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Chronic urinary retention is chronic or repeated inability to empty the bladder, despite the ability to pass some urine. High-pressure chronic retention is a risk factor for renal failure. If renal failure is present, catheterization is needed to recover renal function. Catheterization may be followed by post-obstructive diuresis, due to the impaired concentrating ability of the renal tubules, and the accumulated salt, water, and toxins. Consequently, fluid replacement might be needed while renal function improves. Once stabilized, a period of intermittent catheterization may help improve function. Urodynamic testing enables determination of likelihood of response to surgical intervention. Where bladder outlet obstruction is the cause of chronic urinary retention, there is a good chance of responding to procedures such as transurethral resection of the prostate and laser prostatectomy. If detrusor underactivity is the cause, there may be minimal benefit from interventional procedures.

Evolved stars are excellent targets for precision radial velocity studies for three main reasons: i) they are cooler and rotate slower than their former main-sequence progenitor, which allows us to achieve a radial velocity precision at the m/s level for intermediate-mass stars, ii) we can study the dynamical evolution of planetary orbits due to the interaction with the expanding stellar envelope, and iii) they are significantly brighter than main sequence stars at a similar distance, allowing high SNRs to be acquired in an efficient manner. Since 2009, we have been conducting a radial velocity survey called EXPRESS (EXoPlanets aRound Evolved StarS) aimed at detecting giant planets and studying the correlation between their orbital parameters and the host star properties (mass, metallicity, age) and to investigate whether planets can survive the stellar evolution after the main-sequence. For this, we have obtained thousands of individual radial velocities epochs over the course of 13 years, for a sample of 166 bright giant stars, resulting in the detection of ~30 planetary systems, including multi-planet systems, brown dwarfs in the desert, a gas giant in a compact binary system, among others. Figure 1 shows the orbital period versus host star mass, for planets detected among the EXPRESS sample. In addition, we have detected more than 20 stellar binary companions, resolved with RVs, astrometry and high-contrast direct imaging observations. Moreover, we have recently combined our data with those obtained by the PPPS and the Lick Surveys. Among our results we highlight: 1) Giant planets are more frequent around metal-rich giant stars.2) The fraction of giant planets increases with the stellar mass, up to a maximum at ~1.7 Msun. Beyond ~3.0 Msun, no planets are found.3) A surprisingly high fraction of giant planets around low-luminosity RGB stars of 39.4 +/- 8.0%.4) An overall higher fraction of giant planet around RGB stars (f~14%) compared to post-RGB Horizontal-branch stars (f ~ 7%). In this talk I will describe our project, including observations, data reduction, planet detections and the results in collaboration with the PPPS and the Lick Survey.Finally, I will discuss our findings in the context of planetary formation and evolution, particularly after the main-sequence. Figure 1: Orbital period versus stellar mass for EXPRESS planets. The symbol size is proportional to the minimum planet mass (Mp sini). Planets in multiple systems are connected by a dashed line. The red and blue dots correspond to RGB and HB host stars, respectively.

Due to the dangerous nature of construction work, thousands of U.S. construction workers are injured and hundreds are killed on American worksites each year. In 2019, the U.S. construction industry accounted for one thousand and sixty-one (1,061) work-related fatalities, which amounts to a five percent (5%) increase from the previous year and the largest total number of annual worker deaths since 2007. This research study examined the Occupational Safety & Heath Administration (OSHA) construction fatality investigation findings during 2019 and organized these data points into four (4) major fatality-type categories and forty-nine (49) detailed types of events to identify potential trends for these fatal incidents.

The efficiency of carbon sequestration by the biological pump could decline in the coming decades because respiration tends to increase more with temperature than photosynthesis. Despite these differences in the short‐term temperature sensitivities of photosynthesis and respiration, it remains unknown whether the long‐term impacts of global warming on metabolic rates of phytoplankton can be modulated by evolutionary adaptation. We found that respiration was consistently more temperature dependent than photosynthesis across 18 diverse marine phytoplankton, resulting in universal declines in the rate of carbon fixation with short‐term increases in temperature. Long‐term experimental evolution under high temperature reversed the short‐term stimulation of metabolic rates, resulting in increased rates of carbon fixation. Our findings suggest that thermal adaptation may therefore have an ameliorating impact on the efficiency of phytoplankton as primary mediators of the biological carbon pump., The primary production of marine phytoplankton could decline with warming if rates of respiration increase more with rising temperature than photosynthesis. We found that respiration was consistently more temperature dependent than photosynthesis across 18 diverse marine phytoplankton taxa, resulting in universal declines in the rate of carbon fixation with short‐term increases in temperature. Whereas, by contrast, long‐term experimental evolution under high temperature acted to reverse the short‐term stimulation of metabolic rates, resulting in increased rates of carbon fixation.

OBJECTIVES To externally validate a nomogram recently proposed by Larcher et al. (BJU Int. 2017; 120: 490) and to develop a simplified model with comparable accuracy to guide on the need for staging chest computed tomography (CT) for patients with new renal masses. PATIENTS AND METHODS We analysed the data of 1082 consecutive patients with unilateral enhancing renal masses referred to urology multidisciplinary team meetings at two centres between 2011 and 2017. All patients underwent a staging chest CT at diagnosis. We fitted multivariable logistic regression models and tested the Larcher model performance using area under the receiver-operating curve (AUC), calibration and decision curve analysis. RESULTS Forty-two patients (3.9%) had a positive chest CT. The Larcher nomogram had an AUC of 83.8% (95% confidence interval [CI] 77.1-90.6), but was only moderately well calibrated (calibration-in-the-large = -0.61, slope = 0.82). Specifically, the nomogram overestimated the risk of positive chest CT, and the magnitude of miscalibration increased with increasing predicted risks. Using a stepwise backward approach, a new model was developed including tumour size, nodal stage and systemic symptoms. Compared with the Larcher model, the new model had a similar AUC (82.7% [95% CI 75.5-90.0]), but improved calibration and clinical net benefit. The predicted risk of positive chest CT was

The most frequent reason for missed playing time in competitive athletes is low back pain (LBP). Lumbar disk herniation (LDH), degenerative disk disease (DDD), and spondylolysis are the most common lumbar conditions that cause symptoms in this patient population. In elite athletes, most lumbar conditions can be managed nonsurgically with excellent outcomes. Surgical treatment is a viable option for athletes in whom nonsurgical treatment has failed. Many elite athletes are able to return to play even after surgical treatment, especially those who require lumbar diskectomy; high-level evidence has shown that they can return to play in 75–100% of cases. Lumbar fusion is not a contraindication for return to play in contact athletes. Return to play is assessed on an individual basis, but athletes should complete a rehabilitation program and demonstrate resolution of symptoms before medical clearance.

Summary Tissue-resident memory CD8+ T (Trm) cells share core residency gene programs with tumor-infiltrating lymphocytes (TILs). However, the transcriptional, metabolic, and epigenetic regulation of Trm cell and TIL development and function is largely undefined. Here, we found that the transcription factor Bhlhe40 was specifically required for Trm cell and TIL development and polyfunctionality. Local PD-1 signaling inhibited TIL Bhlhe40 expression, and Bhlhe40 was critical for TIL reinvigoration following anti-PD-L1 blockade. Mechanistically, Bhlhe40 sustained Trm cell and TIL mitochondrial fitness and a functional epigenetic state. Building on these findings, we identified an epigenetic and metabolic regimen that promoted Trm cell and TIL gene signatures associated with tissue residency and polyfunctionality. This regimen empowered the anti-tumor activity of CD8+ T cells and possessed therapeutic potential even at an advanced tumor stage in mouse models. Our results provide mechanistic insights into the local regulation of Trm cell and TIL function.

INTRODUCTION Despite the proven success of anterior cervical surgery (ACS) postoperative issues can arise, with retropharyngeal hematoma (RH) being one of the most feared. A recent USA Today article highlighted the failed management and subsequent death of patients developing RH after ACS. The article calls into question the safety of performing ACS in an outpatient setting. METHODS We tested the knowledge of 20 healthcare professionals (4 surgeons, 16 ancillary providers) regarding ways to minimize the post-ACS complications. We used a multiple-choice and fill-in-the-blank test. We then developed a No S.C.A.R.E. safety protocol and after teaching the same professionals, re-administered the examination to assess improvement. RESULTS We identified large gaps in knowledge between spine surgeons and other providers (92.3% versus 31.2%) on preintervention testing. Postintervention testing showed significant improvement in nonsurgeon scores (31.2% to 86.1%, P < 0.01). Improvement was also seen in provider confidence after completion of the education module. CONCLUSION Previous studies demonstrate that the incidence of RH necessitating evacuation after ACS is extremely small (

Opioid use disorders are chronic and relapse is common. Both negative affect and craving have been suggested antecedents of relapse and have been shown to demonstrate within- and between-person variability, as well as association with each other. The present study extends previous research by examining the covariation of negative affect and craving both within-day and at the person-level during 12 days of treatment among opioid-dependent patients. Ecological momentary assessment (EMA) data were collected from 73 participants starting between 10 and 14 days after admission to an inpatient treatment facility. These data were analyzed using multivariate multilevel models and time-varying effect models. Results demonstrated strong association between negative affect and craving. Within-day, negative affect and craving were most associated in the early afternoon. At the person-level, association between negative affect and craving declined during the first week of data collection. Following this initial decline in association, negative affect and craving increasingly covaried during days 8–12 of data collection. To our knowledge, this is the first study to report a lagged increase in the association between negative affect and craving among patients during inpatient treatment for opioid dependence. Implications for research and treatment providers are discussed.

The mammalian SPCA1 and SPCA2 ATPases localize in membranes of the secretory pathway and transport ions of Ca2+ and Mn2+. The role of tissue-specific SPCA2 isoform, highly expressed in lungs, mammary gland and gastrointestinal tract, is poorly understood. To elucidate the function of SPCA2, we studied human colon cancer HCT116 cells, grown under ambient and decreased O2 levels. We found that in contrast with other Ca2+-ATPase isoforms the expression of SPCA2 was up-regulated under hypoxia (3% O2), in both adherent (2D) and spheroid (3D) cultures. In spheroids, experiencing lowest O2 levels (30–50 μM, measured by phosphorescence lifetime imaging microscopy), we observed lower staining with reactive oxygen species (ROS)-specific fluorescent probe, which correlated with increased SPCA2. However, SPCA2 expression was up-regulated in cells exposed to reactive oxygen and nitrogen species donors, and when grown at higher density. We noticed that the culture exposed to hypoxia showed overall increase in S phase-positive cells and hypothesized that SPCA2 up-regulation under hypoxia can be linked to Mn2+-dependent cell cycle arrest. Consequently, we found that SPCA2-transfected cells display a higher number of cells entering S phase. Altogether, our results point at the important role of SPCA2 in regulation of cell cycle in cancer cells.

Numerous physical and psychiatric effects can be attributed to the cessation of psychotropic medications, similar to those agents with abuse potential. In addition, drug withdrawal can exacerbate underlying psychiatric conditions and alter disease course and long-term outcomes. This article discusses specific withdrawal syndromes associated with several classes of psychotropic medications to increase prescriber awareness when tapering and discontinuing psychotropic medications, thereby facilitating discussions with patients about the risks of withdrawal. [ Psychiatr Ann . 2016;46(8):466–472.]

Sensing of intracellular NAD(P)H is important for numerous applications ranging from diagnostic assays to drug discovery. Up to now, investigation of NAD(P)H-dependent pathways in live cells has been restrained by the lack of efficient tools. Toward this goal, we developed a small molecule indicator, which allows both colorimetric and fluorescent NAD(P)H detection in biological samples. The design is based on a cyanine dye scaffold and utilizes a novel two-acceptor “turn-on” mechanism. Consequently, this indicator features unprecedented sensitivity and rapid response toward NAD(P)H at low micromolar levels under physiological conditions. First, we demonstrated the value of this reagent in a diagnostic assay of glucose, through the enzyme-coupling reaction of NAD(P)+-dependent glucose dehydrogenase (GDH). Second, we showed the utility of our indicator for NAD(P)H imaging in live cells. We confirmed its ability to reflect different NAD(P)H levels using the human colon cancer cell line deficient on mitochond...

Marine phytoplankton are responsible for over 45% of annual global net primary production. Ocean warming is expected to drive massive reorganisation of phytoplankton communities, resulting in pole-ward range shifts and sharp declines in species diversity, particularly in the tropics. The impacts of warming on phytoplankton species depend critically on their physiological sensitivity to temperature change, characterised by thermal tolerance curves. Local extinctions arise when temperatures exceed species’ thermal tolerance limits. The mechanisms that determine the characteristics of thermal tolerance curves (e.g. optimal and maximal temperatures) and their variability among the broad physiological diversity of marine phytoplankton are however poorly understood. Here we show that differences in the temperature responses of photosynthesis and respiration establish physiological trade-offs that constrain the thermal tolerance of 18 species of marine phytoplankton, spanning cyanobacteria as well as the red and green super-families. Across all species we found that rates of respiration were more sensitive to increasing temperature and typically had higher optimal temperatures than photosynthesis. Consequently, the fraction of photosynthetic energy available for allocation to growth (carbon-use efficiency) declined exponentially with rising temperatures with a sensitivity that was invariant among the 18 species. Furthermore, the optimal temperature of growth was generally lower than that of photosynthesis and as a result, supra-optimal declines in growth rate were associated with temperature ranges where the carbon-use efficiency exhibited accelerated declines. These highly conserved patterns demonstrate that the limits of thermal tolerance in marine phytoplankton are underpinned by common metabolic constraints linked to the differential temperature responses of photosynthesis and respiration.Significance StatementThe impacts of warming on marine phytoplankton depend on their sensitivity to rising temperatures, yet there is currently limited understanding of the mechanisms that limit thermal tolerance among the diversity of marine phytoplankton. Using a comparative study on the dominant, ecologically important lineages of marine phytoplankton – Bacillariophyceae, Dinophyceae, Cyanophyceae, Prasinophyceae, Prymnesiophyceae – we show that rates of respiration are consistently more sensitive to increasing temperature than photosynthesis. Consequently, the fraction of photosynthetic energy available for growth declines with rising temperatures with a sensitivity that is invariant among species. Our results suggest that declines in phytoplankton performance at high temperatures are driven by universal metabolic constrains linked to rising respiratory costs eventually exceeding the supply of reduced carbon from photosynthesis.

Porous membrane scaffolds are widely used materials for three-dimensional cell cultures and tissue models. Additional functional modification of such scaffolds can significantly extend their use and operational performance. Here we describe hybrid microporous polystyrene-based scaffolds impregnated with a phosphorescent O2-sensitive dye PtTFPP, optimized for live cell fluorescence microscopy and imaging of O2 distribution in cultured cells. Modified scaffolds possess high brightness, convenient spectral characteristics (534 nm excitation, 650 nm emission), stable and robust response to pO2 in phosphorescence intensity and lifetime imaging modes (>twofold response over 21/0% O2), such as confocal PLIM. They are suitable for prolonged use under standard culturing conditions without affecting cell viability, and for multi-parametric imaging analysis of cultured cells and tissue samples. We tested the O2 scaffolds with cultured cancer cells (HCT116), multicellular aggregates (PC12) and rat brain slices and showed that they can inform on tissue oxygenation at different depths and cell densities, changes in respiration activity, viability and responses to drug treatment. Using this method multiplexed with staining of dead cells (CellTox Green) and active mitochondria (TMRM), we demonstrated that decreased O2 (20-24 μM) in scaffold corresponds to highest expression of tyrosine hydroxylase in PC12 cells. Such hypoxia is also beneficial for action of hypoxia-specific anti-cancer drug tirapazamine (TPZ). Thus, O2 scaffolds allow for better control of conditions in 3D tissue cultures, and are useful for a broad range of biomaterials and physiological studies.

A profound understanding of how to tailor surface topographies of electrospun fibers is of great importance for surface sensitive applications including optical sensing, catalysis, drug delivery and tissue engineering. Hereby, a novel approach to comprehend the driving forces for fiber surface topography formation is introduced through inclusion of the dynamic solvent-polymer interaction during fiber formation. Thus, the interplay between polymer solubility as well as computed fiber jet surface temperature changes in function of time during solvent evaporation and the resultant phase separation behavior are studied. The correlation of experimental and theoretical results shows that the temperature difference between the polymer solution jet surface temperature and the dew point of the controlled electrospinning environment are the main influencing factors with respect to water condensation and thus phase separation leading to the final fiber surface topography. As polymer matrices with enhanced surface area are particularly appealing for sensing applications, we further functionalized our nanoporous fibrous membranes with a phosphorescent oxygen-sensitive dye. The hybrid membranes possess high brightness, stability in aqueous medium, linear response to oxygen and hence represent a promising scaffold for cell growth, contactless monitoring of oxygen and live fluorescence imaging in 3-D cell models.

Lars Valerian Ahlfors was arguably the preeminent complex function theorist of the twentieth century. With a career spanning more than sixty years, Ahlfors made decisive contributions to areas ranging from meromorphic curves to value distribution theory, Riemann surfaces, conformal geometry, extremal length, quasiconformal mappings, and Kleinian groups ([7] serves as a map of Ahlfors’s contributions to the subject). Ahlfors was both role model and mentor to his graduate students and to the many mathematicians around the world who learned from his example. He is remembered warmly, both as a mathematician and as a man.

Live cells function within narrow limits of physiological temperature (T) and O2 and metabolite concentrations. We have designed a cell-permeable T-sensitive fluorescence lifetime-based nanoprobe based on lipophilic sulforhodamine, which stains 2D and 3D cell models, shows cytoplasmic localization, and has a robust response to T (∼0.037 ns/K). Subsequently, we evaluated the probe and fluorescence lifetime imaging microscopy (FLIM) technique for combined imaging of T and O2 gradients in metabolically active cells. We found that in adherent 2D culture of HCT116 cells intracellular T and O2 are close to ambient values. However, in 3D spheroid structures having size >200 μm, T and O2 gradients become pronounced. These microgradients can be enhanced by treatment with mitochondrial uncouplers or dissipated by drug-induced disaggregation of the spheroids. Thus, we demonstrate the existence of local microgradients of T in 3D cell models and utility of combined imaging of O2 and T.

Background Fenestrated endovascular aneurysm repair (FEVAR) allows endovascular treatment of thoracoabdominal and juxtarenal aneurysms previously outside the indications of use for standard devices. However, because of considerable device costs and increased procedure time, FEVAR is thought to result in financial losses for medical centers and physicians. We hypothesized that surgeon leadership in the coding, billing, and contractual negotiations for FEVAR procedures will increase medical center contribution margin (CM) and physician reimbursement. Methods At the UMass Memorial Center for Complex Aortic Disease, a vascular surgeon with experience in medical finances is supported to manage the billing and coding of FEVAR procedures for medical center and physician reimbursement. A comprehensive financial analysis was performed for all FEVAR procedures (2011-2015), independent of insurance status, patient presentation, or type of device used. Medical center CM (actual reimbursement minus direct costs) was determined for each index FEVAR procedure and for all related subsequent procedures, inpatient or outpatient, 3 months before and 1 year subsequent to the index FEVAR procedure. Medical center CM for outpatient clinic visits, radiology examinations, vascular laboratory studies, and cardiology and pulmonary evaluations related to FEVAR were also determined. Surgeon reimbursement for index FEVAR procedure, related adjunct procedures, and assistant surgeon reimbursement were also calculated. All financial analyses were performed and adjudicated by the UMass Department of Finance. Results The index hospitalization for 63 FEVAR procedures incurred $2,776,726 of direct costs and generated $3,027,887 in reimbursement, resulting in a positive CM of $251,160. Subsequent related hospital procedures (n = 26) generated a CM of $144,473. Outpatient clinic visits, radiologic examinations, and vascular laboratory studies generated an additional CM of $96,888. Direct cost analysis revealed that grafts accounted for the largest proportion of costs (55%), followed by supplies (12%), bed (12%), and operating room (10%). Total medical center CM for all FEVAR services was $492,521. Average surgeon reimbursements per FEVAR from 2011 to 2015 increased from $1601 to $2480 while the surgeon payment denial rate declined from 50% to 0%. Surgeon-led negotiations with the Centers for Medicare & Medicaid Services during 2015 resulted in a 27% increase in physician reimbursement for the remainder of 2015 ($2480 vs $3068/case) and a 91% increase in reimbursement from 2011 ($1601 vs $3068). Assistant surgeon reimbursement also increased ($266 vs $764). Concomitant FEVAR-related procedures generated an additional $27,347 in surgeon reimbursement. Conclusions Physician leadership in the coding, billing, and contractual negotiations for FEVAR results in a positive medical center CM and increased physician reimbursement.

Context: Burnout is a known problem among physicians in training but has not been extensively studied in osteopathic residents. Objective: To evaluate the relationship of burnout to sex, age, marital status, and residency program type (surgical vs nonsurgical) and length (3, 4, 5, or 6 years) across 12 residency programs at Doctors Hospital in Columbus, Ohio. Methods: An anonymous, voluntary questionnaire was given to residents in their 10th month of residency. Thirty questions were provided, including general background questions and the Maslach Burnout Inventory Human Services Survey to assess burnout. Results: A total of 131 of 180 residents (72.8%) returned the questionnaire, and 129 provided complete responses in most categories. Of the 129 respondents, 89 (69.0%) reported emotional exhaustion and 96 (74.4%) reported depersonalization at a moderate or high level. In addition, 70 respondents (54.4%) experienced a moderate or high level of burnout in relation to personal accomplishment. No statistically significant association was found between the burnout factors (emotional exhaustion, depersonalization, and personal accomplishment) and sex, age, marital status, and residency program type and length. Conclusions: The majority of the osteopathic residents surveyed reported experiencing burnout. More data on burnout among osteopathic residents and associated factors are needed.

School of Biochemistry and Cell Biology, University College Cork, Cork, IrelandThe mammalian SPCA1 and SPCA2 ATPases located in membranes of the secretory pathway (Golgi complex, endosomes, secretory vesicles) transport Ca2+ and Mn2+ ions with different affinities. The role of tissue-specific SPCA2 isoform in the cytosolic Mn2+ homeostasis is poorly understood. To elucidate the function of SPCA2, we studied HCT116 cells, grown with in situ control by O2 imaging. In contrast to other Ca2+-ATPase isoforms the expression of SPCA2 was upregulated under hypoxia (3% O2), in both adherent (2D) and spheroid (3D) cultures. In the spheroid cores, experiencing lowest O2 levels (30-50 μM, measured with O2-sensitive phosphorescent nanoparticles) we also saw upregulation of SPCA2. Such increase of SPCA2 was in line with observed lower staining with reactive oxygen species (ROS)-specfic fluorescent probe in spheroids. Using fluorescent sensor GPP130-GFP we confirmed that SPCA2 decreases cytosolic Mn2+. We hypothesized that SPCA2 upregulation under hypoxia may help preventing Mn2+-dependent cell cycle arrest. Using BrdU-labeling and overexpression of SPCA2 and SERCA2b (control) with and without elevated Mn2+, we found that SPCA2-transfected cells display higher amount of cells entering S-phase. This correlated with the overall increase of S-phase positive cells exposed to hypoxia in non-transfected cells. In summary, our results point at the important role in Mn2+ regulation of cell cycle in cancer cells, with possible links to tumour progression and ROS production.Supported by Science Foundation Ireland grants 13/SIRG/2144 and 12/RC/2276.

Compared with recent advances in treatment of serious cardiovascular diseases, such as myocardial infarction and stroke, the treatment and outcome of acute pulmonary embolism (PE) have remained relatively unchanged over the last few decades. This has prompted several experts to call for the formation of multidisciplinary PE response teams with a more proactive approach to the treatment of PE. In the current document, we discuss the formation of such teams and describe the available treatment options beyond anticoagulation, with a focus on the interventional approach. Acknowledging the paucity of data to support widespread adoption of such techniques, we call for the collection of outcomes data in multicenter registries and support for randomized trials to evaluate interventional treatments in patients with high-risk PE.

Multi-modal probes allow for flexible choice of imaging equipment when performing quenched-phosphorescence O2 measurements: one- or two-photon, PLIM or intensity-based ratiometric read-outs. Spectral and temporal (e.g. FLIMPLIM) discrimination can be used to image O 2 together with pH, Ca 2+ , mitochondrial membrane potential, cell death markers or cell/organelle specific markers. However, the main challenge of existing nanoparticle probes is their limited diffusion across thick (> 20-50 μm) 3D cell models such as tumor spheroids. Here, we present new class of polymeric nanoparticle probes having tunable size, charge, cell-penetrating ability, and reporter dyes. Being spectrally similar to the recently described MM2, PA2 and other O 2 probes, they are 5-10 times brighter, demonstrate improved ratiometric response and their surface chemistry can be easily modified. With cultures of 2D and 3D cell models (fibroblasts, PC12 aggregates, HCT116 human colon cancer spheroids) we found cell-specific staining by these probes. However, the efficient staining of model of interest can be tuned by changing number of positive and negative surface groups at nanoparticle, to allow most efficient loading. We also demonstrate how real-time monitoring of oxygenation can be used to select optimal spheroid production with low variability in size and high cell viability.

We describe a technique of imaging tissue oxygen using phosphorescence based probes and TCSPC-PLIM method. Included is a brief overview of the significance of biological oxygen imaging, the theory behind the phosphorescence quenching method, the main O2 sensitive probes (mostly intracellular, cell-permeable) and imaging modalities currently available, highlighting their merits and limitations. In the practical part, the live cell microscopy imaging and TCSPC-PLIM hardware and software are described, along with the detailed experimental procedures of preparation of tissue samples, their staining with intracellular O2 probes, acquisition of PLIM images and their processing to produce 2D and 3D maps of O2 concentration. Several examples demonstrate practical use of O2 imaging with different models of mammalian tissue, including cell mono-layers (2D model), multi-cellular spheroids, scaffold structures and tissue slices (3D models). Physiological experiments and multi-parametric analysis of these samples with some other fluorescent imaging probes are also presented.

Porous three-dimensional membrane scaffolds are useful biomaterials that mimic physiological interactions between cells and the environment in complex multi-cellular systems and tissue models. In contrast to simple 2D cultures, they provide increased cell-cell interaction as well as nutrient, drug and O2 gradients. In order to further enhance functionality, we impregnated a highly porous scaffold made from polystyrene with a phosphorescent indicator dye Pt(II)-meso-tetra(pentafluorophenyl)porphine (PtTFPP) making it sensitive to local O2 concentrations and compatible with phosphorescence lifetime imaging microscopy (PLIM). These hybrid scaffold materials retain the dye during prolonged culturing (at least 7 days) without effects on cell viability, provide high intensity signals for high-resolution PLIM microscopy thus allowing reliable intensity and lifetime-based O2 calibrations. We evaluated the O2 sensitive scaffolds with cancer HCT116 cells and multi-cellular aggregates (PC12 cells) and found that sample oxygenation depends on cell number, cell respiration activity and viability. We also monitored the effect of O2 gradient in scaffold on cell differentiation and reaction to chemotherapeutic drugs, with particular emphasis on those effective during hypoxia. Altogether, hybrid O2-sensitive polystyrene scaffold allows us to analyse oxygenation under mild conditions (534 nm excitation, 650 nm emission), with multiplexed detection of other fluorescent labels and probes. They are useful for a broad range of physiological applications of biomaterials.Supported by Science Foundation Ireland (SFI) grants 13/SIRG/2144 and 12/RC/2276.

We study the existence of solutions of the generalized Beltrami equation fz = μ(z)fz, ||μ(z)||∞ = 1, in a plane domain Δ, under general conditions that include previously known results.