NKG2C+ NK Cells for Immunotherapy of Glioblastoma Multiforme.

In glioblastoma, non-classical human leucocyte antigen E (HLA-E) and HLA-G are frequently overexpressed. HLA-E loaded with peptides derived from HLA class I and from HLA-G contributes to inhibition of natural killer (NK) cells with expression of the inhibitory receptor CD94/NKG2A. We investigated whether NK cells expressing the activating CD94/NKG2C receptor counterpart were able to exert anti-glioma effects. NKG2C+ subsets were preferentially expanded by a feeder cell line engineered to express an artificial disulfide-stabilized trimeric HLA-E ligand (HLA-E*spG). NK cells expanded by a feeder cell line, which facilitates outgrowth of conventional NKG2A+, and fresh NK cells, were included for comparison. Expansion via the HLA-E*spG feeder cells selectively increased the fraction of NKG2C+ NK cells, which displayed a higher frequency of KIR2DL2/L3/S2 and CD16 when compared to expanded NKG2A+ NK cells. NKG2C+ NK cells exhibited increased cytotoxicity against K562 and KIR:HLA-matched and -mismatched primary glioblastoma multiforme (GBM) cells when compared to NKG2A+ NK cells and corresponding fresh NK cells. Cytotoxic responses of NKG2C+ NK cells were even more pronounced when utilizing target cells engineered with HLA-E*spG. These findings support the notion that NKG2C+ NK cells have potential therapeutic value for treating gliomas.

The impact of long-term non-pharmaceutical interventions on COVID-19 epidemic dynamics and control: the value and limitations of early models.

Mathematical models of epidemics are important tools for predicting epidemic dynamics and evaluating interventions. Yet, because early models are built on limited information, it is unclear how long they will accurately capture epidemic dynamics. Using a stochastic SEIR model of COVID-19 fitted to reported deaths, we estimated transmission parameters at different time points during the first wave of the epidemic (March-June, 2020) in Santa Clara County, California. Although our estimated basic reproduction number ([Formula: see text]) remained stable from early April to late June (with an overall median of 3.76), our estimated effective reproduction number ([Formula: see text]) varied from 0.18 to 1.02 in April before stabilizing at 0.64 on 27 May. Between 22 April and 27 May, our model accurately predicted dynamics through June; however, the model did not predict rising summer cases after shelter-in-place orders were relaxed in June, which, in early July, was reflected in cases but not yet in deaths. While models are critical for informing intervention policy early in an epidemic, their performance will be limited as epidemic dynamics evolve. This paper is one of the first to evaluate the accuracy of an early epidemiological compartment model over time to understand the value and limitations of models during unfolding epidemics.

Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic ß-Cells of the Line INS-1.

In pancreatic ß-cells of the line INS-1, glucose uptake and metabolism induce the openings of Ca2+-permeable TRPM3 channels that contribute to the elevation of the intracellular Ca2+ concentration and the fusion of insulin granules with the plasma membrane. Conversely, glucose-induced Ca2+ signals and insulin release are reduced by the activity of the serine/threonine kinase CK2. Therefore, we hypothesized that TRPM3 channels might be regulated by CK2 phosphorylation. We used recombinant TRPM3α2 proteins, native TRPM3 proteins from INS-1 ß-cells, and TRPM3-derived oligopeptides to analyze and localize CK2-dependent phosphorylation of TRPM3 channels. The functional consequences of CK2 phosphorylation upon TRPM3-mediated Ca2+ entry were investigated in Fura-2 Ca2+-imaging experiments. Recombinant TRPM3α2 channels expressed in HEK293 cells displayed enhanced Ca2+ entry in the presence of the CK2 inhibitor CX-4945 and their activity was strongly reduced after CK2 overexpression. TRPM3α2 channels were phosphorylated by CK2 in vitro at serine residue 1172. Accordingly, a TRPM3α2 S1172A mutant displayed enhanced Ca2+ entry. The TRPM3-mediated Ca2+ entry in INS-1 ß-cells was also strongly increased in the presence of CX-4945 and reduced after overexpression of CK2. Our study shows that CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 ß-cells.

Is Endovascular Therapy for Stroke Cost-Effective Globally? A Systematic Review of the Literature.

OBJECTIVES: Cost-effectiveness of endovascular therapy (EVT) is a key consideration for broad use of this approach for emergent large vessel occlusion stroke. We evaluated the evidence on cost-effectiveness of EVT in comparison with best medical management from a global perspective. MATERIALS AND METHODS: This systematic review of studies published between January 2010 and May 2020 evaluated the cost effectiveness of EVT for patients with large vessel occlusion acute ischemic stroke. The gain in quality adjusted life year (QALY) and incremental cost-effectiveness ratio (ICER), expressed as cost per QALY resulting from EVT, were recorded. The study setting (country, economic perspective), decision model, and data sources used in economic models of EVT cost-effectiveness were recorded. RESULTS: Twenty-five original studies from 12 different countries were included in our review. Five of these studies were reported from a societal perspective; 18 were reported from a healthcare system perspective. Two studies used real-world data. The time horizon varied from 1 year to a lifetime; however, 18 studies reported a time horizon of >10 years. Twenty studies reported using outcome data from randomized, controlled clinical trials for their models. Nineteen studies reported using a Markov model. Incremental QALYs ranged from 0.09-3.5. All studies but 1 reported that EVT was cost-effective. CONCLUSIONS: Evidence from different countries and economic perspectives suggests that EVT for stroke treatment is cost-effective. Most cost-effectiveness studies are based on outcome data from randomized clinical trials. However, there is a need to study the cost-effectiveness of EVT based solely on real-world outcome data.

CT as a first-line modality in elderly patients with stable blunt chest trauma.

PURPOSE: Blunt thoracic injuries are common among elderly patients and may be a common cause of morbidity and death from blunt trauma injuries. We aimed to examine the impact of chest CT on the diagnosis and change of management plan in elderly patients with stable blunt chest trauma. We hypothesized that chest CT may play an important role in providing optimal management to this subgroup of trauma patients. METHODS: A retrospective analysis was performed on all the admitted adult blunt trauma patients between January 2014 and December 2018. Stable blunt chest trauma patients with abbreviated injury severity (AIS) < 3 for extra-thoracic injuries confirmed with chest X-ray (CXR) and chest CT on admission or during hospitalization were included in the study. The AIS is an international scale for grading the severity of anatomic injury following blunt trauma. Primary outcome variables were occult injuries, change in management, need for surgical procedures, missed injuries, readmission rate, intensive care unit (ICU) and length of hospital stay. RESULTS: There are 473 patients with blunt chest trauma included in the study. The study patients were divided into two groups according to the age range: group 1: 289 patients were included and aged 18-64 years; group 2: 184 patients were included and aged 65-99 years . Elderly patients in group 2 more often required ICU admission (11.4% vs. 5.2%), had a longer length of ICU stay (days) (median 11 vs. 6, p = 0.01), and the length of hospital stay (days) (median 14 vs. 6, p = 0.04). Injuries identified on chest CT has led to a change of management in 4.4% of young patients in group 1 and in 10.9% of elderly patients in group 2 with initially normal CXR. Chest CT resulted in a change of management in 12.8% of young patients in group 1 and in 25.7% of elderly patients in group 2 with initially abnormal CXR. CONCLUSION: Chest CT led to a change of management in a substantial proportion of elderly patients. Therefore, we recommend chest CT as a first-line imaging modality in patients aged over 65 years with isolated blunt chest trauma.

Control of Insulin Release by Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels.

BACKGROUND/AIMS: The release of insulin in response to increased levels of glucose in the blood strongly depends on Ca2+ influx into pancreatic beta cells by the opening of voltage-gated Ca2+ channels. Transient Receptor Potential Melastatin 3 proteins build Ca2+ permeable, non-selective cation channels serving as pain sensors of noxious heat in the peripheral nervous system. TRPM3 channels are also strongly expressed in pancreatic beta cells that respond to the TRPM3 agonist pregnenolone sulfate with Ca2+ influx and increased insulin release. Therefore, we hypothesized that in beta cells TRPM3 channels may contribute to pregnenolone sulfate- as well as to glucose-induced insulin release. METHODS: We used INS-1 cells as a beta cell model in which we analysed the occurrence of TRPM3 isoformes by immunoprecipitation and western blotting and by cloning of RT-PCR amplified cDNA fragments. We applied pharmacological as well as CRISPR/Cas9-based strategies to analyse the interplay of TRPM3 and voltage-gated Ca2+ channels in imaging experiments (FMP, Fura-2) and electrophysiological recordings. In immunoassays, we examined the contribution of TRPM3 channels to pregnenolone sulfate- and glucose-induced insulin release. To confirm our findings, we generated beta cell-specific Trpm3-deficient mice and compared their glucose clearance with the wild type in glucose tolerance tests. RESULTS: TRPM3 channels triggered the activity of voltage-gated Ca2+ channels and both channels together contributed to insulin release after TRPM3 activation. Trpm3-deficient INS-1 cells lacked pregnenolone sulfate-induced Ca2+ signals just like the pregnenolone sulfate-induced insulin release. Both, glucose-induced Ca2+ signals and the glucose-induced insulin release were strongly reduced. Accordingly, Trpm3-deficient mice displayed an impaired decrease of the blood sugar concentration after intraperitoneal or oral administration of glucose. CONCLUSION: The present study suggests an important role for TRPM3 channels in the control of glucose-dependent insulin release.

Coexisting attractors in the context of cross-scale population dynamics: measles in London as a case study.

Patterns of measles infection in large urban populations have long been considered the paradigm of synchronized nonlinear dynamics. Indeed, recurrent epidemics appear approximately mass-action despite underlying heterogeneity. However, using a subset of rich, newly digitized mortality data (1897-1906), we challenge that proposition. We find that sub-regions of London exhibited a mixture of simultaneous annual and biennial dynamics, while the aggregate city-level dynamics appears firmly annual. Using a simple stochastic epidemic model and maximum-likelihood inference methods, we show that we can capture this observed variation in periodicity. We identify agreement between theory and data, indicating that both changes in periodicity and epidemic coupling between regions can follow relatively simple rules; in particular we find local variation in seasonality drives periodicity. Our analysis underlines that multiple attractors can coexist in a strongly mixed population and follow theoretical predictions.

Long-term dynamics of measles in London: Titrating the impact of wars, the 1918 pandemic, and vaccination.

A key question in ecology is the relative impact of internal nonlinear dynamics and external perturbations on the long-term trajectories of natural systems. Measles has been analyzed extensively as a paradigm for consumer-resource dynamics due to the oscillatory nature of the host-pathogen life cycle, the abundance of rich data to test theory, and public health relevance. The dynamics of measles in London, in particular, has acted as a prototypical test bed for such analysis using incidence data from the pre-vaccination era (1944-1967). However, during this timeframe there were few external large-scale perturbations, limiting an assessment of the relative impact of internal and extra demographic perturbations to the host population. Here, we extended the previous London analyses to include nearly a century of data that also contains four major demographic changes: the First and Second World Wars, the 1918 influenza pandemic, and the start of a measles mass vaccination program. By combining mortality and incidence data using particle filtering methods, we show that a simple stochastic epidemic model, with minimal historical specifications, can capture the nearly 100 years of dynamics including changes caused by each of the major perturbations. We show that the majority of dynamic changes are explainable by the internal nonlinear dynamics of the system, tuned by demographic changes. In addition, the 1918 influenza pandemic and World War II acted as extra perturbations to this basic epidemic oscillator. Our analysis underlines that long-term ecological and epidemiological dynamics can follow very simple rules, even in a non-stationary population subject to significant perturbations and major secular changes.

Hypotension on admission in patients with isolated traumatic brain injury: contemporary examination of the incidence and outcomes using a national registry.

PURPOSE: This study was primarily aimed at establishing the incidence and impact of hypotension in patients with blunt traumatic brain injury based on National Trauma Registry Database. METHODS: A retrospective cohort study using the National Trauma Registry was conducted. Patients with TBI following blunt mechanisms of injury were examined, comparing those with and without hypotension (SBP < 90 mm Hg) on arrival. RESULTS: During the period from 1998 to 2017, the registry included 437.354 blunt trauma patients. Of them, 7818 patients were hemodynamically unstable (SBP < 90 mm Hg) on admission. 513 met the inclusion criteria. Significant percentages of patients with high grade injures (ISS≥16) and low admission's GCS 3-12 (46% vs 16.4%), were found in the group of hypotensive TBI patients (p<0.0001). 323 (62.9%) patients had head AIS score 3-4 and only 190 (37.1%) patients AIS 5-6 (p<0.0001). Mortality in the hypotensive TBI group was 32.3%, whereas 6.1% patients died in the TBI hemodynamically stable group (p<0.0001). CONCLUSION: TBI patients presenting with hypotension represent an appreciable portion blunt trauma patients. Prompt brain CT, expedient efforts at optimal resuscitation and possibly early inotropic and vasopressors agents use may have an impact on final outcome in these patients.

Protein Kinase CK2 Controls CaV2.1-Dependent Calcium Currents and Insulin Release in Pancreatic ß-Cells.

The regulation of insulin biosynthesis and secretion in pancreatic ß-cells is essential for glucose homeostasis in humans. Previous findings point to the highly conserved, ubiquitously expressed serine/threonine kinase CK2 as having a negative regulatory impact on this regulation. In the cell culture model of rat pancreatic ß-cells INS-1, insulin secretion is enhanced after CK2 inhibition. This enhancement is preceded by a rise in the cytosolic Ca2+ concentration. Here, we identified the serine residues S2362 and S2364 of the voltage-dependent calcium channel CaV2.1 as targets of CK2 phosphorylation. Furthermore, co-immunoprecipitation experiments revealed that CaV2.1 binds to CK2 in vitro and in vivo. CaV2.1 knockdown experiments showed that the increase in the intracellular Ca2+ concentration, followed by an enhanced insulin secretion upon CK2 inhibition, is due to a Ca2+ influx through CaV2.1 channels. In summary, our results point to a modulating role of CK2 in the CaV2.1-mediated exocytosis of insulin.

Is computed tomography cystography indicated in children with pelvic fractures?

PURPOSE: Pelvic fracture evaluation with abdominopelvic computed tomography (CT) and formal CT cystography for rule out of urine bladder injury have been commonly employed in pediatric trauma patients. The additional delayed imaging required to obtain optimal CT cystography is, however, associated with increased doses of ionizing radiation to pelvic organs and represent a significant risk in the pediatric population for future carcinogenic risk. We hypothesized that avoidance of routine CT cystography among pediatric pelvic fracture victims would not result in an appreciable rate of missed bladder injuries and would aid in mitigating the radiation exposure risk associated with these additional images. METHODS: A retrospective cohort study involving blunt trauma pelvic fractures among pediatric trauma patients (age<14) between the years 1997 and 2016 was conducted utilizing the Israeli National Trauma Registry. Statistical analysis was performed using SAS statistical software version 9.4 via the tests of Chi-square test and two-sided Fisher's exact test. A p value of less than 0.05 was considered statistically significant. RESULTS: A total of 1072 children were identified from the registry for inclusion. Mean age of patients was 7.7 years (range 0-14) and 713 (66.5%) were male. Overall mortality in this population was 4.1% (44/1072). Only 2.1% (23) of pediatric patients with pelvic fractures had bladder injury identified, with just 9 children having intraperitoneal bladder rupture (0.8% of all the patients). CONCLUSION: The vast majority of blunt pediatric trauma victims with pelvic fractures do not have urine bladder injuries. Based on our study results we do not recommend the routine utilization of CT cystography in this unique population.

Estimating enhanced prevaccination measles transmission hotspots in the context of cross-scale dynamics.

A key question in clarifying human-environment interactions is how dynamic complexity develops across integrative scales from molecular to population and global levels. Apart from its public health importance, measles is an excellent test bed for such an analysis. Simple mechanistic models have successfully illuminated measles dynamics at the city and country levels, revealing seasonal forcing of transmission as a major driver of long-term epidemic behavior. Seasonal forcing ties closely to patterns of school aggregation at the individual and community levels, but there are few explicit estimates of school transmission due to the relative lack of epidemic data at this scale. Here, we use data from a 1904 measles outbreak in schools in Woolwich, London, coupled with a stochastic Susceptible-Infected-Recovered model to analyze measles incidence data. Our results indicate that transmission within schools and age classes is higher than previous population-level serological data would suggest. This analysis sheds quantitative light on the role of school-aged children in measles cross-scale dynamics, as we illustrate with references to the contemporary vaccination landscape.

The emergency department length of stay: Is the time running out?

PURPOSE: To examine the relationships between emergency department length of stay (EDLOS) with hospital length of stay (HLOS) and clinical outcome in hemodynamically stable trauma patients. METHODS: Prospective data collected for 2 years from consecutive trauma patients admitted to the trauma resuscitation bay. Only stable blunt trauma patients with appropriate trauma triage criteria requiring trauma team activation were included in the study. EDLOS was determined short if patient spent less than 2 h in the emergency department (ER) and long for more than 2 h. RESULTS: A total of 248 patients were enrolled in the study. The mean total EDLOS was 125 min (range 78-180). Injury severity score (ISS) were significantly higher in the long EDLOS group (17 ± 13 versus 11 ± 9, p < 0.001). However, when leveled according to ISS, there were no differences in mean in diagnostic workup, admission rate to intensive care unit (ICU) or HLOS between the short and long EDLOS groups. CONCLUSION: EDLOS is not a significant parameter for HLOS in stable trauma patients.

Top-down proteomics for the analysis of proteolytic events - Methods, applications and perspectives.

Mass spectrometry based proteomics is an indispensable tool for almost all research areas relevant for the understanding of proteolytic processing, ranging from the identification of substrates, products and cleavage sites up to the analysis of structural features influencing protease activity. The majority of methods for these studies are based on bottom-up proteomics performing analysis at peptide level. As this approach is characterized by a number of pitfalls, e.g. loss of molecular information, there is an ongoing effort to establish top-down proteomics, performing separation and MS analysis both at intact protein level. We briefly introduce major approaches of bottom-up proteomics used in the field of protease research and highlight the shortcomings of these methods. We then discuss the present state-of-the-art of top-down proteomics. Together with the discussion of known challenges we show the potential of this approach and present a number of successful applications of top-down proteomics in protease research. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Analysis of incidence of traumatic brain injury in blunt trauma patients with Glasgow Coma Scale of 12 or less.

PURPOSE: Early diagnosis of traumatic brain injury (TBI) is important for improving survival and neurologic outcome in trauma victims. The purpose of this study was to assess whether Glasgow Coma Scale (GCS) of 12 or less can predict the presence of TBI and the severity of associated injuries in blunt trauma patients. METHODS: A retrospective cohort study including 303,435 blunt trauma patients who were transferred from the scene to hospital from 1998 to 2013. The data was obtained from the records of the National Trauma Registry maintained by Israel's National Center for Trauma and Emergency Medicine Research, in the Gertner Institute for Epidemiology and Health Policy Research. All blunt trauma patients with GCS 12 or less were included in this study. Data collected in the registry include age, gender, mechanism of injury, GCS, initial blood pressure, presence of TBI and incidence of associated injuries. Patients younger than 14 years old and trauma victims with GCS 13-15 were excluded from the study. Statistical analysis was performed by using Statistical Analysis Software Version 9.2. Statistical tests performed included Chi-square tests. A p-value less than 0.05 was considered statistically significant. RESULTS: There were 303,435 blunt trauma patients, 8731 (2.9%) of them with GCS of 3-12 that including 6351 (72%) patients with GCS of 3-8 and 2380 (28%) patient with GCS of 9-12. In these 8731 patients with GCS of 3-12, 5372 (61.5%) patients had TBI. There were total 1404 unstable patients in all the blunt trauma patients with GCS of 3-12, 1256 (89%) patients with GCS 3-8, 148 (11%) patients with GCS 9-12. In the 5095 stable blunt trauma patients with GCS 3-8, 32.4% of them had no TBI. The rate in the 2232 stable blunt trauma patients with GCS 9-12 was 50.1%. In the unstable patients with GCS 3-8, 60.5% of them had TBI, and in subgroup of patients with GCS 9-12, only 37.2% suffered from TBI. CONCLUSION: The utility of a GCS 12 and less is limited in prediction of brain injury in multiple trauma patients. Significant proportion of trauma victims with low GCS had no TBI and their impaired neurological status is related to severe extra-cranial injuries. The findings of this study showed that using of GCS in initial triage and decision making processes in blunt trauma patients needs to be re-evaluated.

Rivaroxaban-Induced Gastrointestinal Bleeding Presenting as Acute Colon Obstruction.

BACKGROUND: Rivaroxaban is a member of the novel target-specific oral anticoagulants (TSOACs) family of drugs recently approved for the prevention and treatment of venous thromboembolism events. A major drawback of the drug is its potential for causing severe hemorrhagic events, which may be difficult to treat in an emergency setting due to lack of effective antidote. Here, we describe a case of acute gastrointestinal (GI) hemorrhage leading to complete colon obstruction in a patient treated with rivaroxaban. Summary and Key Messages: The case presented here demonstrates a chain of events originating from an unprovoked intramural bleeding in a patient using rivaroxaban, leading to an organized giant clot formation, and to complete colon obstruction. In the available literature, the specific site of the GI bleeding has not been discussed. A further study is recommended and re-examination of bleeding events and exploration of new cases due to the use of TSOACs can help predict the course and the outcomes of such complications.

Early pneumonia and timing of antibiotic therapy in patients after nontraumatic out-of-hospital cardiac arrest.

BACKGROUND: While early pneumonia is common in patients after out-of-hospital cardiac arrest (OHCA), little is known about the impact of pneumonia and the optimal timing of antibiotic therapy after OHCA. METHODS: We conducted a 5-year retrospective cohort study, including patients who suffered from OHCA and were treated with therapeutic hypothermia. ICU treatment was strictly standardized with defined treatment goals and procedures. Medical records, chest radiographic images and microbiological findings were reviewed. RESULTS: Within the study period, 442 patients were admitted to our medical ICU after successfully resuscitated cardiac arrest. Of those, 174 patients fulfilled all inclusion and no exclusion criteria and were included into final analysis. Pneumonia within the first week could be confirmed in 39 patients (22.4%) and was confirmed or probable in 100 patients (57.5%), without a difference between survivors and non-survivors (37.8% vs. 23.1% confirmed pneumonia, p = 0.125). In patients with confirmed pneumonia a tracheotomy was performed more frequently (28.2 vs. 12.6%, p = 0.026) compared to patients without confirmed pneumonia. Importantly, patients with confirmed pneumonia had a longer ICU- (14.0 [8.5-20.0] vs. 8.0 [5.0-14.0] days, p < 0.001) and hospital stay (23.0 [11.5-29.0] vs. 15.0 [6.5-25.0] days, p = 0.016). A positive end expiratory pressure (PEEP) > =10.5 mbar on day 1 of the hospital stay was identified as early predictor of confirmed pneumonia (odds ratio 2.898, p = 0.006). No other reliable predictor could be identified. Median time to antibiotic therapy was 8.7 [5.4-22.8] hours, without a difference between patients with or without confirmed pneumonia (p = 0.381) and without a difference between survivors and non-survivors (p = 0.264). Patients receiving antibiotics within 12 hours after admission had a shorter ICU- (8.0 [4.0-14.0] vs. 10.5 [6.0-16.0] vs. 13.5 [8.0-20.0] days, p = 0.004) and hospital-stay (14.0 [6.0-25.0] vs. 16.5 [11.0-27.0] vs. 21.0 [17.0-28.0] days, p = 0.007) compared to patients receiving antibiotics after 12 to 36 or more than 36 hours, respectively. CONCLUSIONS: Early pneumonia may extend length of ICU- and hospital-stay after OHCA and its occurrence is difficult to predict. A delayed initiation of antibiotic therapy in OHCA patients may increase the duration of the ICU- and hospital-stay.

Identification and relative quantification of phosphopeptides by a combination of multi-protease digestion and isobaric labeling.

RATIONALE: The identification and the determination of the extent of protein phosphorylation are major prerequisites for the comparative analysis of this important posttranslational modification of proteins in different biological situations. High sequence coverages and the availability of straightforward quantification methods are necessary to achieve these goals. METHODS: Phosphoproteins and non-phosphorylated analogues separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were digested using four different proteases (trypsin, chymotrypsin, elastase and GluC) and the digests were isobarically labeled using eight-plex iTRAQ. The combined labeled digests were subsequently enriched using titanium dioxide and both the phosphorylated and non-phosphorylated fractions were analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The influence of different data analysis algorithms (Percolator or False Discovery Rate) on the outcome of analysis was investigated. RESULTS: Almost complete sequence coverage could be achieved upon application of a multi-protease approach. The formation of peptides of different lengths and physicochemical properties allowed the identification and the mapping of all phosphorylation sites in the investigated model proteins. The introduction of isobaric labels allowed quantification of different peptides of the same phosphorylation site with more than one peptide, leading to significantly improved statistical confidence. CONCLUSIONS: A workflow for the straightforward comparative analysis of protein phosphorylation in samples of low complexity, e.g. isolated proteins, was developed. The workflow is transferable to other posttranslational modifications.

Distribution of coronary calcifications in patients with suspected coronary heart disease.

BACKGROUND: Coronary calcifications are a marker of coronary atherosclerosis. The role of coronary calcium scoring (CS) as part of the initial evaluation of patients with suspected coronary heart disease (CHD) is controversially discussed. The primary goal of this study was to characterize the coronary calcium distribution in this particular patient population. In a second step, we aimed to establish a possible clinical implication using CS for the diagnosis of CHD. METHODS: Calcium scoring procedure was performed by either using a multidetector or a dual-source computed tomographic scanner. All patients underwent invasive coronary angiography (ICA) as the current criterion standard for CHD detection. A total of 4,137 (2,780 men, mean age 60.5 ± 12.4 years) consecutive patients were included. RESULTS: Mean CS was 288 ± 446 (range 0-5,252). Overall coronary artery calcifications significantly increased with patients' age. In 2,048 patients (mean CS 101 ± 239, range 0-5252), significant CHD (≥50% stenosis) was excluded by ICA (1,939 patients without calcifications). In remaining 2,089 patients (51%, mean CS 607 ± 821, range 0-5,252), significant CHD was documented leading to intervention in 732 patients. A threshold of zero calcifications (existence of calcified tissue) had the best overall sensitivity and negative predictive value with 99%. Overall specificity with 34% and overall positive predictive value with 24% were rather low. CONCLUSION: Coronary calcium scoring is able to exclude significant CHD in patients with suspected CHD with a high negative predictive value and, therefore, possibly reduce the number of invasive diagnostic examinations. Because of the low specificity and positive predictive value, CS cannot be used to indicate ICA.

Rapid ammonia gas transport accounts for futile transmembrane cycling under NH3/NH4+ toxicity in plant roots.

Futile transmembrane NH3/NH4(+) cycling in plant root cells, characterized by extremely rapid fluxes and high efflux to influx ratios, has been successfully linked to NH3/NH4(+) toxicity. Surprisingly, the fundamental question of which species of the conjugate pair (NH3 or NH4(+)) participates in such fluxes is unresolved. Using flux analyses with the short-lived radioisotope (13)N and electrophysiological, respiratory, and histochemical measurements, we show that futile cycling in roots of barley (Hordeum vulgare) seedlings is predominately of the gaseous NH3 species, rather than the NH4(+) ion. Influx of (13)NH3/(13)NH4(+), which exceeded 200 µmol g(-1) h(-1), was not commensurate with membrane depolarization or increases in root respiration, suggesting electroneutral NH3 transport. Influx followed Michaelis-Menten kinetics for NH3 (but not NH4(+)), as a function of external concentration (Km = 152 µm, Vmax = 205 µmol g(-1) h(-1)). Efflux of (13)NH3/(13)NH4(+) responded with a nearly identical Km. Pharmacological characterization of influx and efflux suggests mediation by aquaporins. Our study fundamentally revises the futile-cycling model by demonstrating that NH3 is the major permeating species across both plasmalemma and tonoplast of root cells under toxicity conditions.