Cotinine influences the effect of high and low nicotine concentrations on planarian motility differently.

Previous work from our laboratory showed that cotinine, a nicotine metabolite, reverses three nicotine-induced behavioral effects in freshwater planarians: motility decrease, seizure-like movements, and withdrawal-like behaviors. The present work explored whether cotinine, a nicotine metabolite, antagonized the nicotine-induced effects on planarian motility in a concentration-dependent manner. We found that nicotine decreased planarian motility at nicotine concentrations above 60 µM but increased planarian velocity at concentrations equal to or below 50 µM, in agreement with previous data. Cotinine did not affect planarian motility at a concentration range between 250 and 2750 µM. Furthermore, we found that cotinine alleviated the 100 µM nicotine-induced motility decrease in a concentration-dependent manner and reversed the low nicotine concentration motility increase, albeit in a concentration-independent manner. The apparent concentration-dependent alleviation of >60 µM nicotine-induced motility decrease by cotinine suggests an orthosteric relationship between nicotine and cotinine. On the other hand, the evident concentration-independent cotinine alleviation of the increase in motility induced by 50 µM nicotine suggests an allosteric relationship. Our data is consistent with the existing literature about the relationship between nicotine and cotinine in various models, reinforcing the case for the usefulness of the planarian model in pharmacological studies.

Implementation of Extracorporeal CPR Programs for Out-of-Hospital Cardiac Arrest: Another Tale of Two County Hospitals.

Extracorporeal cardiopulmonary resuscitation (ECPR) is a form of intensive life support that has seen increasing use globally to improve outcomes for patients who experience out-of-hospital cardiac arrest (OHCA). Hospitals with advanced critical care capabilities may be interested in launching an ECPR program to offer this support to the patients they serve; however, to do so, they must first consider the significant investment of resources necessary to start and sustain the program. The existing literature describes many single-center ECPR programs and often focuses on inpatient care and patient outcomes in hospitals with cardiac surgery capabilities. However, building a successful ECPR program and using this technology to support an individual patient experiencing refractory cardiac arrest secondary to a shockable rhythm depends on efficient out-of-hospital and emergency department (ED) management. This article describes the process of implementing 2 intensivist-led ECPR programs with limited cardiac surgery capability. We focus on emergency medical services and ED clinician roles in identifying patients, mobilizing resources, initiation and management of veno-arterial extracorporeal membrane oxygenation (VA-ECMO) in the ED, and ongoing efforts to improve ECPR program quality. Each center experienced a significant learning curve to reach goals of arrest-to-flow times of cannulation for ECPR. Building consensus from multidisciplinary stakeholders, including out-of-hospital stakeholders; establishing shared expectations of ECPR outcomes; and ensuring adequate resource support for ECPR activation were all key lessons in improving our ECPR programs.

Implementation of Extracorporeal Membrane Oxygenation Without On-Site Cardiac Surgery or Perfusion Support: A Tale of Two County Hospitals.

Patients with refractory respiratory and cardiac failure may present to noncardiac surgery centers. Prior studies have demonstrated that acute care surgeons, intensivists, and emergency medicine physicians can safely cannulate and manage patients receiving extracorporeal membrane oxygenation (ECMO). Harborview Medical Center (Harborview) and Hennepin County Medical Center (Hennepin) are both urban, county-owned, level 1 trauma centers that implemented ECMO without direct, on-site cardiac surgery or perfusion support. Both centers 1) use an ECMO specialist model staffed by specially trained nurses and respiratory therapists and 2) developed comparable training curricula for ECMO specialists, intensivists, surgeons, and trainees. Each program began with venovenous ECMO to provide support for refractory hypoxemic respiratory failure and subsequently expanded to venoarterial ECMO support. The coronavirus disease 2019 (COVID-19) pandemic created an impetus for restructuring, with each program creating a consulting service to facilitate ECMO delivery across multiple intensive care units (ICUs) and to promote fellow and resident training and experience. Both Harborview and Hennepin, urban county hospitals 1,700 miles apart in the United States, independently implemented and operate adult ECMO programs without involvement from cardiovascular surgery or perfusion services. This experience further supports the role of ECMO specialists in the delivery of extracorporeal life support.

Traffic-generated air pollution - Exposure mediated expression of factors associated with demyelination in a female apolipoprotein E-/- mouse model.

Epidemiology studies suggest that exposure to ambient air pollution is associated with demyelinating diseases in the central nervous system (CNS), including multiple sclerosis (MS). The pathophysiology of MS results from an autoimmune response involving increased inflammation and demyelination in the CNS, which is higher in young (adult) females. Exposure to traffic-generated air pollution is associated with neuroinflammation and other detrimental outcomes in the CNS; however, its role in the progression of pathologies associated with demyelinating diseases has not yet been fully characterized in a female model. Thus, we investigated the effects of inhalation exposure to mixed vehicle emissions (MVE) in the brains of both ovary-intact (ov+) and ovariectomized (ov-) female Apolipoprotein (ApoE-/-) mice. Ov + and ov- ApoE-/- mice were exposed via whole-body inhalation to either filtered air (FA, controls) or mixed gasoline and diesel vehicle emissions (MVE: 200 PM µg/m3) for 6 h/d, 7 d/wk., for 30 d. We then analyzed MVE-exposure mediated alterations in myelination, the presence of CD4+ and CD8+ T cells, reactive oxygen species (ROS), myelin oligodendrocyte protein (MOG), and expression of estrogen (ERα and ERß) and progesterone (PROA/B) receptors in the CNS. MVE-exposure mediated significant alterations in myelination across multiple regions in the cerebrum, as well as increased CD4+ and CD8+ staining. There was also an increase in ROS production in the CNS of MVE-exposed ov- and ov + ApoE-/- mice. Ov- mice displayed a reduction in cerebral ERα mRNA expression, compared to ov + mice; however, MVE exposure resulted in an even further decrease in ERα expression, while ERß and PRO A/B were unchanged across groups. These findings collectively suggest that inhaled MVE-exposure may mediate estrogen receptor expression alterations associated with increased CD4+/CD8+ infiltration, regional demyelination, and ROS production in the CNS of female ApoE-/- mice.

Acute interstitial nephritis secondary to vedolizumab.

We present a patient with an acute kidney injury thought secondary to acute interstitial nephritis as a result of vedolizumab maintenance therapy for Crohn's disease. This appears to be a rare but serious side effect in patients receiving this treatment which clinicians should consider in the event of renal dysfunction.

Phosphodiesterase 1C integrates store-operated calcium entry and cAMP signaling in leading-edge protrusions of migrating human arterial myocytes.

In addition to maintaining cellular ER Ca2+ stores, store-operated Ca2+ entry (SOCE) regulates several Ca2+-sensitive cellular enzymes, including certain adenylyl cyclases (ADCYs), enzymes that synthesize the secondary messenger cyclic AMP (cAMP). Ca2+, acting with calmodulin, can also increase the activity of PDE1-family phosphodiesterases (PDEs), which cleave the phosphodiester bond of cAMP. Surprisingly, SOCE-regulated cAMP signaling has not been studied in cells expressing both Ca2+-sensitive enzymes. Here, we report that depletion of ER Ca2+ activates PDE1C in human arterial smooth muscle cells (HASMCs). Inhibiting the activation of PDE1C reduced the magnitude of both SOCE and subsequent Ca2+/calmodulin-mediated activation of ADCY8 in these cells. Because inhibiting or silencing Ca2+-insensitive PDEs had no such effects, these data identify PDE1C-mediated hydrolysis of cAMP as a novel and important link between SOCE and its activation of ADCY8. Functionally, we showed that PDE1C regulated the formation of leading-edge protrusions in HASMCs, a critical early event in cell migration. Indeed, we found that PDE1C populated the tips of newly forming leading-edge protrusions in polarized HASMCs, and co-localized with ADCY8, the Ca2+ release activated Ca2+ channel subunit, Orai1, the cAMP-effector, protein kinase A, and an A-kinase anchoring protein, AKAP79. Because this polarization could allow PDE1C to control cAMP signaling in a hyper-localized manner, we suggest that PDE1C-selective therapeutic agents could offer increased spatial specificity in HASMCs over agents that regulate cAMP globally in cells. Similarly, such agents could also prove useful in regulating crosstalk between Ca2+/cAMP signaling in other cells in which dysregulated migration contributes to human pathology, including certain cancers.

Exposure to traffic-generated air pollution promotes alterations in the integrity of the brain microvasculature and inflammation in female ApoE-/- mice.

Traffic-generated air pollutants have been correlated with alterations in blood-brain barrier (BBB) integrity, which is associated with pathologies in the central nervous system (CNS). Much of the existing literature investigating the effects of air pollution in the CNS has predominately been reported in males, with little known regarding the effects in females. As such, this study characterized the effects of inhalation exposure to mixed vehicle emissions (MVE), as well as the presence of female sex hormones, in the CNS of female ApoE-/- mice, which included cohorts of both ovariectomized (ov-) and ovary-intact (ov+) mice. Ov + and ov- were placed on a high-fat diet and randomly grouped to be exposed to either filtered-air (FA) or MVE (200 PM/m3: 50 µg PM/m3 gasoline engine + 150 µg PM/m3 from diesel engine emissions) for 6 h/d, 7d/wk, for 30d. MVE-exposure resulted in altered cerebral microvascular integrity and permeability, as determined by the decreased immunofluorescent expression of tight junction (TJ) proteins, occludin, and claudin-5, and increased IgG extravasation into the cerebral parenchyma, compared to FA controls, regardless of ovary status. Associated with the altered cerebral microvascular integrity, we also observed an increase in matrix metalloproteinases (MMPs) -2/9 activity in the MVE ov+, MVE ov-, and FA ov- groups, compared to FA ov+. There was also elevated expression of intracellular adhesion molecule (ICAM)-1, inflammatory interleukins (IL-1, IL-1ß), and tumor necrosis factor (TNF-α) mRNA in the cerebrum of MVE ov + and MVE ov- animals. IκB kinase (IKK) subunits IKKα and IKKß mRNA expressions were upregulated in the cerebrum of MVE ov- and FA ov- mice. Our findings indicate that MVE exposure mediates altered integrity of the cerebral microvasculature correlated with increased MMP-2/9 activity and inflammatory signaling, regardless of female hormones present.

Emergency Department Management of Out-of-Hospital Laryngeal Tubes.

STUDY OBJECTIVE: Laryngeal tubes are commonly used by emergency medical services (EMS) personnel for out-of-hospital advanced airway management. The emergency department (ED) management of EMS-placed laryngeal tubes is unknown. We seek to describe ED airway management techniques, success, and complications of patients receiving EMS laryngeal tubes. METHODS: Using a keyword text search of ED notes, we identified patients who arrived at our ED with a laryngeal tube from 2010 through 2017. We performed structured chart and video reviews for all eligible patients. In our ED, emergency physicians perform all airway management, and there is no protocol dictating airway management for patients arriving with a laryngeal tube. Using descriptive methods, we report the techniques, success, and complications of ED airway management. RESULTS: We analyzed data on 647 patients receiving out-of-hospital laryngeal tubes, including 472 (73%) with cardiac arrest from medical causes, 75 (21%) with cardiac arrest from trauma, and 100 (15%) with other conditions. For 580 patients (89%), emergency physicians exchanged the laryngeal tube for a definitive airway in the ED. Of the 67 patients not intubated in the ED, 66 died in the ED without further airway management. Of the 580 patients intubated in the ED, orotracheal intubation was the first method attempted for 578 (>99%) and was successful on the first attempt for 515 of 578 (89%). Macintosh video laryngoscopy (88% of initial attempts) and a bougie (68% of initial attempts) were commonly used adjuncts. For 345 of 578 patients (60%), the laryngeal tube was removed before intubation attempts. For 112 of 578 patients (19%), the first intubation attempt occurred with the deflated laryngeal tube left in place. Three patients (<1%) required a surgical airway. CONCLUSION: In this cohort, emergency physicians successfully exchanged an out-of-hospital laryngeal tube for an endotracheal tube, using commonly available airway management techniques. ED clinicians should be familiar with techniques for exchanging out-of-hospital extraglottic airways for an endotracheal tube.

A prospective observational study of prevalence and outcomes of patients with Gold Standard Framework criteria in a tertiary regional Australian Hospital.

OBJECTIVES: Report the use of an objective tool, UK Gold Standards Framework (GSF) criteria, to describe the prevalence, recognition and outcomes of patients with palliative care needs in an Australian acute health setting. The rationale for this is to enable hospital doctors to identify patients who should have a patient-centred discussion about goals of care in hospital. DESIGN: Prospective, observational, cohort study. PARTICIPANTS: Adult in-patients during two separate 24 h periods. MAIN OUTCOME MEASURES: Prevalence of in-patients with GSF criteria, documentation of treatment limitations, hospital and 1 year survival, admission and discharge destination and multivariate regression analysis of factors associated with the presence of hospital treatment limitations and 1 year survival. RESULTS: Of 626 in-patients reviewed, 171 (27.3%) had at least one GSF criterion, with documentation of a treatment limitation discussion in 60 (30.5%) of those patients who had GSF criteria. Hospital mortality was 9.9%, 1 year mortality 50.3% and 3-year mortality 70.2% in patients with GSF criteria. One-year mortality was highest in patients with GSF cancer (73%), renal failure (67%) and heart failure (60%) criteria. Multivariate analysis revealed age, hospital length of stay and presence of the GSF chronic obstructive pulmonary disease criteria were independently associated with the likelihood of an in-hospital treatment limitation. Non-survivors at 3 years were more likely to have a GSF cancer (25% vs 6%, p=0.004), neurological (10% vs 3%, p=0.04), or frailty (45% vs 3%, p=0.04) criteria. After multivariate logistic regression GSF cancer criteria, renal failure criteria and the presence of two or more GSF clinical criteria were independently associated with increased risk of death at 3 years. Patients returning home to live reduced from 69% (preadmission) to 27% after discharge. CONCLUSIONS: The use of an objective clinical tool identifies a high prevalence of patients with palliative care needs in the acute tertiary Australian hospital setting, with a high 1 year mortality and poor return to independence in this population. The low rate of documentation of discussions about treatment limitations in this population suggests palliative care needs are not recognised and discussed in the majority of patients. TRIAL REGISTRATION NUMBER: 11/121.

Effect of communication skills training on outcomes in critically ill patients with life-limiting illness referred for intensive care management: a before-and-after study.

OBJECTIVES: To describe the effect of a communication skills training programme on patient-centred goals of care documentation and clinical outcomes in critically ill patients with life-limiting illnesses (LLI) referred for intensive care management. METHODS: Prospective before-and-after cohort study in a tertiary teaching hospital in Australia. The population was 222 adult patients with LLI referred to the intensive care unit (ICU). The study was divided into two periods, before (1 May to 31 July 2015) and after (15 September to 15December 2015) the intervention. The intervention was a 2-day, small group, simulated-patient, communication skills course, and process of care for patients with LLI. The primary outcome was documentation of patient-centred goals of care discussion (PCD) within 48 hours of referral to the ICU. Secondary outcomes included clinical outcomes and 90-day mortality. RESULTS: The intervention was associated with increased documentation of a PCD from 50% to 69% (p=0.004) and 43% to 94% (p<0.0001) in patients deceased by day 90. A significant decrease in critical care as the choice of resuscitation goal (61% vs 42%, p=0.02) was observed. Although there was no decrease in admission to ICU, there was a significant decrease in medical emergency team call prevalence (87% vs 73%, p=0.009). The cancer and organ failure groups had a significant decrease in 90-day mortality (75% vs 44%, p=0.02; 42% vs 16%, p=0.01), and the frailty group had a significant decrease in 90-day readmissions (48% vs 19%, p=0.003). CONCLUSIONS: The intervention was associated with increased PCD documentation and decrease in the choice of critical care as the resuscitation goal. Admissions to ICU did not decrease, and although limited by study design, condition-specific trajectory changes, clinical interventions and outcomes warrant further study.

Comparison of pulmonary artery catheter, echocardiography, and arterial waveform analysis monitoring in predicting the hemodynamic state during and after cardiac surgery.

OBJECTIVE: The aim of this trial was to determine whether Flotrac Vigileo™ (FV™) provides a reliable representation of the hemodynamic state of a cardiac surgical patient population when compared to pulmonary artery catheter (PAC) and echocardiography in the peril-operative period. DESIGN: This was a prospective observational trial comparing perioperative hemodynamic states using transesophageal echocardiography (TEE), transthoracic echocardiography (TTE), FV™ and PAC during and post cardiothoracic surgery. SETTING: Tertiary regional hospital Intensive Care Unit (ICU). PARTICIPANTS: 50 consecutive adult cardiothoracic patients with written consent provided. INTERVENTION: Comparison of the perioperative hemodynamic states using echocardiography, FV™ and PAC was performed. Evaluation of the hemodynamic state (HDS) was performed using TEE, TTE, PAC and FV™ during and after cardiac surgery. Data were compared between the three hemodynamic assessment modalities. MAIN OUTCOME MEASURE: Predicted hemodynamic state. RESULTS: FV™ and PAC were shown to correlate poorly with TEE/TTE assessment of the hemodynamic state. Both PAC and FV™ showed significant discordance with echocardiographic assessment of the hemodynamic state. CONCLUSIONS: In this trial, FV™ and PAC were shown to agree poorly with TTE/TEE assessment of the HDS in an adult cardiothoracic population. Agreement between the FV™ and PAC was also poor. Caution is recommended in interpreting isolated hemodynamic monitoring data. All hemodynamic monitoring devices have inherent sources of error. Caution is advised in interpreting any single device or measurement as a gold standard. We suggest that hemodynamic measuring devices such as FV™/PAC may act as triggers for a global hemodynamic assessment including consideration of TTE/TEE.

LY3009120, a panRAF inhibitor, has significant anti-tumor activity in BRAF and KRAS mutant preclinical models of colorectal cancer.

Activating mutations in the KRAS and BRAF genes, leading to hyperactivation of the RAS/RAF/MAPK oncogenic signaling cascade, are common in patients with colorectal cancer (CRC). While selective BRAF inhibitors are efficacious in BRAFmut melanoma, they have limited efficacy in BRAFmut CRC patients. In a RASmut background, selective BRAF inhibitors are contraindicated due to paradoxical activation of the MAPK pathway through potentiation of CRAF kinase activity. A way to overcome such paradoxical activation is through concurrent inhibition of the kinase activity of both RAF isoforms. Here, we further examined the effects of LY3009120, a panRAF and RAF dimer inhibitor, in human models of CRC with various mutational backgrounds. We demonstrate that LY3009120 induced anti-proliferative effects in BRAFmut and KRASmut CRC cell lines through G1-cell cycle arrest. The anti-proliferative effects of LY3009120 in KRASmut CRC cell lines phenocopied molecular inhibition of RAF isoforms by simultaneous siRNA-mediated knockdown of ARAF, BRAF and CRAF. Additionally, LY3009120 displayed significant activity in in vivo BRAFmut and KRASmut CRC xenograft models. Examination of potential resistance to LY3009120 demonstrated RAF-independent ERK and AKT activation in the KRASmut CRC cell line HCT 116. These findings describe the preclinical activity of a panRAF inhibitor in a BRAFmut and KRASmut CRC setting.

Prevalence, goals of care and long-term outcomes of patients with life-limiting illness referred to a tertiary ICU.

OBJECTIVE: To describe the prevalence, characteristics, long-term outcomes and goals-of-care discussions of patients with objective indicators of life-limiting illnesses (LLIs) referred to the intensive care unit. DESIGN, SETTING AND PATIENTS: A prospective, observational, cohort study of all adult inpatients referred to the ICU by the medical emergency team or by direct referral, during the period 30 August 2012 to 1 February 2013, at a tertiary teaching hospital in Australia. MAIN OUTCOME MEASURES: Mortality, LLIs, discharge destination and documentation on goals of care in medical record. RESULTS: A total of 649 of 1024 patients referred to the ICU had an LLI, and only 34.4% of these patients had goals of care documented. Overall, 49.2% were admitted to the ICU, 48.4% were discharged home, and the 1-year mortality was 35.1%. The most common LLI criteria were heart disease (52.2%), chronic obstructive pulmonary disease (24.8%) and frailty (23.7%). The highest 1-year mortality was associated with pre-hospital residence in a nursing home (64.9%), dementia (63.3%), cancer (60.8%) and frailty (50.6%). Analysis of patients by clinical trajectory showed that 1-year mortality was significantly higher for patients with cancer (59.6%), combined organ failure and frailty (47.3%), frailty (43.8%) and organ failure (23.6%), compared with patients with no LLI (P < 0.0001). CONCLUSIONS: A high proportion of patients referred to the ICU have an LLI, and this is associated with prolonged hospital length of stay and a high 1-year mortality, and only one-quarter have documented discussions on goals of care. Patients with cancer-related and frailty-related LLIs have the worst survival trajectories.

In Situ Transplantation of Alginate Bioencapsulated Adipose Tissues Derived Stem Cells (ADSCs) via Hepatic Injection in a Mouse Model.

OBJECTIVE: Adipose tissue derived stem cells (ADSCs) transplantation has recently gained widespread enthusiasm, particularly in the perspective to use them as potential alternative cell sources for hepatocytes in cell based therapy, mainly because of their capability of hepatogenic differentiation in vitro and in vivo. But some challenges remain to be addressed, including whether ADSCs can be provided effectively to the target organ and whether subsequent proliferation of transplanted cells can be achieved. To date, intrasplenic injection is the conventional method to deliver ADSCs into the liver; however, a number of donor cells retained in the spleen has been reported. In this study, our objective is to evaluate a novel route to transplant ADSCs specifically to the liver. We aimed to test the feasibility of in situ transplantation of ADSCs by injecting bioencapsulated ADSCs into the liver in mouse model. METHODS: The ADSCs isolated from human alpha 1 antitrypsin (M-hAAT) transgenic mice were used to allow delivered ADSCs be readily identified in the liver of recipient mice, and alginate was selected as a cell carrier. We first evaluated whether alginate microspheres are implantable into the liver tissue by injection and whether ADSCs could migrate from alginate microspheres (study one). Once proven, we then examined the in vivo fate of ADSCs loaded microspheres in the liver. Specifically, we evaluated whether transplanted, undifferentiated ASDCs could be induced by the local microenvironment toward hepatogenic differentiation and the distribution of surviving ADSCs in major tissue organs (study two). RESULTS: Our results indicated ADSCs loaded alginate microspheres were implantable into the liver. Both degraded and residual alginate microspheres were observed in the liver up to three weeks. The viable ADSCs were detectable surrounding degraded and residual alginate microspheres in the liver and other major organs such as bone marrow and the lungs. Importantly, transplanted ADSCs underwent hepatogenic differentiation to become cells expressing albumin in the liver. These findings improve our understanding of the interplay between ADSCs (donor cells), alginate (biomaterial), and local microenvironment in a hepatectomized mouse model, and might improve the strategy of in situ transplantation of ADSCs in treating liver diseases.

Glucose oxidation modulates anoikis and tumor metastasis.

Cancer cells exhibit altered glucose metabolism characterized by a preference for aerobic glycolysis or the Warburg effect, and the cells resist matrix detachment-induced apoptosis, which is called anoikis, a barrier to metastasis. It remains largely unclear whether tumor metabolism influences anoikis and metastasis. Here we show that when detached from the matrix, untransformed mammary epithelial cells undergo metabolic reprogramming by markedly upregulating pyruvate dehydrogenase (PDH) kinase 4 (PDK4) through estrogen-related receptor gamma (ERRγ), thereby inhibiting PDH and attenuating the flux of glycolytic carbon into mitochondrial oxidation. To decipher the significance of this metabolic response, we found that depletion of PDK4 or activation of PDH increased mitochondrial respiration and oxidative stress in suspended cells, resulting in heightened anoikis. Conversely, overexpression of PDKs prolonged survival of cells in suspension. Therefore, decreased glucose oxidation following cell detachment confers anoikis resistance. Unlike untransformed cells, most cancer cells demonstrate reduced glucose oxidation even under attached conditions, and thus they inherently possess a survival advantage when suspended. Normalization of glucose metabolism by stimulating PDH in cancer cells restores their susceptibility to anoikis and impairs their metastatic potential. These results suggest that the Warburg effect, more specifically, diminished glucose oxidation, promotes anoikis resistance and metastasis and that PDKs are potential targets for antimetastasis therapy.

In vivo MR studies of glycine and glutathione metabolism in a rat mammary tumor.

The metabolism of glycine into glutathione was monitored noninvasively in vivo in intact rat mammary adenocarcinomas (R3230Ac) by MRI and MRS. Metabolism was tracked by following the isotope label from intravenously infused [2-(13)C]-glycine into the glycinyl residue of glutathione. Signals from [2-(13)C]-glycine and γ-glutamylcysteinyl-[2-(13)C]-glycine ((13)C-glutathione) were detected by nonlocalized (13)C spectroscopy, as these resonances are distinct from background signals. In addition, using spectroscopic imaging methods, heterogeneity in the in vivo tumor distribution of glutathione was observed. In vivo spectroscopy also detected isotope incorporation from [2-(13)C]-glycine into both the 2- and 3-carbons of serine. Analyses of tumor tissue extracts showed single- and multiple-label incorporation from [2-(13)C]-glycine into serine from metabolism through the serine hydroxymethyltransferase and glycine cleavage system pathways. Mass spectrometric analysis of extracts also showed that isotope-labeled serine is further metabolized via the trans-sulfuration pathway, as (13)C isotope labels appear in both the glycinyl and cysteinyl residues of glutathione. Our studies demonstrate the use of MRI and MRS for the monitoring of tumor metabolic processes central to oxidative stress defense.

Dual perfluorocarbon method to noninvasively monitor dissolved oxygen concentration in tissue engineered constructs in vitro and in vivo.

Noninvasive in vivo monitoring of tissue implants provides important correlations between construct function and the observed physiologic effects. As oxygen is a key parameter affecting cell and tissue function, we established a monitoring method that utilizes (19) F nuclear magnetic resonance (NMR) spectroscopy, with perfluorocarbons (PFCs) as oxygen concentration markers, to noninvasively monitor dissolved oxygen concentration (DO) in tissue engineered implants. Specifically, we developed a dual PFC method capable of simultaneously measuring DO within a tissue construct and its surrounding environment, as the latter varies among animals and with physiologic conditions. In vitro studies using an NMR-compatible bioreactor demonstrated the feasibility of this method to monitor the DO within alginate beads containing metabolically active murine insulinoma ßTC-tet cells, relative to the DO in the culture medium, under perfusion and static conditions. The DO profiles obtained under static conditions were supported by mathematical simulations of the system. In vivo, the dual PFC method was successful in tracking the oxygenation state of entrapped ßTC-tet cells and the surrounding peritoneal DO over 16 days in normal mice. DO measurements correlated well with the extent of cell growth and host cell attachment examined postexplantation. The peritoneal oxygen environment was found to be variable and hypoxic, and significantly lower in the presence of metabolically active cells. The significance of the dual PFC system in providing critical DO measurements for entrapped cells and other tissue constructs, in vitro and in vivo, is discussed.

Diverse Helotiales associated with the roots of three species of Arctic Ericaceae provide no evidence for host specificity.

Ericoid mycorrhizal fungi differ in their abilities to use nitrogen sources and may be integral to maintaining fungal and plant diversity in ecosystems in which Ericaceae occur. In this study, we tested whether the fungal communities differ among three species of co-occurring Ericaceae. Fungi colonizing Cassiope tetragona, Empetrum nigrum and Vaccinium vitis-idaea roots in the Arctic tundra were characterized via culture-dependent and culture-independent techniques. The cultured fungi were tested for their ability to colonize Vaccinium uliginosum in laboratory-based assays. The pure-cultured Helotiales were grouped into eight clades and dominated by the Phialocephala-Acephala complex. Representatives of these clades, plus an unknown basidiomycete with affinity to the genus Irpex (Polyporales), colonized V. uliginosum intracellularly. The Helotiales detected by direct PCR, cloning and sequencing were assigned to 14 clades and dominated by members of the Rhizoscyphus ericae complex. Ordination analyses indicated that culture-dependent and culture-independent assays provided distinct views of root fungal communities, but no evidence for host specificity. These data suggest that ericaceous roots host diverse fungal communities dominated by the Helotiales. However, these fungal communities are unlikely to be controlled by fungal host preferences. The mechanisms maintaining high diversity in root-symbiotic communities remain to be elucidated.

Non-invasive monitoring of L-2-oxothiazolidine-4-carboxylate metabolism in the rat brain by in vivo 13C magnetic resonance spectroscopy.

The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5-(13)C]-thiazolidine-4-carboxylate, (13)C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by (13)C magnetic resonance spectroscopy. Although uptake and clearance of (13)C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of (13)C-OTZ over 20 h, however, resulted in (13)C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.

Limited beneficial effects of perfluorocarbon emulsions on encapsulated cells in culture: experimental and modeling studies.

Due to the high solubility of oxygen in perfluorocarbons (PFCs), these compounds have been explored for improved cell and tissue oxygenation. The goal of this study is to investigate the effects of a PFC emulsion on cellular growth and function in a tissue engineered construct. A perfluorotributylamine (PFTBA) emulsion was co-encapsulated at 10 vol% with mouse ßTC-tet insulinoma cells in calcium alginate beads and cultured under normoxic and severely hypoxic conditions. The number of metabolically active cells and the induced insulin secretion rate were measured over time for up to 16 days. Results showed no significant effect of PFTBA relative to the PFTBA-free control. The alginate-PFC-cell system was also modeled mathematically, and simulations tracked the number of viable cells over time under the same conditions used experimentally. Simulations revealed only a small, likely experimentally undetectable difference in cell density between the PFC-containing and PFC-free control beads. It is concluded that PFTBA up to 10 vol% has no significant effect on the growth and function of encapsulated ßTC-tet cells under normoxic and hypoxic conditions.