXCR4 mRNA expression. Moreover, immunocytochemical analysis indicated that SDF1 and CXCR4 proteins were expressed in the SDP11 and UE7T-13 cells. SDF1 was also detected in the cell lysates (CLs) and conditioned medium (CM) collected from the SDP11 and UE7T-13 cells, and AMD3100, a specific antagonist of CXCR4, inhibited the migration of the UE7T-13 cells; this migration was induced by treatment with CM, which was collected from the SDP11 cells. In addition, real-time PCR showed that the expression of SDF1 in the SDP11 cells was inhibited by treatment with 20 ng/ml fibroblast growth factor (FGF)-2, and exposure to AZD4547, an inhibitor of the FGF receptor, blocked this inhibition. Collectively, these data suggest that SDF1 produced by DP plays an important role in homeostasis, repair and regeneration via the recruitment of MSCs.

The Effects of Perchlorates on the Permafrost Methanogens: Implication for Autotrophic Life on Mars.

The terrestrial permafrost represents a range of possible cryogenic extraterrestrial ecosystems on Earth-like planets without obvious surface ice, such as Mars. The autotrophic and chemolithotrophic psychrotolerant methanogens are more likely than aerobes to function as a model for life forms that may exist in frozen subsurface environments on Mars, which has no free oxygen, inaccessible organic matter, and extremely low amounts of unfrozen water. Our research on the genesis of methane, its content and distribution in permafrost horizons of different ages and origin demonstrated the presence of methane in permanently frozen fine-grained sediments. Earlier, we isolated and described four strains of methanogenic archaea of Methanobacterium and Methanosarcina genera from samples of Pliocene and Holocene permafrost from Eastern Siberia. In this paper we study the effect of sodium and magnesium perchlorates on growth of permafrost and nonpermafrost methanogens, and present evidence that permafrost hydogenotrophic methanogens are more resistant to the chaotropic agent found in Martian soil. In this paper we study the effect of sodium and magnesium perchlorates on the growth of permafrost and nonpermafrost methanogens, and present evidence that permafrost hydogenotrophic methanogens are more resistant to the chaotropic agent found in Martian soil. Furthermore, as shown in the studies strain M2(T) M. arcticum, probably can use perchlorate anion as an electron acceptor in anaerobic methane oxidation. Earth's subzero subsurface environments are the best approximation of environments on Mars, which is most likely to harbor methanogens; thus, a biochemical understanding of these pathways is expected to provide a basis for designing experiments to detect autotrophic methane-producing life forms on Mars.

Distribution of antibiotic resistance genes in glacier environments.

Antibiotic resistance genes are biologically transmitted from microorganism to microorganism in particular micro-environments where dense microbial communities are often exposed to an intensive use of antibiotics, such as intestinal microflora, and the soil microflora of agricultural fields. However, recent studies have detected antibiotic-resistant bacteria and/or antibiotic resistance genes in the natural environment geographically isolated from such areas. Here we sought to examine the prevalence of antibiotic resistance genes in 54 snow and ice samples collected from the Arctic, Antarctic, Central Asia, North and South America and Africa, to evaluate the level of these genes in environments supposedly not affected by anthropogenic factors. We observed a widespread distribution of antibiotic resistance genes in samples from various glaciers in Central Asia, North and South America, Greenland and Africa. In contrast, Antarctic glaciers were virtually free from these genes. Antibiotic resistance genes, of both clinical (i.e. aac(3), blaIMP) and agricultural (i.e. strA and tetW) origin, were detected. Our results show regional geographical distribution of antibiotic resistance genes, with the most plausible modes of transmission through airborne bacteria and migrating birds.