Senescent Phenotype Induced by p90RSK-NRF2 Signaling Sensitizes Monocytes and Macrophages to Oxidative Stress in HIV-Positive Individuals.

BACKGROUND: The incidence of cardiovascular disease is higher in HIV-positive (HIV+) patients than it is in the average population, and combination antiretroviral therapy (cART) is a recognized risk factor for cardiovascular disease. However, the molecular mechanisms that link cART and cardiovascular disease are currently unknown. Our study explores the role of the activation of p90RSK, a reactive oxygen species-sensitive kinase, in engendering senescent phenotype in macrophages and accelerating atherogenesis in patients undergoing cART. METHODS: Peripheral whole blood from cART-treated HIV+ individuals and nontreated HIV-negative individuals was treated with H2O2 (200 µmol/L) for 4 minutes, and p90RSK activity in CD14+ monocytes was measured. Plaque formation in the carotids was also analyzed in these individuals. Macrophage senescence was determined by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. The involvement of p90RSK-NRF2 signaling in cART-induced senescence was assessed by p90RSK-specific inhibitor (FMK-MEA) or dominant-negative p90RSK (DN-p90RSK) and NRF2 activator (NRF2A). Further, the severity of atherosclerosis was determined in myeloid cell-specific wild-type and DN-p90RSK transgenic mice. RESULTS: Monocytes from HIV+ patients exhibited higher levels of p90RSK activity and were also more sensitive to reactive oxygen species than monocytes from HIV-negative individuals. A multiple linear regression analysis involving cART, Reynolds cardiovascular risk score, and basal p90RSK activity revealed that cART and basal p90RSK activity were the 2 significant determinants of plaque formation. Many of the antiretroviral drugs individually activated p90RSK, which simultaneously triggered all components of the macrophage senescent phenotype. cART inhibited antioxidant response element reporter activity via ERK5 S496 phosphorylation. NRF2A reversed the H2O2-induced overactivation of p90RSK in cART-treated macrophages by countering the induction of senescent phenotype. Last, the data obtained from our gain- or loss-of-function mice conclusively showed the crucial role of p90RSK in inducing senescent phenotype in macrophages and atherogenesis. CONCLUSIONS: cART increased monocyte/macrophage sensitivity to reactive oxygen species- in HIV+ individuals by suppressing NRF2-ARE activity via p90RSK-mediated ERK5 S496 phosphorylation, which coordinately elicited senescent phenotypes and proinflammatory responses. As such, our report underscores the importance of p90RSK regulation in monocytes/macrophages as a viable biomarker and therapeutic target for preventing cardiovascular disease, especially in HIV+ patients treated with cART.

A Simple Ex Vivo Semiquantitative Fluorescent Imaging Utilizing Planar Laser Scanner: Detection of Reactive Oxygen Species Generation in Mouse Brain and Kidney.

OBJECTIVE: Oxidative stress plays an important role in the onset of many neuronal and peripheral disorders. We examined the feasibility of obtaining semiquantitative fluorescent images of reactive oxygen species (ROS) generation in mouse brain and kidney utilizing a planar laser scanner and dihydroethidium (DHE). METHODS: To investigate ROS generation in brain, sodium nitroprusside was injected into the striatum. Dihydroethidium was injected into the tail vein. After DHE injection, tissue slices were analyzed utilizing a planar laser scanner. For kidney study, cis-diamminedichloroplatinum [II] (cisplatin) was intraperitoneally administrated into mice. RESULTS: Clear and semiquantitative fluorescent images of ROS generation in the mouse brain and kidney were obtained. Furthermore, the fluorescence intensity was stable and not affected by fading. Sodium nitroprusside induced approximately 6 times the fluorescence accumulation in the brain. Cisplatin caused renal injury in all mice, and in comparison with control mice, more than 10 times fluorescence accumulation was observed in the renal medulla with tubular necrosis and vacuolization. CONCLUSIONS: We successfully obtained ex vivo semiquantitative fluorescent images of ROS generation utilizing a planar laser scanner and DHE. This simple method is useful for ROS detection in several ROS-related animal models and would be applicable to a variety of biochemical processes.

Some inconvenient truths about biosignatures involving two chemical species on Earth-like exoplanets.

The detection of strong thermochemical disequilibrium in the atmosphere of an extrasolar planet is thought to be a potential biosignature. In this article we present a previously unidentified kind of false positive that can mimic a disequilibrium or any other biosignature that involves two chemical species. We consider a scenario where the exoplanet hosts a moon that has its own atmosphere and neither of the atmospheres is in chemical disequilibrium. Our results show that the integrated spectrum of the planet and the moon closely resembles that of a single object in strong chemical disequilibrium. We derive a firm limit on the maximum spectral resolution that can be obtained for both directly imaged and transiting planets. The spectral resolution of even idealized space-based spectrographs that might be achievable in the next several decades is in general insufficient to break the degeneracy. Both chemical species can only be definitively confirmed in the same object if absorption features of both chemicals can be unambiguously identified and their combined depth exceeds 100%.

Zfp296 is a novel Klf4-interacting protein and functions as a negative regulator.

Pluripotency and self-renewing ability of embryonic stem (ES) cells are regulated by several transcription factors, including Oct3/4, Sox2, Kruppel-like factor 4 (Klf4), and c-Myc. These transcription factors reprogram somatic cells into induced pluripotent stem (iPS) cells. Zinc finger protein (Zfp) 296 has been reported to enhance iPS cell formation. Here we found that Zfp296 interacts with Klf4. A maltose-binding protein pull-down assay demonstrated that Klf4 binds to the Zfp296 158-483 amino acid region, and that Zfp296 binds to the Klf4 DNA-binding domain (DBD). A quantitative reverse transcription-polymerase chain reaction analysis revealed that expression of Zfp296 and Klf4 decreased during differentiation of E14 and ZHBTc4 ES cells. We also found that green fluorescent protein-labeled Zfp296 and Klf4 were localized to the nucleus. Because Zfp296 bound to the Klf4 DBD, we next examined the influence of Zfp296 on Klf4 DNA-binding activity. A biotin DNA pull-down assay showed that Klf4 binds to the Lefty1 promoter region, and that binding activity was sustained even in the presence of Zfp296. In contrast, a reporter assay showed that the Lefty1 promoter was activated by Klf4, and that the enhanced activity was repressed by Zfp296. These findings suggest that Zfp296 is a functional regulator of Klf4 in ES cells.

Serum soluble triggering receptor expressed on myeloid cells-2 was not altered by rTMS in patients with treatment-resistant depression.

AIM: Repetitive transcranial magnetic stimulation (rTMS) is one of the most effective and minimally invasive treatments for treatment-resistant depression (TRD). However, the mechanism underlying the therapeutic effects of rTMS in patients with TRD remains unclear. In recent years, the pathogenesis of depression has been closely associated with chronic inflammation and microglia are believed to play an important role in chronic inflammation. Triggering receptor expressed on myeloid cells-2 (TREM2) plays an important role in microglial neuroinflammatory regulation. In this study, we investigated the changes in peripheral soluble TREM2 (sTREM2) before and after rTMS treatment in patients with TRD. METHODS: Twenty-six patients with TRD were enrolled in this frequency (10 Hz) rTMS study. Depressive symptoms, cognitive function, and serum sTREM2 concentrations were measured at baseline and the end of the 6-week rTMS treatment. RESULTS: This study showed that rTMS ameliorated depressive symptoms and partially improved cognitive dysfunction in TRD. However, rTMS treatment did not alter serum sTREM2 levels. CONCLUSIONS: This is the first sTREM2 study in patients with TRD who underwent rTMS treatment. These results suggest that serum sTREM2 may not be relevant for the mechanism underlying the therapeutic effect of rTMS in patients with TRD. Future studies should confirm the present findings using a larger patient sample and a sham rTMS procedure, as well as CSF sTREM2. Furthermore, a longitudinal study should be conducted to clarify the effects of rTMS on sTREM2 levels.

Changes in the metabolites of cerebrospinal fluid induced by rTMS in treatment-resistant depression: A pilot study.

Repetitive transcranial magnetic stimulation (rTMS) improves depressive symptoms in treatment-resistant depression (TRD). This study aimed to analyze changes in cerebrospinal fluid (CSF) metabolites in patients with TRD after rTMS. Five patients with TRD were enrolled in a high frequency (10-Hz) rTMS study. The concentration of 72 CSF metabolites were measured at baseline and at the end of the 6-week rTMS treatment. rTMS significantly increased CSF niacinamide, kynurenine, and creatinine levels and significantly decreased CSF cystine levels, but not the levels of the other 68 CSF metabolites. This is the first CSF metabolomics study on patients with TRD who underwent rTMS.

Ultrastructural Analysis of Inflammatory Breast Cancer Cell Clusters in an Ex Vivo Environment Mechanically Mimicking the Lymph Vascular System.

BACKGROUND: Inflammatory breast cancer (IBC) is a rare form of breast cancer with a poor prognosis. IBC is characterized by florid lymphovascular tumor emboli in the skin and the parenchyma of the breast. We hypothesized that the formation of these emboli/clusters plays a pivotal role in IBC metastasis and its rapid progression, and that their structure and function may be a key to identifying molecular biological differences between IBC and non IBC. METHODS: Mechanical methods were used to mimic the lymph fluid viscosity by adding 2.25% of PEG8000 to the media. Clusters were obtained for IBC tumor cell lines (SUM149 and IBC-3), non IBC tumor cell lines (MDA-MB-231, MDA-MB-468, and MCF7), and a non-tumorigenic human mammary epithelial cell line (MCF10A). Clusters were analyzed by light microscopy, and then prepared for and observed by transmission electron microscopy (TEM). RESULTS: Significant differences were seen between IBC and non IBC clusters. The TEM analysis revealed that IBC cells harbored numerous microvilli and microvesicles, both on the free outer surface and inside the cluster. Microvilli from IBC cell clusters were noted at higher density and were longer than those of non IBC cell clusters. CONCLUSIONS: IBC tumor cell clusters exhibited distinct ultrastructural features characterized by the presence of long, crowded microvilli and numerous microvesicles. These microvilli may play an important role in the biology and aggressiveness of IBC.

Evaluation of intracellular processes in quinolinic acid-induced brain damage by imaging reactive oxygen species generation and mitochondrial complex I activity.

PURPOSE: Our study aimed to elucidate the intracellular processes associated with quinolinic acid (QA)-induced brain injury by acquiring semiquantitative fluorescent images of reactive oxygen species (ROS) generation and positron emission tomography (PET) images of mitochondrial complex I (MC-I) activity. METHODS: Ex vivo fluorescent imaging with dihydroethidium (DHE) and PET scans with 18F-BCPP-EF were conducted at 3 h and 24 h after QA injection into the rat striatum. Immunohistochemical studies were performed 24 h after QA injection into the rat brain using monoclonal antibodies against neuronal nuclei (NeuN) and CD11b. RESULTS: A strong DHE-derived fluorescent signal was detected in a focal area within the QA-injected striatum 3 h after QA injection, and increased fluorescent signal spread throughout the striatum and parts of the cerebral cortex after 24 h. By contrast, 18F-BCPP-EF uptake in the QA-injected rat brain was unchanged after 3 h and markedly decreased after 24 h, not only in the striatum but also in the cerebral hemisphere. The fluorescent signal in the striatum 24 h after QA injection colocalised with microglial marker expression. CONCLUSIONS: We successfully obtained functional images of focal ROS generation during the early period of excitotoxic injury, and microglial ROS generation and mitochondrial dysfunction were observed during the progression of the inflammatory response. Both ex vivo DHE imaging and in vivo 18F-BCPP-EF-PET were sufficiently sensitive to detect the respective processes of QA-induced brain damage. Our study contributes to the functional imaging of multiple events during the pathological process.

The changes in kynurenine metabolites induced by rTMS in treatment-resistant depression: A pilot study.

BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive brain stimulation technique that is considered a valuable and promising technique for improving depressive symptoms in treatment-resistant depression (TRD). However, the exact mechanism by which rTMS ameliorates depressive symptoms remains to be clarified. OBJECTIVE: The aim of the present study was to analyzed the changes in metabolites of patients with TRD in the rTMS treatment, especially focusing on the kynurenine (KYN) pathway. METHODS: Thirteen participants with TRD were enrolled in a high-frequency (10 Hz) rTMS study. Cognitive function, depressive symptoms and the concentration of plasma tryptophan (TRP) metabolites were measured at baseline and at the endpoint of rTMS treatment. RESULTS: rTMS treatment significantly improved depressive symptom scores and some subscales of cognitive dysfunction. The present study has demonstrated that rTMS treatment significantly increased plasma TRP levels and significantly decreased plasma serotonin levels, while plasma KYN and kynurenic acid level as well as KYN/TRP ratio remained unchanged. CONCLUSIONS: This is the first metabolomic study of patients with TRD undergoing rTMS treatment. To validate the present results, it is necessary to increase the number of cases including controls, use a sample of cerebrospinal fluid, and measure blood concentration over time in the course of rTMS treatment.

Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice.

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.

Reduction of Excessive Visceral Fat and Safety with 52-Week Administration of Lipase Inhibitor Orlistat in Japanese: Long-Term Clinical Study.

INTRODUCTION: Orlistat is an inhibitor of pancreatic lipase and is used as an anti-obesity drug in many countries. However, there are no data available regarding the effects of orlistat on visceral fat (VF) accumulation in Japanese individuals. Therefore, this study aimed to analyze the efficacy and safety of 52 weeks of orlistat administration in Japanese individuals. METHODS: Orlistat 60 mg was administered orally three times daily for 52 weeks to Japanese participants with excessive VF accumulation and without dyslipidemia, diabetes mellitus, and hypertension (metabolic diseases). Participants were also counseled to improve their diet and to maintain exercise habits. We defined excessive VF accumulation as a waist circumference (WC) of ≥ 85 cm for males and ≥ 90 cm for females, which corresponds to a VF area of 100 cm2. Adverse reactions, clinical laboratory tests, VF, WC, body weight (BW), etc., were monitored throughout the study period. RESULTS: VF, WC, and BW were significantly reduced at week 52 from baseline; the mean ± standard error rate of change was - 21.52% ± 1.89%, - 4.89% ± 0.45%, and - 5.36% ± 0.56%, respectively, and continued to reduce throughout the 52 weeks; these significantly reduced at whole term compared with baseline. Most adverse reactions were defecation-related symptoms such as oily spotting and flatus with discharge (flatus with small amounts of stool or oil) due to the pharmacologic effects of the lipase inhibitor. These symptoms were mostly mild, reversible, and recognizable by the participants; none were serious or severe. No participants discontinued by medical judgment about adverse reactions, and the drug could be administered continuously. CONCLUSION: VF, WC, and BW were reduced from week 4 to week 52, indicating the effect of long-term orlistat administration. Moreover, it was well tolerated with an acceptable safety profile. Long-term administration of orlistat may be efficacious in reducing VF accumulation with safety when used in combination with diet and exercise. TRIAL REGISTRATION: This study is registered with the Japan Pharmaceutical Information Center (identifier: JapicCTI-184004). FUNDING: Funding for this study was provided by Taisho Pharmaceutical Co., Ltd.

Efficacy and Safety of Lipase Inhibitor Orlistat in Japanese with Excessive Visceral Fat Accumulation: 24-Week, Double-Blind, Randomized, Placebo-Controlled Study.

INTRODUCTION: Orlistat is an inhibitor of pancreatic lipase and is used as an anti-obesity drug in many countries. However, there are no data available regarding the effects of orlistat on visceral fat accumulation in Japanese subjects. Therefore, this comparative, placebo-controlled, double-blind, randomized study aimed to evaluate the efficacy and safety of orlistat in Japanese participants with excessive visceral fat accumulation and without dyslipidemia, diabetes mellitus, and hypertension ("metabolic diseases"). METHODS: The study population included Japanese participants with excessive visceral fat accumulation (waist circumference ≥ 85 cm in males and ≥ 90 cm in females, which corresponds to a visceral fat area of 100 cm2) and without metabolic diseases. Following a 12-week observation term, participants were randomized to the orlistat 60 mg group (n = 100) or placebo group (n = 100). Both drugs were administered orally three times daily for 24 weeks. Participants were also counseled to improve their diet and to maintain exercise throughout the study. Visceral fat area, subcutaneous fat area, waist circumference, body weight, body mass index, adverse reactions, laboratory tests, and blood pressure were regularly assessed. RESULTS: Visceral fat area, waist circumference, and body weight were significantly reduced in the orlistat group (mean ± standard error, - 13.50 ± 1.52%, - 2.51 ± 0.25%, and - 2.79 ± 0.30%, respectively) compared to the placebo group (- 5.45 ± 1.50%, - 1.55 ± 0.26%, and - 1.22 ± 0.28%, respectively) at the last assessment. The main adverse reactions were defecation-related symptoms including oily spotting and flatus with discharge, resulting from the pharmacological effects of orlistat. Most adverse reactions were mild, and none were serious or severe. CONCLUSION: Orlistat administration reduced visceral fat area, waist circumference, and body weight in Japanese participants with excessive visceral fat and without metabolic diseases. In addition, safety was confirmed with a tolerable profile. Orlistat may be useful to reduce excessive visceral fat accumulation when used in combination with diet and exercise. TRIAL REGISTRATION: Japan Pharmaceutical Information Center identifier, JapicCTI-184005. FUNDING: Taisho Pharmaceutical Co., Ltd.

Albedos, Equilibrium Temperatures, and Surface Temperatures of Habitable Planets.

The potential habitability of known exoplanets is often categorized by a nominal equilibrium temperature assuming a Bond albedo of either ∼0.3, similar to Earth, or 0. As an indicator of habitability, this leaves much to be desired, because albedos of other planets can be very different, and because surface temperature exceeds equilibrium temperature due to the atmospheric greenhouse effect. We use an ensemble of general circulation model simulations to show that for a range of habitable planets, much of the variability of Bond albedo, equilibrium temperature and even surface temperature can be predicted with useful accuracy from incident stellar flux and stellar temperature, two known parameters for every confirmed exoplanet. Earth's Bond albedo is near the minimum possible for habitable planets orbiting G stars, because of increasing contributions from clouds and sea ice/snow at higher and lower instellations, respectively. For habitable M star planets, Bond albedo is usually lower than Earth's because of near-IR H2O absorption, except at high instellation where clouds are important. We apply relationships derived from this behavior to several known exoplanets to derive zeroth-order estimates of their potential habitability. More expansive multivariate statistical models that include currently non-observable parameters show that greenhouse gas variations produce significant variance in albedo and surface temperature, while increasing length of day and land fraction decrease surface temperature; insights for other parameters are limited by our sampling. We discuss how emerging information from global climate models might resolve some degeneracies and help focus scarce observing resources on the most promising planets.

Endothelial senescence is induced by phosphorylation and nuclear export of telomeric repeat binding factor 2-interacting protein.

The interplay among signaling events for endothelial cell (EC) senescence, apoptosis, and activation and how these pathological conditions promote atherosclerosis in the area exposed to disturbed flow (d-flow) in concert remain unclear. The aim of this study was to determine whether telomeric repeat-binding factor 2-interacting protein (TERF2IP), a member of the shelterin complex at the telomere, can regulate EC senescence, apoptosis, and activation simultaneously, and if so, by what molecular mechanisms. We found that d-flow induced p90RSK and TERF2IP interaction in a p90RSK kinase activity-dependent manner. An in vitro kinase assay revealed that p90RSK directly phosphorylated TERF2IP at the serine 205 (S205) residue, and d-flow increased TERF2IP S205 phosphorylation as well as EC senescence, apoptosis, and activation by activating p90RSK. TERF2IP phosphorylation was crucial for nuclear export of the TERF2IP-TRF2 complex, which led to EC activation by cytosolic TERF2IP-mediated NF-κB activation and also to senescence and apoptosis of ECs by depleting TRF2 from the nucleus. Lastly, using EC-specific TERF2IP-knockout (TERF2IP-KO) mice, we found that the depletion of TERF2IP inhibited d-flow-induced EC senescence, apoptosis, and activation, as well as atherosclerotic plaque formation. These findings demonstrate that TERF2IP is an important molecular switch that simultaneously accelerates EC senescence, apoptosis, and activation by S205 phosphorylation.

MAGI1 as a link between endothelial activation and ER stress drives atherosclerosis.

The possible association between the membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1) and inflammation has been suggested, but the molecular mechanisms underlying this link, especially during atherogenesis, remain unclear. In endothelial cells (ECs) exposed to disturbed flow (d-flow), p90 ribosomal S6 kinase (p90RSK) bound to MAGI1, causing MAGI1-S741 phosphorylation and sentrin/SUMO-specific protease 2 T368 phosphorylation-mediated MAGI1-K931 deSUMOylation. MAGI1-S741 phosphorylation upregulated EC activation via activating Rap1. MAGI1-K931 deSUMOylation induced both nuclear translocation of p90RSK-MAGI1 and ATF-6-MAGI1 complexes, which accelerated EC activation and apoptosis, respectively. Microarray screening revealed key roles for MAGI1 in the endoplasmic reticulum (ER) stress response. In this context, MAGI1 associated with activating transcription factor 6 (ATF-6). MAGI1 expression was upregulated in ECs and macrophages found in atherosclerotic-prone regions of mouse aortas as well as in the colonic epithelia and ECs of patients with inflammatory bowel disease. Further, reduced MAGI1 expression in Magi1-/+ mice inhibited d-flow-induced atherogenesis. In sum, EC activation and ER stress-mediated apoptosis are regulated in concert by two different types of MAGI1 posttranslational modifications, elucidating attractive drug targets for chronic inflammatory disease, particularly atherosclerosis.

[Neurosyphilis with cerebellar ataxia, personality change and cognitive decline one year after onset of cerebral infarction].

A 51-year-old man with a cerebral lacunar infarction of the midbrain that had occurred two years before, was transferred from a regional psychiatric hospital with chronic progressive psychiatric symptoms including cognitive decline, irritability and hallucinations. Neurological examinations upon admission revealed cerebellar ataxia including dysarthria, ataxic gait and bilateral intention tremor. Brain FLAIR MRI on day 2 revealed abnormal hyperintense lesions in the bilateral insular cortex and temporal pole. Treponemal and non-treponemal specific antibodies were positive in both serum and cerebrospinal fluid (CSF), indicating a diagnosis of neurosyphilis. Treatment with intravenous penicillin (24 × 106 units/day × 28 days) improved his psychiatric symptoms, ataxia, imaging abnormalities and inflammatory CSF findings. Cerebellar ataxia is a rare symptom of neurosyphilis. Nonetheless, the possibility of neurosyphilis should be considered if a young adult ataxia accompanied by psychiatric symptoms.

Exoplanet Biosignatures: At the Dawn of a New Era of Planetary Observations.

The rapid rate of discoveries of exoplanets has expanded the scope of the science possible for the remote detection of life beyond Earth. The Exoplanet Biosignatures Workshop Without Walls (EBWWW) held in 2016 engaged the international scientific community across diverse scientific disciplines, to assess the state of the science and technology in the search for life on exoplanets, and to identify paths for progress. The workshop activities resulted in five major review papers, which provide (1) an encyclopedic review of known and proposed biosignatures and models used to ascertain them (Schwieterman et al., 2018 in this issue); (2) an in-depth review of O2 as a biosignature, rigorously examining the nuances of false positives and false negatives for evidence of life (Meadows et al., 2018 in this issue); (3) a Bayesian framework to comprehensively organize current understanding to quantify confidence in biosignature assessments (Catling et al., 2018 in this issue); (4) an extension of that Bayesian framework in anticipation of increasing planetary data and novel concepts of biosignatures (Walker et al., 2018 in this issue); and (5) a review of the upcoming telescope capabilities to characterize exoplanets and their environment (Fujii et al., 2018 in this issue). Because of the immense content of these review papers, this summary provides a guide to their complementary scope and highlights salient features. Strong themes that emerged from the workshop were that biosignatures must be interpreted in the context of their environment, and that frameworks must be developed to link diverse forms of scientific understanding of that context to quantify the likelihood that a biosignature has been observed. Models are needed to explore the parameter space where measurements will be widespread but sparse in detail. Given the technological prospects for large ground-based telescopes and space-based observatories, the detection of atmospheric signatures of a few potentially habitable planets may come before 2030. Key Words: Exoplanets-Biosignatures-Remote observation-Spectral imaging-Bayesian analysis. Astrobiology 18, 619-626.

Exoplanet Biosignatures: Observational Prospects.

Exoplanet hunting efforts have revealed the prevalence of exotic worlds with diverse properties, including Earth-sized bodies, which has fueled our endeavor to search for life beyond the Solar System. Accumulating experiences in astrophysical, chemical, and climatological characterization of uninhabitable planets are paving the way to characterization of potentially habitable planets. In this paper, we review our possibilities and limitations in characterizing temperate terrestrial planets with future observational capabilities through the 2030s and beyond, as a basis of a broad range of discussions on how to advance "astrobiology" with exoplanets. We discuss the observability of not only the proposed biosignature candidates themselves but also of more general planetary properties that provide circumstantial evidence, since the evaluation of any biosignature candidate relies on its context. Characterization of temperate Earth-sized planets in the coming years will focus on those around nearby late-type stars. The James Webb Space Telescope (JWST) and later 30-meter-class ground-based telescopes will empower their chemical investigations. Spectroscopic studies of potentially habitable planets around solar-type stars will likely require a designated spacecraft mission for direct imaging, leveraging technologies that are already being developed and tested as part of the Wide Field InfraRed Survey Telescope (WFIRST) mission. Successful initial characterization of a few nearby targets will be an important touchstone toward a more detailed scrutiny and a larger survey that are envisioned beyond 2030. The broad outlook this paper presents may help develop new observational techniques to detect relevant features as well as frameworks to diagnose planets based on the observables. Key Words: Exoplanets-Biosignatures-Characterization-Planetary atmospheres-Planetary surfaces. Astrobiology 18, 739-778.

Cushing's Syndrome and Psychosis: A Case Report and Literature Review.

Psychiatric complications of Cushing's syndrome include irritability, anxiety, depressed mood, and cognitive impairment. Psychosis is a rare manifestation of Cushing's syndrome; therefore, the literature on the subject is limited and consists mainly of clinical case reports. We report a case of Cushing's syndrome misdiagnosed as schizophrenia-like psychosis for more than 10 years. Transsphenoidal adenomectomy resulted in amelioration of psychiatric symptoms as well as improvement of cognitive ability. Clinicians should consider the presence of psychiatric symptoms predating the diagnosis of Cushing's syndrome, especially when these symptoms are persistent and treatment-resistant, as seen in the present case.

Ionizing Radiation Induces Endothelial Inflammation and Apoptosis via p90RSK-Mediated ERK5 S496 Phosphorylation.

Adverse cardiovascular events are a leading nonmalignant cause of morbidity and mortality among cancer survivors who have been exposed to ionizing radiation (IR), but the exact mechanism of the cardiovascular complications induced by IR remains unclear. In this study we investigated the potential role of the p90RSK-ERK5 module in regulating IR-induced endothelial cell inflammation and apoptosis. Whole body radiation of mice with 2 Gy γ-ray significantly increased endothelial VCAM-1 expression; especially in the disturbed flow area in vivo. In vitro studies showed that IR increased p90RSK activation as well as subsequent ERK5 S496 phosphorylation in cultured human endothelial cells (ECs). A specific p90RSK inhibitor, FMK-MEA, significantly inhibited both p90RSK activation and ERK5 S496 phosphorylation, but it had no effect on IR-induced ERK5 TEY motif phosphorylation, suggesting that p90RSK regulates ERK5 transcriptional activity, but not its kinase activity. In fact, we found that IR-induced NF-kB activation and VCAM-1 expression in ECs were significantly inhibited by the over-expression of S496 phosphorylation site mutant of ERK5 (ERK5 S496A) compared to overexpression of wild type ERK5. Furthermore, when ECs were exposed to IR, the number of annexin V positive cells increased, and overexpression of ERK5 S496A, but not wild type ERK5, significantly inhibited this increase. Our results demonstrate that IR augmented disturbed flow-induced VCAM-1 expression in vivo. Endothelial p90RSK was robustly activated by IR and subsequently up-regulated ERK5 S496 phosphorylation, inflammation, and apoptosis in ECs. The EC p90RSK-ERK5 signaling axis can be a good target to prevent cardiovascular events after radiation therapy in cancer patients.