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Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

This review makes a case for describing many of the flows observed in our oceans, simply based on the Euler equation, with (piecewise) constant density and with suitable boundary conditions. The analyses start from the Euler and mass conservation equations, expressed in a rotating, spherical coordinate system (but the f -plane and β -plane approximations are also mentioned); five examples are discussed. For three of them, a suitable non-dimensionalization is introduced, and a single small parameter is identified in each case. These three examples lead straightforwardly and directly to new results for: waves on the Pacific Equatorial Undercurrent (EUC) with a thermocline (in the f -plane); a nonlinear, three-dimensional model for EUC-type flows (in the β -plane); and a detailed model for large gyres. The other two examples are exact solutions of the complete system: a flow which corresponds to the underlying structure of the Pacific EUC; and a flow based on the necessary requirement to use a non-conservative body force, which produces the type of flow observed in the Antarctic Circumpolar Current. (All these examples have been discussed in detail in the references cited.) This review concludes with a few comments on how these solutions can be extended and expanded.This article is part of the theme issue 'Nonlinear water waves'., (© 2017 The Author(s).)

Cytotoxic T cells infiltrating tumors are thought to utilize HIF transcription factors during adaptation to the hypoxic tumor microenvironment. Deletion analyses of the two key HIF isoforms found that HIF-1α, but not HIF-2α, was essential for the effector state in CD8 + T cells. Furthermore, loss of HIF-1α in CD8 + T cells reduced tumor infiltration and tumor cell killing, and altered tumor vascularization. Deletion of VEGF-A, an HIF target gene, in CD8 + T cells accelerated tumorigenesis while also altering vascularization. Analyses of human breast cancer showed inverse correlations between VEGF-A expression and CD8 + T cell infiltration, and a link between T cell infiltration and vascularization. These data demonstrate that the HIF-1α/VEGF-A axis is an essential aspect of tumor immunity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Systemic vascular pressure in vertebrates is regulated by a range of factors: one key element of control is peripheral resistance in tissue capillary beds. Many aspects of the relationship between central control of vascular flow and peripheral resistance are unclear. An important example of this is the relationship between hypoxic response in individual tissues, and the effect that response has on systemic cardiovascular adaptation to oxygen deprivation. We show here how hypoxic response via the HIF transcription factors in one large vascular bed, that underlying the skin, influences cardiovascular response to hypoxia in mice. We show that the response of the skin to hypoxia feeds back on a wide range of cardiovascular parameters, including heart rate, arterial pressures, and body temperature. These data represent the first demonstration of a dynamic role for oxygen sensing in a peripheral tissue directly modifying cardiovascular response to the challenge of hypoxia.

Clinical Scenario: It is hypothesized that cognitive activity following a concussion may potentially hinder patient recovery. While the recommendation of cognitive rest is often maintained and rationalized, a causal relationship between cognitive activity and symptom duration has yet to be established., Clinical Question: Does the implementation of cognitive rest as part of the postconcussion management plan reduce the number of days until the concussed adolescent patient is symptom free compared to a postconcussion management plan that does not incorporate cognitive rest? Summary of Key Findings: A thorough literature search returned 7 possible studies; 5 studies met the inclusion criteria and were included. Three studies indicated that increased cognitive activity is associated with longer recovery from a concussion, and, therefore, supported the use of cognitive rest. One study indicated that the recommendation for cognitive rest was not significantly associated with time to concussion symptom resolution. One study indicated that strict rest, defined as 5 days of no school, work, or physical activity; might prolong symptom duration. Clinical Bottom Line: There is moderate evidence to support the prescription of moderate cognitive rest for concussed patients. Clinicians who intend on implementing cognitive rest in their concussion protocols should be aware of inconsistencies and be open-minded to alternative treatment progressions while taking into consideration each individual patient and maintaining adequate patient-centered care principles. Strength of Recommendation: Grade B evidence exists that prescription of moderate cognitive rest for concussed patients may be beneficial as a supplement to physical rest as treatment for symptom reduction in adolescents.

Aim: Experiments have indicated that skin perfusion in mice is sensitive to reductions in environmental O 2 availability. Specifically, a reduction in skin-surface PO 2 attenuates transcutaneous O 2 diffusion, and hence epidermal O 2 supply. In response, epidermal HIF-1α expression increases and facilitates initial cutaneous vasoconstriction and subsequent nitric oxide-dependent vasodilation. Here, we investigated whether the same mechanism exists in humans., Methods: In a first experiment, eight males rested twice for 8 h in a hypobaric chamber. Once, barometric pressure was reduced by 50%, while systemic oxygenation was preserved by O 2 -enriched (42%) breathing gas (Hypoxia Skin ), and once barometric pressure and inspired O 2 fraction were normal (Control 1 ). In a second experiment, nine males rested for 8 h with both forearms wrapped in plastic bags. O 2 was expelled from one bag by nitrogen flushing (Anoxia Skin ), whereas the other bag was flushed with air (Control 2 ). In both experiments, skin blood flux was assessed by laser Doppler on the dorsal forearm, and HIF-1α expression was determined by immunohistochemical staining in forearm skin biopsies., Results: Skin blood flux during Hypoxia Skin and Anoxia Skin remained similar to the corresponding Control trial (P = 0.67 and P = 0.81). Immunohistochemically stained epidermal HIF-1α was detected on 8.2 ± 6.1 and 5.3 ± 5.7% of the analysed area during Hypoxia Skin and Control 1 (P = 0.30) and on 2.3 ± 1.8 and 2.4 ± 1.8% during Anoxia Skin and Control 2 (P = 0.90) respectively., Conclusion: Reductions in skin-surface PO 2 do not affect skin perfusion in humans. The unchanged epidermal HIF-1α expression suggests that epidermal O 2 homoeostasis was not disturbed by Hypoxia Skin /Anoxia Skin , potentially due to compensatory increases in arterial O 2 extraction., (© 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter. TCP4 induces CO expression around dusk by directly associating with the CO promoter in vivo. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces CO expression in a GI-dependent manner. The physical association of TCP4 with the CO promoter was reduced in the gi mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the in vivo TCP4 complex, and the gi mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is known as a mediator of toxic responses. Recently, it was shown that the AhR has dual functions. Besides being a transcription factor, it also possesses an intrinsic E3 ubiquitin ligase function that targets, e.g., the steroid receptors for proteasomal degradation. The aim of this study was to identify the molecular switch that determines whether the AhR acts as a transcription factor or an E3 ubiquitin ligase. To do this, we used the breast cancer cell line MCF7, which expresses a functional estrogen receptor alpha (ERα) signaling pathway. Our data suggest that aryl hydrocarbon receptor nuclear translocator (ARNT) plays an important role in the modulation of the dual functions of the AhR. ARNT knockdown dramatically impaired the transcriptional activation properties of the ligand-activated AhR but did not affect its E3 ubiquitin ligase function. The availability of ARNT itself is modulated by another basic helix-loop-helix (bHLH)-Per-ARNT-SIM (PAS) protein, the repressor of AhR function (AhRR). MCF7 cells overexpressing the AhRR showed lower ERα protein levels, reduced responsiveness to estradiol, and reduced growth rates. Importantly, when these cells were used to produce estrogen-dependent xenograft tumors in SCID mice, we also observed lower ERα protein levels and a reduced tumor mass, implying a tumor-suppressive-like function of the AhR in MCF7 xenograft tumors., (Copyright © 2017 American Society for Microbiology.)

The Himalayan Sherpas, a human population of Tibetan descent, are highly adapted to life in the hypobaric hypoxia of high altitude. Mechanisms involving enhanced tissue oxygen delivery in comparison to Lowlander populations have been postulated to play a role in such adaptation. Whether differences in tissue oxygen utilization (i.e., metabolic adaptation) underpin this adaptation is not known, however. We sought to address this issue, applying parallel molecular, biochemical, physiological, and genetic approaches to the study of Sherpas and native Lowlanders, studied before and during exposure to hypobaric hypoxia on a gradual ascent to Mount Everest Base Camp (5,300 m). Compared with Lowlanders, Sherpas demonstrated a lower capacity for fatty acid oxidation in skeletal muscle biopsies, along with enhanced efficiency of oxygen utilization, improved muscle energetics, and protection against oxidative stress. This adaptation appeared to be related, in part, to a putatively advantageous allele for the peroxisome proliferator-activated receptor A ( PPARA ) gene, which was enriched in the Sherpas compared with the Lowlanders. Our findings suggest that metabolic adaptations underpin human evolution to life at high altitude, and could have an impact upon our understanding of human diseases in which hypoxia is a feature., Competing Interests: Conflict of interest statement: E.G. is Chief Executive Officer and V.L. is Chief Operating Officer of Oroboros Instruments.

Thrombosis can cause localized ischemia and tissue hypoxia, and both of these are linked to cancer metastasis. Vascular micro-occlusion can occur as a result of arrest of circulating tumour cells in small capillaries, giving rise to microthrombotic events that affect flow, creating localized hypoxic regions. To better understand the association between metastasis and thrombotic events, we generated an experimental strategy whereby we modelled the effect of microvascular occlusion in metastatic efficiency by using inert microbeads to obstruct lung microvasculature before, during and after intravenous tumour cell injection. We found that controlled induction of a specific number of these microthrombotic insults in the lungs caused an increase in expression of the hypoxia-inducible transcription factors (HIFs), a pro-angiogenic and pro-tumorigenic environment, as well as an increase in myeloid cell infiltration. Induction of pulmonary microthrombosis prior to introduction of tumour cells to the lungs had no effect on tumorigenic success, but thrombosis at the time of tumour cell seeding increased number and size of tumours in the lung, and this effect was strikingly more pronounced when the micro-occlusion occurred on the day following introduction of tumour cells. The tumorigenic effect of microbead treatment was seen even when thrombosis was induced five days after tumour cell injection. We also found positive correlations between thrombotic factors and expression of HIF2α in human tumours. The model system described here demonstrates the importance of thrombotic insult in metastatic success and can be used to improve understanding of thrombosis-associated tumorigenesis and its treatment., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Starting from the Euler equation expressed in a rotating frame in spherical coordinates, coupled with the equation of mass conservation and the appropriate boundary conditions, a thin-layer (i.e. shallow water) asymptotic approximation is developed. The analysis is driven by a single, overarching assumption based on the smallness of one parameter: the ratio of the average depth of the oceans to the radius of the Earth. Consistent with this, the magnitude of the vertical velocity component through the layer is necessarily much smaller than the horizontal components along the layer. A choice of the size of this speed ratio is made, which corresponds, roughly, to the observational data for gyres; thus the problem is characterized by, and reduced to an analysis based on, a single small parameter. The nonlinear leading-order problem retains all the rotational contributions of the moving frame, describing motion in a thin spherical shell. There are many solutions of this system, corresponding to different vorticities, all described by a novel vorticity equation: this couples the vorticity generated by the spin of the Earth with the underlying vorticity due to the movement of the oceans. Some explicit solutions are obtained, which exhibit gyre-like flows of any size; indeed, the technique developed here allows for many different choices of the flow field and of any suitable free-surface profile. We comment briefly on the next order problem, which provides the structure through the layer. Some observations about the new vorticity equation are given, and a brief indication of how these results can be extended is offered., Competing Interests: We declare we have no competing interests.

Hypoxia and bacterial infection frequently co-exist, in both acute and chronic clinical settings, and typically result in adverse clinical outcomes. To ameliorate this morbidity, we investigated the interaction between hypoxia and the host response. In the context of acute hypoxia, both S. aureus and S. pneumoniae infections rapidly induced progressive neutrophil mediated morbidity and mortality, with associated hypothermia and cardiovascular compromise. Preconditioning animals through longer exposures to hypoxia, prior to infection, prevented these pathophysiological responses and profoundly dampened the transcriptome of circulating leukocytes. Specifically, perturbation of HIF pathway and glycolysis genes by hypoxic preconditioning was associated with reduced leukocyte glucose utilisation, resulting in systemic rescue from a global negative energy state and myocardial protection. Thus we demonstrate that hypoxia preconditions the innate immune response and determines survival outcomes following bacterial infection through suppression of HIF-1α and neutrophil metabolism. The therapeutic implications of this work are that in the context of systemic or tissue hypoxia therapies that target the host response could improve infection associated morbidity and mortality., Competing Interests: Competing Financial Interests The authors declare no competing financial interests.

Objective: Significant variability exists regarding the criteria and procedures used by different veterans' courts (VCs) across the country. Limited guidance is available regarding which VC model has the most successful outcomes. The purpose of this study was to examine factors associated with incarceration during VC participation., Methods: This study used data for 1,224 veterans collected from the HOMES (Homeless Operations Management and Evaluation System) database of the Department of Veterans Affairs, as well as data from a national phone survey inventory of all U.S. VCs. To identify variables associated with incarceration during VC participation, four backward conditional logistic regressions were performed., Results: The following variables were associated with higher rates of incarceration because of a veteran's noncompletion of the VC program: charges of probation or parole violations, longer stays in the VC program, end of VC participation because of incarceration for a new arrest or case transfer by the legal system, and requiring mental health follow-up but not undergoing treatment. The following variables were associated with lower rates of incarceration: stable housing and participating in a VC program that referred veterans for substance abuse treatment., Conclusions: This study offers VCs a thorough review of an extensive set of recidivism data. Further investigation is necessary to understand the impact of VCs.

Sensing the level of oxygen in the external and internal environments is essential for survival. Organisms have evolved multiple mechanisms to sense oxygen. No function in oxygen sensing has been attributed to any mammalian olfactory system. Here, we demonstrate that low environmental oxygen directly activates a subpopulation of sensory neurons in the mouse main olfactory epithelium. These neurons express the soluble guanylate cyclase Gucy1b2 and the cation channel Trpc2. Low oxygen induces calcium influx in these neurons, and Gucy1b2 and Trpc2 are required for these responses. In vivo exposure of a mouse to low environmental oxygen causes Gucy1b2-dependent activation of olfactory bulb neurons in the vicinity of the glomeruli formed by axons of Gucy1b2+ sensory neurons. Low environmental oxygen also induces conditioned place aversion, for which Gucy1b2 and Trpc2 are required. We propose that this chemosensory function enables a mouse to rapidly assess the oxygen level in the external environment., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

In this issue of Cancer Cell, Cantelmo et al. describe how reduction of PFKFB3 enzyme activity can promote vascular normalization. The authors show in turn how this affects vascular permeability and can ultimately improve the efficacy of chemotherapeutic agents., (Copyright © 2016 Elsevier Inc. All rights reserved.)

R-2-hydroxyglutarate accumulates to millimolar levels in cancer cells with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both metabolite enantiomers, R- and S-2-hydroxyglutarate, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that 2-hydroxyglutarate accumulates in mouse CD8 + T cells in response to T-cell receptor triggering, and accumulates to millimolar levels in physiological oxygen conditions through a hypoxia-inducible factor 1-alpha (HIF-1α)-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8 + T-cell differentiation in response to this metabolite. Modulation of histone and DNA demethylation, as well as HIF-1α stability, mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8 + T cells. Thus, S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, through a metabolic-epigenetic axis, to immune fate and function., Competing Interests: The authors declare no conflict of interest.

Competing Interests: The author declares no conflict of interest.

Phloem localization of plant viruses is advantageous for acquisition by sap-sucking vectors but hampers host-virus protein interaction studies. In this study, Potato leafroll virus (PLRV)-host protein complexes were isolated from systemically infected potato, a natural host of the virus. Comparing two different co-immunoprecipitation (co-IP) support matrices coupled to mass spectrometry (MS), we identified 44 potato proteins and one viral protein (P1) specifically associated with virus isolated from infected phloem. An additional 142 proteins interact in complex with virus at varying degrees of confidence. Greater than 80% of these proteins were previously found to form high confidence interactions with PLRV isolated from the model host Nicotiana benthamiana. Bioinformatics revealed that these proteins are enriched for functions related to plasmodesmata, organelle membrane transport, translation, and mRNA processing. Our results show that model system proteomics experiments are extremely valuable for understanding protein interactions regulating infection in recalcitrant pathogens such as phloem-limited viruses.

Extensive metabolic changes accompany T cell activation, including a switch to glycolytic energy production and increased biosynthesis. Recent studies suggest that subsequent return to reliance on oxidative phosphorylation and increasing spare respiratory capacity are essential for the differentiation of memory CD8 + T cells. In contrast, we found that constitutive glycolytic metabolism and suppression of oxidative phosphorylation in CD8 + T cells, achieved by conditional deletion of hypoxia-inducible factor regulator Vhl, accelerated CD8 + memory cell differentiation during viral infection. Despite sustained glycolysis, CD8 + memory cells emerged that upregulated key memory-associated cytokine receptors and transcription factors and showed a heightened response to secondary challenge. In addition, increased glycolysis not only permitted memory formation, but it also favored the formation of long-lived effector-memory CD8 + T cells. These data redefine the role of cellular metabolism in memory cell differentiation, showing that reliance on glycolytic metabolism does not hinder formation of a protective memory population., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In recent years, nonmammographic breast imaging devices, such as thermography, electrical impedance scanning and elastography, have been promoted directly to consumers, which has captured the attention of governments, researchers and health organisations. These devices are not supported by evidence and risk undermining existing mammographic breast cancer screening services. During a 5-year period, Cancer Council Western Australia (CCWA) used strategic research combined with legal, policy and media advocacy to contest claims that these devices were proven alternatives to mammography for breast cancer screening. The campaign was successful because it had input from people with public health, academic, clinical and legal backgrounds, and took advantage of existing legal and regulatory avenues. CCWA's experience provides a useful advocacy model for public health practitioners who are concerned about unsafe consumer products, unproven medical devices, and misleading health information and advertising.

Vascular endothelial growth factor A (VEGF) is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration, and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2). We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO) synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell homeostasis in normoxia and hypoxia.

Objective: The affect regulation theory suggests that people binge eat to regulate negative emotional states. In this study, we used a basic emotions perspective to consider the role of perceived threat of emotions, emotional suppression and reduced emotional expressiveness in predicting binge eating behaviours in people who are obese., Method: Treatment-seeking participants with obesity (N = 51, body mass index range from 30.8 to 60.2 kg m -2 ) completed measures of 'perception of threat from emotion' as well as 'emotional expressiveness' and binge eating., Results: The results demonstrated that perceived threat of sadness predicted binge eating (β = .55, p < .05). Additionally, a mediation analysis revealed that reduced emotional expressiveness mediated the relationship between perceived threat of fear and binge eating (β = .25, 95%)., Discussion: These findings are contextualized within a theoretical perspective that suggests that individuals who binge eat are threatened by certain emotional states and they use binge eating to suppress certain, but not all, emotional states. Copyright © 2015 John Wiley & Sons, Ltd., Key Practitioner Message: Considering basic emotions within binge eating should be a part of a psychological assessment and treatment. This should consider how emotions could often be perceived as being threatening and their expression is limited. It is possible that the emotions of fear and sadness appear to be particularly threatening within binge eating/obese populations., (Copyright © 2015 John Wiley & Sons, Ltd.)

Hypoxic pulmonary vasoconstriction is correlated with pulmonary vascular remodeling. The hypoxia-inducible transcription factors (HIFs) HIF-1α and HIF-2α are known to contribute to the process of hypoxic pulmonary vascular remodeling; however, the specific role of pulmonary endothelial HIF expression in this process, and in the physiological process of vasoconstriction in response to hypoxia, remains unclear. Here we show that pulmonary endothelial HIF-2α is a critical regulator of hypoxia-induced pulmonary arterial hypertension. The rise in right ventricular systolic pressure (RVSP) normally observed following chronic hypoxic exposure was absent in mice with pulmonary endothelial HIF-2α deletion. The RVSP of mice lacking HIF-2α in pulmonary endothelium after exposure to hypoxia was not significantly different from normoxic WT mice and much lower than the RVSP values seen in WT littermate controls and mice with pulmonary endothelial deletion of HIF-1α exposed to hypoxia. Endothelial HIF-2α deletion also protected mice from hypoxia remodeling. Pulmonary endothelial deletion of arginase-1, a downstream target of HIF-2α, likewise attenuated many of the pathophysiological symptoms associated with hypoxic pulmonary hypertension. We propose a mechanism whereby chronic hypoxia enhances HIF-2α stability, which causes increased arginase expression and dysregulates normal vascular NO homeostasis. These data offer new insight into the role of pulmonary endothelial HIF-2α in regulating the pulmonary vascular response to hypoxia.

The kinetics of the reaction CH3OCH3 + Fe(+) has been studied between 250 and 600 K in the buffer gas He at pressures between 0.4 and 1.6 Torr. Total rate constants and branching ratios for the formation of Fe(+)O(CH3)2 adducts and of Fe(+)OCH2 + CH4 products were determined. Quantum-chemical calculations provided the parameters required for an analysis in terms of statistical unimolecular rate theory. The analysis employed a recently developed simplified representation of the rates of complex-forming bimolecular reactions, separating association and chemical activation contributions. Satisfactory agreement between experimental results and kinetic modeling was obtained that allows for an extrapolation of the data over wide ranges of conditions. Possible reaction pathways with or without spin-inversion are discussed in relation to the kinetic modeling results.

In severely hypoxic condition, HIF-1α-mediated induction of Pdk1 was found to regulate glucose oxidation by preventing the entry of pyruvate into the tricarboxylic cycle. Monocyte-derived macrophages, however, encounter a gradual decrease in oxygen availability during its migration process in inflammatory areas. Here we show that HIF-1α-PDK1-mediated metabolic changes occur in mild hypoxia, where mitochondrial cytochrome c oxidase activity is unimpaired, suggesting a mode of glycolytic reprogramming. In primary macrophages, PKM2, a glycolytic enzyme responsible for glycolytic ATP synthesis localizes in filopodia and lammelipodia, where ATP is rapidly consumed during actin remodelling processes. Remarkably, inhibition of glycolytic reprogramming with dichloroacetate significantly impairs macrophage migration in vitro and in vivo. Furthermore, inhibition of the macrophage HIF-1α-PDK1 axis suppresses systemic inflammation, suggesting a potential therapeutic approach for regulating inflammatory processes. Our findings thus demonstrate that adaptive responses in glucose metabolism contribute to macrophage migratory activity.

Despite the increased risk of thrombosis in cancer patients compared with healthy individuals, mechanisms that regulate cancer-induced hypercoagulation are incompletely understood. The aim of this study was to investigate whether cell-specific hypoxia-inducible factor (HIF) 1α regulates cancer-associated hypercoagulation, using in vitro clotting assays and in vivo cancer models. In mouse lung and mammary tumor cells, hypoxia led to increases in cell adhesion, clotting, and fibrin deposition; these increases were eliminated in HIF1α null cells. Increased levels of HIF1α were also associated with increased tissue factor expression in human breast tumor samples. Conversely, deletion of endothelial (but not myeloid) cell-specific HIF1α doubled pulmonary fibrin deposition, and trebled thrombus formation compared with wildtype littermates in tumor-bearing mice. Our data suggest that tumor and endothelial cell-specific HIF1α may have opposing roles in cancer-associated coagulation and thrombosis. Off-target effects of manipulating the HIF1 axis in cancer patients should be carefully considered when managing thrombotic complications., (© 2016 by The American Society of Hematology.)

Borderline personality disorder (BPD) is an internationally recognized disorder, although it is slightly varied in its nosology in the International Classification of Diseases, 10th Revision (ICD-10), the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5), and the Chinese Classification of Mental Disorders (CCMD). While it is recognized by genetic and neurobiological patterns, instability of affect, impaired interpersonal relationships, and unstable sense of self, its manifestation is extremely varied based on environmental factors, particularly culture. Several studies of the manifestation of BPD between and across countries, particularly in immigrant populations, identify variations in symptom prevalence based on culture. These findings reveal a need for more unified dimensional-based categorization of BPD to reduce cross-cultural bias and improve identification.

This study used data from a phone survey inventory of US veterans' courts to provide descriptive information on the current status of their various elements. To identify which items were most predictive of a court's percentage of subjects terminated from their program, a linear regression was performed. The following were associated with higher rates of termination from the veterans' court (VC) program: (a) programs that offered phase progression based on measurable goals, (b) programs that conduct frequent drug and alcohol testing, and (c) programs for which sanctions are more severe for failing immediate goals (sobriety) versus long-term ones (completion of training). The following were associated with lower rates of termination from the VC program: (a) programs in which later phases permit less stringent testing, (b) programs utilizing behavioral contracts, (c) programs utilizing brief incarcerations. This inventory provides nationwide empirical data that may be used in the development of veterans' courts.

In targeted proteomics, the development of robust methodologies is dependent upon the selection of a set of optimal peptides for each protein-of-interest. Unfortunately, predicting which peptides and respective product ion transitions provide the greatest signal-to-noise ratio in a particular assay matrix is complicated. Using in vitro synthesized proteins as analytical standards, we report here an empirically driven method for the selection of said peptides in a human plasma assay matrix.

Purpose of Review: Textbook theory holds that blood pressure (BP) is regulated by the brain, by blood vessels, or by the kidney. Recent evidence suggests that BP could be regulated in the skin., Recent Findings: The skin holds a complex capillary counter current system, which controls body temperature, skin perfusion, and apparently systemic BP. Epidemiological data suggest that sunlight exposure plays a role in controlling BP. Ultraviolet A radiation produces vasodilation and a fall in BP. Keratinocytes and immune cells control blood flow in the extensive countercurrent loop system of the skin by producing nitric oxide, a key regulator of vascular tone. The balance between hypoxia-inducible factor-1α and hypoxia-inducible factor-2α activity in keratinocytes controls skin perfusion, systemic thermoregulation, and systemic BP by nitric oxide-dependent mechanisms. Furthermore, the skin accumulates Na which generates a barrier to promote immunological host defense. Immune cells control skin Na metabolism and the clearance of Na via the lymphatic system. Reduced lymphatic clearance increases BP., Summary: Apart from the well-known role of the brain, blood vessels, and the kidney, the skin is important for systemic BP control in humans and in experimental animals.

Background: Insulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of β-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms., Results: Herein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. We show that nitrate induces FA oxidation through a soluble guanylate cyclase (sGC)/cGMP-mediated PPARβ/δ- and PPARα-dependent mechanism. Enhanced PPARβ/δ and PPARα expression and DNA binding induces expression of FA oxidation enzymes, increasing muscle carnitine and lowering tissue malonyl-CoA concentrations, thereby supporting intra-mitochondrial pathways of FA oxidation and enhancing mitochondrial respiration. At higher doses, nitrate induces mitochondrial biogenesis, further increasing FA oxidation and lowering long-chain FA concentrations. Meanwhile, nitrate did not affect mitochondrial FA oxidation in PPARα(-/-) mice. In C2C12 myotubes, nitrate increased expression of the PPARα targets Cpt1b, Acadl, Hadh and Ucp3, and enhanced oxidative phosphorylation rates with palmitoyl-carnitine; however, these changes in gene expression and respiration were prevented by inhibition of either sGC or protein kinase G. Elevation of cGMP, via the inhibition of phosphodiesterase 5 by sildenafil, also increased expression of Cpt1b, Acadl and Ucp3, as well as CPT1B protein levels, and further enhanced the effect of nitrate supplementation., Conclusions: Nitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle.

Few if any publications discuss the effectiveness of voluntary versus mandated treatment for impaired physicians. This retrospective case-control study compared the recovery rates of physicians whose treatment was mandated or coerced by either licensure boards or employers (mandated physicians) with the rates for physicians admitted voluntarily (voluntary physicians) to the Menninger Clinic's Professionals in Crisis program from 2009 through 2012. Beck Depression Inventory (BDI)-II scores served as the primary outcome measure. At the time of admission, voluntary physicians were more depressed, but the improvement rates in the voluntary and mandated groups did not differ significantly. In addition, the two groups differed neither in rates of return to the healthy range of BDI-II scores, nor in whether BDI-II scores had decreased by at least two standard deviations by the time of discharge. These findings suggest that state physician health programs can continue to mandate physicians into treatment despite concerns that mandatory treatment may be less efficacious than voluntary treatment., (© 2015 American Academy of Psychiatry and the Law.)

'Candidatus Liberibacter asiaticus' (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid. Interactions among D. citri and its microbial endosymbionts, including 'Candidatus Profftella armatura', are likely to impact transmission of CLas. We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri, and found that proteins involved in polyketide biosynthesis by the endosymbiont Profftella were up-regulated in CLas(+) insects. Mass spectrometry analysis of the Profftella polyketide diaphorin in D. citri metabolite extracts revealed the presence of a novel diaphorin-related polyketide and the ratio of these two polyketides was changed in CLas(+) insects. Insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins involved in endocytosis, cellular adhesion, and cytoskeletal remodeling which are associated with microbial invasion of host cells. Insight into the metabolic interdependence between the insect vector, its endosymbionts, and the citrus greening pathogen reveals novel opportunities for control of this disease, which is currently having a devastating impact on citrus production worldwide.

Multiple protein subcomplexes of the kinetochore cooperate as a cohesive molecular unit that forms load-bearing microtubule attachments that drive mitotic chromosome movements. There is intriguing evidence suggesting that central kinetochore components influence kinetochore-microtubule attachment, but the mechanism remains unclear. Here, we find that the conserved Mis12/MIND (Mtw1, Nsl1, Nnf1, Dsn1) and Ndc80 (Ndc80, Nuf2, Spc24, Spc25) complexes are connected by an extensive network of contacts, each essential for viability in cells, and collectively able to withstand substantial tensile load. Using a single-molecule approach, we demonstrate that an individual MIND complex enhances the microtubule-binding affinity of a single Ndc80 complex by fourfold. MIND itself does not bind microtubules. Instead, MIND binds Ndc80 complex far from the microtubule-binding domain and confers increased microtubule interaction of the complex. In addition, MIND activation is redundant with the effects of a mutation in Ndc80 that might alter its ability to adopt a folded conformation. Together, our results suggest a previously unidentified mechanism for regulating microtubule binding of an outer kinetochore component by a central kinetochore complex.

Background: Pericytes are members of the tumor stroma; however, little is known about their origin, function, or interaction with other tumor components. Emerging evidence suggest that pericytes may regulate leukocyte transmigration. Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with powerful inhibitory effects on T-cell-mediated antitumor reactivity., Methods: We generated subcutaneous tumors in a genetic mouse model of pericyte deficiency (the pdgfb (ret/ret) mouse) and littermate control mice (n = 6-25). Gene expression profiles from 253 breast cancer patients (stage I-III) were evaluated for clinic-pathological parameters and survival using Cox proportional hazard ratios (HRs) and 95% confidence intervals (CIs) based on a two-sided Wald test., Results: We report that pericyte deficiency leads to increased transmigration of Gr1(+)/CD11b(+) cells in experimentally induced tumors. Pericyte deficiency produced defective tumor vasculature, resulting in a more hypoxic microenvironment promoting IL-6 upregulation in the malignant cells. Silencing IL-6 expression in tumor cells attenuated the observed differences in MDSC transmigration. Restoring the pericyte coverage in tumors abrogated the increased MDSC trafficking to pericyte-deficient tumors. MDSC accumulation in tumors led to increases in tumor growth and in circulating malignant cells. Finally, gene expression analysis from human breast cancer patients revealed increased expression of the human MDSC markers CD33 and S100A9 with concomitant decreased expression of pericyte genes and was associated with poor prognosis (HR = 1.88, 95% CI = 1.08 to 3.25, P = .03)., Conclusions: Our data uncovers a novel paracrine interaction between tumor pericytes and inflammatory cells and delineates the cellular events resulting in the recruitment of MDSC to tumors. Furthermore, we propose for the first time a role for tumor pericytes in modulating the expression of immune mediators in malignant cells by promoting a hypoxic microenvironment., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The rates of the reactions Fe(+) + N2O → FeO(+) + N2 and FeO(+) + CO → Fe(+) + CO2 are modeled by statistical rate theory accounting for energy- and angular momentum-specific rate constants for formation of the primary and secondary cationic adducts and their backward and forward reactions. The reactions are both suggested to proceed on sextet and quartet potential energy surfaces with efficient, but probably not complete, equilibration by spin-inversion of the populations of the sextet and quartet adducts. The influence of spin-inversion on the overall reaction rate is investigated. The differences of the two reaction rates mostly are due to different numbers of entrance states (atom + linear rotor or linear rotor + linear rotor, respectively). The reaction Fe(+) + N2O was studied either with (6)Fe(+) or with (4)Fe(+) reactants. Differences in the rate constants of (6)Fe(+) and (4)Fe(+) reacting with N2O are attributed to different contributions from electronically excited potential energy surfaces, such as they originate from the open-electronic shell reactants.

The reactions of FeO(+) with H2 and of Fe(+) with N2O were studied with respect to the production and reactivity of electronically excited (4)Fe(+) cations. The reaction of electronic ground state (6)FeO(+) with H2 was found to predominantly produce electronically excited (4)Fe(+) as opposed to electronic ground state (6)Fe(+) corresponding to a spin-allowed reaction. (4)Fe(+) was observed to react with N2O with a rate constant of 2.3 (+0.3/-0.8) × 10(-11) cm(3) molecule(-1) s(-1), smaller than the ground state (6)Fe(+) rate constant of 3.2 (±0.5) × 10(-11) cm(3) molecule(-1) s(-1) (at room temperature). While the overall reaction of (6)FeO(+) with H2 within the Two-State-Reactivity concept is governed by efficient sextet-quartet spin-inversion in the initial reaction complex, the observation of predominant (4)Fe(+) production in the reaction is attributed to a much less efficient quartet-sextet back-inversion in the final reaction complex. Average spin-inversion probabilities are estimated by statistical modeling of spin-inversion processes and related to the properties of spin-orbit coupling along the reaction coordinate. The reaction of FeO(+) with H2 served as a source for (4)Fe(+), subsequently reacting with N2O. The measured rate constant has allowed for a more detailed understanding of the ground state (6)Fe(+) reaction with N2O, leading to a significantly improved statistical modeling of the previously measured temperature dependence of the reaction. In particular, evidence for the participation of electronically excited states of the reaction complex was found. Deexcitation of (4)Fe(+) by He was found to be slow, with a rate constant <3 × 10(-14) cm(3) molecule(-1) s(-1).

Unlabelled: Hypoxia-inducible transcription factors (HIFs) control cellular adaptation to low oxygen. In the kidney, activation of HIF is beneficial during injury; however, the specific contribution of HIF-1α in renal endothelial cells (EC) remains elusive. Since EC display tissue-specific heterogeneity, we investigated how HIF-1α affects key functions of glomerular EC in vitro and its contribution to renal development and pathophysiological adaptation to acute or chronic renal injury in vivo. Loss of HIF-1α in glomerular EC induces hypoxic cell death and reduces hypoxic adhesion of macrophages in vitro. In vivo, HIF-1α expression in EC in mouse kidneys is detectable but limited. Accordingly, EC-specific ablation of HIF-1α does not lead to developmental or phenotypical abnormalities in the kidney. Renal function and expression of adhesion molecules during acute ischemic kidney injury is independent of HIF-1α in EC. Likewise, inflammation and development of fibrosis after unilateral ureteric obstruction is not influenced by endothelial HIF-1α. Taken together, although HIF-1α exerts effects on glomerular EC in vitro, endothelial HIF-1α does not influence renal development and pathophysiological adaptation to kidney injury in vivo. This implies a profound difference of the hypoxic response of the renal vascular bed compared to other organs, such as the heart. This has implications for the development of pharmacological strategies targeting the endothelial hypoxic response pathways., Key Message: HIF-1α controls hypoxic survival and adhesion on endothelial cells (EC) in vitro. In vivo, HIF-1α expression in renal EC is low. Deletion of HIF-1α in EC does not affect kidney development and function in mice. Renal function after acute and chronic kidney injury is independent of HIF-1α in EC. Data suggest organ-specific regulation of HIF-1α function in EC.

Transcriptional mediators of cell stress pathways, including HIF1α, ATF4, and p53, are key to normal development and play critical roles in disease, including ischemia and cancer. Despite their importance, mechanisms by which pathways mediated by these transcription factors interact with one another are not fully understood. In addressing the controversial role of HIF1α in cardiomyocytes (CMs) during heart development, we discovered a mid-gestational requirement for HIF1α for proliferation of hypoxic CMs, involving metabolic switching and a complex interplay among HIF1α, ATF4, and p53. Loss of HIF1α resulted in activation of ATF4 and p53, the latter inhibiting CM proliferation. Bioinformatic and biochemical analyses revealed unexpected mechanisms by which HIF1α intersects with ATF4 and p53 pathways. Our results highlight previously undescribed roles of HIF1α and interactions among major cell stress pathways that could be targeted to enhance proliferation of CMs in ischemia and may have relevance to other diseases, including cancer., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Qiu and colleagues describe how a structural component of lipid droplets is markedly induced in pseudohypoxic renal tumors, where it maintains endoplasmic reticulum (ER) homeostasis. This adaptation is indispensable in tumor cells-where growth demands and a fluctuating blood supply place unnatural stresses on ER function-and is therefore an attractive therapeutic target., (©2015 American Association for Cancer Research.)

It has been suggested that physicians are particularly vulnerable to being stalked. Our goal was to examine the prevalence of physicians who have been stalked and the associated consequences for the victims. We conducted multiple searches of PubMed and PsycINFO for articles in English from 1950 to 2013, using the terms stalker, stalking, aggression, assaults, patient, physician, resident, registrar, intern, and trainee. Reference lists of relevant articles were also searched. We developed and used a five-point evaluation tool for critical appraisal of the articles. We found 12 prevalence studies on the stalking of physicians, of which 8 were national surveys and 4 were focused exclusively on stalking. The studies varied in their methodological quality with common limitations including the lack of a national sample, the lack of construct validity of the survey tool and of the provision of a formal definition of stalking, and low response rates. Prevalence rates ranged from 2 to 25 percent, although one study found a prevalence rate of 68.5 percent. Information on the physical and psychological consequences of having been stalked was also limited. Although a substantial minority of physicians reported having been stalked, there remains a dearth of high-quality studies on the topic., (© 2015 American Academy of Psychiatry and the Law.)

This descriptive analysis is an examination of the extent to which a veteran's mental health diagnosis or the initial criminal charge committed before program enrollment relate to a greater propensity for sanctions, harsher sanctions, higher rates of relapse on substances, or overall program compliance. This is a retrospective descriptive analysis that focuses on those participants in the Harris County (Texas) Veterans' Court Program from June 2010 through April 2012 for whom the court had issued sanctions. The most clinically relevant association (p = .014) was found between veterans with substance use relapse and subsequent discharge from the program. Furthermore, the following four infractions were associated with a subsequent jail sanction: unexcused absence (p = .014), failure to complete a task (p = .010), substance use relapse (p = .001), and missing a hearing (p = .012). Given these findings, veterans with relapses in substance use during the course of the program are at greatest risk of noncompletion of the program and may represent a subpopulation of veterans who require greater or different types of assistance., (© 2015 American Academy of Psychiatry and the Law.)

Protein chemical cross-linking and mass spectrometry enable the analysis of protein-protein interactions and protein topologies; however, complicated cross-linked peptide spectra require specialized algorithms to identify interacting sites. The Kojak cross-linking software application is a new, efficient approach to identify cross-linked peptides, enabling large-scale analysis of protein-protein interactions by chemical cross-linking techniques. The algorithm integrates spectral processing and scoring schemes adopted from traditional database search algorithms and can identify cross-linked peptides using many different chemical cross-linkers with or without heavy isotope labels. Kojak was used to analyze both novel and existing data sets and was compared to existing cross-linking algorithms. The algorithm provided increased cross-link identifications over existing algorithms and, equally importantly, the results in a fraction of computational time. The Kojak algorithm is open-source, cross-platform, and freely available. This software provides both existing and new cross-linking researchers alike an effective way to derive additional cross-link identifications from new or existing data sets. For new users, it provides a simple analytical resource resulting in more cross-link identifications than other methods.

Inflammation is closely related to the extent of damage following cerebral ischaemia, and the targeting of this inflammation has emerged as a promising therapeutic strategy. Here, we present that hypoxia-induced glial T-cell immunoglobulin and mucin domain protein (TIM)-3 can function as a modulator that links inflammation and subsequent brain damage after ischaemia. We find that TIM-3 is highly expressed in hypoxic brain regions of a mouse cerebral hypoxia-ischaemia (H/I) model. TIM-3 is distinctively upregulated in activated microglia and astrocytes, brain resident immune cells, in a hypoxia-inducible factor (HIF)-1-dependent manner. Notably, blockade of TIM-3 markedly reduces infarct size, neuronal cell death, oedema formation and neutrophil infiltration in H/I mice. Hypoxia-triggered neutrophil migration and infarction are also decreased in HIF-1α-deficient mice. Moreover, functional neurological deficits after H/I are significantly improved in both anti-TIM-3-treated mice and myeloid-specific HIF-1α-deficient mice. Further understanding of these insights could serve as the basis for broadening the therapeutic scope against hypoxia-associated brain diseases.

This study aims to examine the extent to which a veteran's propensity for arrest following separation from veterans' court is associated with that veteran's length of stay within the program, type of discharge, or number of judicial sanctions issued. This is a retrospective chart review that focuses on the first 100 participants in the Harris County Veterans' Court Program. After controlling for a number of demographic factors, both arrests during enrollment in the veterans' court program (p = .031) and Factor Score 1 (unsuccessful discharge, fewer months in the veterans' court program, and more months of follow up) (p = .042) were predictive of arrest following separation from the veterans' court program. In addition, a prior diagnosis of opiate misuse was also predictive of arrest following separation (p < .001). Given these findings, veterans' court judges and program administrators might examine ways of continuing enrollment for veterans at highest risk for recidivism.

In mammals, hypoxia-triggered erythropoietin release increases red blood cell mass to meet tissue oxygen demands. Using male Wistar rats, we unmask a previously unrecognized regulatory pathway of erythropoiesis involving suppressor control by the NO metabolite and ubiquitous dietary component nitrate. We find that circulating hemoglobin levels are modulated by nitrate at concentrations achievable by dietary intervention under normoxic and hypoxic conditions; a moderate dose of nitrate administered via the drinking water (7 mg NaNO3/kg body weight/d) lowered hemoglobin concentration and hematocrit after 6 d compared with nonsupplemented/NaCl-supplemented controls. The underlying mechanism is suppression of hepatic erythropoietin expression associated with the downregulation of tissue hypoxia markers, suggesting increased pO2. At higher nitrate doses, however, a partial reversal of this effect occurred; this was accompanied by increased renal erythropoietin expression and stabilization of hypoxia-inducible factors, likely brought about by the relative anemia. Thus, hepatic and renal hypoxia-sensing pathways act in concert to modulate hemoglobin in response to nitrate, converging at an optimal minimal hemoglobin concentration appropriate to the environmental/physiologic situation. Suppression of hepatic erythropoietin expression by nitrate may thus act to decrease blood viscosity while matching oxygen supply to demand, whereas renal oxygen sensing could act as a brake, averting a potentially detrimental fall in hematocrit., (© FASEB.)

Sunshine virus is a paramyxovirus of pythons associated with neurorespiratory disease and mortalities. This report provides evidence for its vertical transmission. In a collection of over 200 Australian pythons, a dam and a sire, both carpet pythons (Morelia spilota), were PCR-positive for Sunshine virus at a time when the dam was likely to have been gravid. A clutch of 21 eggs was laid and three non-viable eggs were tested for the presence of Sunshine virus by PCR. One egg had been incubating for 34 days while the other two had been incubating for 49 days. The surface of all three eggs was negative for Sunshine virus but swabs of the allantois and amnion were positive in all three eggs. Embryo tissue samples were tested from the two 49 day old eggs. From one embryo, a sample of brain and a pooled sample of lung, liver, kidney and intestine were positive, while for the other embryo, a pooled sample of lung, liver, kidney, intestine and brain was positive. Fourteen of the 21 eggs hatched and all hatchlings were tested by PCR at least once between the ages of 53 and 229 days old. All hatchlings were PCR-negative for Sunshine virus., (Copyright © 2014 Elsevier B.V. All rights reserved.)