Alkali Metal Cationization of Tumor-associated Antigen Peptides for Improved Dissociation and Measurement by Differential Ion Mobility-Mass Spectrometry.

Tandem mass spectrometry (MS/MS) is a highly sensitive and selective method for the detection of tumor-associated peptide antigens. These short, nontryptic sequences may lack basic residues, resulting in the formation of predominantly [peptide + H]+ ions in electrospray. These singly charged ions tend to undergo inefficient dissociation, leading to issues in sequence determination. Addition of alkali metal salts to the electrospray solvent can drive the formation of [peptide + H + metal]2+ ions that have enhanced dissociation characteristics relative to [peptide + H]+ ions. Both previously identified tumor-associated antigens and predicted neoantigen sequences were investigated. The previously reported rearrangement mechanism in MS/MS of sodium-cationized peptides is applied here to demonstrate complete C-terminal sequencing of tumor-associated peptide antigens. Differential ion mobility spectrometry (DIMS) is shown to selectively enrich [peptide + H + metal]2+ species by filtering out singly charged interferences at relatively low field strengths, offsetting the decrease in signal intensity associated with the use of alkali metal cations.

Hemodynamic targets during therapeutic hypothermia after cardiac arrest: A prospective observational study.

AIM: In analogy with sepsis, current post-cardiac arrest (CA) guidelines recommend to target mean arterial pressure (MAP) above 65 mmHg and SVO2 above 70%. This is unsupported by mortality or cerebral perfusion data. The aim of this study was to explore the associations between MAP, SVO2, cerebral oxygenation and survival. METHODS: Prospective, observational study during therapeutic hypothermia (24h - 33 °C) in 82 post-CA patients monitored with near-infrared spectroscopy. RESULTS: Forty-three patients (52%) survived in CPC 1-2 until 180 days post-CA. The mean MAP range associated with maximal survival was 76-86 mmHg (OR 2.63, 95%CI [1.01; 6.88], p = 0.04). The mean SVO2 range associated with maximal survival was 67-72% (OR 8.23, 95%CI [2.07; 32.68], p = 0.001). In two separate multivariate models, a mean MAP (OR 3.72, 95% CI [1.11; 12.50], p=0.03) and a mean SVO2 (OR 10.32, 95% CI [2.03; 52.60], p = 0.001) in the optimal range persisted as independently associated with increased survival. Based on more than 1625000 data points, we found a strong linear relation between SVO2 (range 40-90%) and average cerebral saturation (R(2) 0.86) and between MAP and average cerebral saturation for MAP's between 45 and 101 mmHg (R(2) 0.83). Based on our hemodynamic model, the MAP and SVO2 ranges associated with optimal cerebral oxygenation were determined to be 87-101 mmHg and 70-75%. CONCLUSION: we showed that a MAP range between 76-86 mmHg and SVO2 range between 67% and 72% were associated with maximal survival. Optimal cerebral saturation was achieved with a MAP between 87-101 mmHg and a SVO2 between 70% and 75%. Prospective interventional studies are needed to investigate whether forcing MAP and SVO2 in the suggested range with additional pharmacological support would improve outcome.

An observational near-infrared spectroscopy study on cerebral autoregulation in post-cardiac arrest patients: time to drop 'one-size-fits-all' hemodynamic targets?

AIMS: A subgroup of patients with ROSC after cardiac arrest (CA) with disturbed cerebral autoregulation might benefit from higher mean arterial pressures (MAP). We aimed to (1) phenotype patients with disturbed autoregulation, (2) investigate whether these patients have a worse prognosis, (3) define an individual optimal MAP per patient and (4) investigate whether time under this individual optimal MAP is associated with outcome. METHODS: Prospective observational study in 51 post-CA patients monitored with near infrared spectroscopy. RESULTS: (1) 18/51 patients (35%) had disturbed autoregulation. Phenotypically, a higher proportion of patients with disturbed autoregulation had pre-CA hypertension (31±47 vs. 65±49%, p=0.02) suggesting that right shifting of autoregulation is caused by chronic adaptation of cerebral blood flow to higher blood pressures. (2) In multivariate analysis, patients with preserved autoregulation (n=33, 65%) had a significant higher 180-days survival rate (OR 4.62, 95% CI [1.06:20.06], p=0.04]. Based on an index of autoregulation (COX), the average COX-predicted optimal MAP was 85 mmHg in patients with preserved and 100 mmHg in patients with disturbed autoregulation. (3) An individual optimal MAP could be determined in 33/51 patients. (4) The time under the individual optimal MAP was negatively associated with survival (OR 0.97, 95% CI [0.96:0.99], p=0.02). The time under previously proposed fixed targets (65, 70, 75, 80 mmHg) was not associated with a differential survival rate. CONCLUSION: Cerebral autoregulation showed to be disturbed in 35% of post-CA patients of which a majority had pre-CA hypertension. Disturbed cerebral autoregulation within the first 24h after CA is associated with a worse outcome. In contrast to uniform MAP goals, the time spent under a patient tailored optimal MAP, based on an index of autoregulation, was negatively associated with survival.

Gender differences in the psychosocial correlates of suicidal ideation among adolescents.

Gender differences in the psychosocial correlates of suicidal ideation were studied. A sample of 613 high school students (ages 14-19) completed measures of suicidal ideation, depression, hopelessness, life stress, loneliness, alcohol and drug use, and reasons for living. The results of a discriminant function analysis indicated that males reported higher loneliness and substance abuse scores than females whereas females reported greater suicidal ideation, depression, and reasons for living. The results of multiple regression analyses found that, although the same four variables, depression, hopelessness, substance abuse, and few reasons for living emerged as significant predictors of suicidal ideation in both samples, the predictive equation accounted for more of the variance in ideation scores in females (57%) than in males (46%). In a final analysis a discriminant function analysis of the subscales of the reasons for living inventory revealed that females have a greater fear of death and injury whereas males have a greater fear of social disapproval over having suicidal thoughts. This may account for the greater rate of suicide completing among males. Fear of social disapproval, more anger and impulsivity, and less help-seeking behavior among males are offered as potential variables to explain the observed gender differences.

Accuracy of continuous thermodilution cardiac output monitoring by pulmonary artery catheter during therapeutic hypothermia in post-cardiac arrest patients.

PURPOSE: Thermodilution continuous cardiac output measurements (TDCCO) by pulmonary artery catheter (PAC) have not been validated during therapeutic hypothermia in post-cardiac arrest patients. The calculated cardiac output based on the indirect Fick principle (FCO) using pulmonary artery blood gas mixed venous oxygen saturation (FCO-BG-SvO2) is considered as the gold standard. Continuous SvO2 by PAC (PAC-SvO2) has also not been validated previously during hypothermia. The aims of this study were (1) to compare FCO-BG-SvO2 with TDCCO, (2) to compare PAC-SvO2 with BG-SvO2 and finally (3) to compare FCO with SvO2 obtained via PAC or blood gas. METHODS: We analyzed 102 paired TDCCO/FCO-BG-SvO2 and 88 paired BG-SvO2/PAC-SvO2 measurements in 32 post-cardiac arrest patients during therapeutic hypothermia. RESULTS: TDCCO was significantly although poorly correlated with FCO-BG-SvO2 (R2 0.21, p<0.01) without systematic bias (-0.15±1.76 l/min). Analysis according to Bland and Altman however showed broad limits of agreement ([-3.61; 3.45] l/min) and an unacceptable high percentage error (105%). None of the criteria for clinical interchangeability were met. Concordance analysis showed that TDCCO had limited trending ability (R2 0.03). FCO based on PAC-SvO2 was highly correlated with FCO-BG-SvO2 (R2 0.72) with a small bias (-0.08±0.72 l/min) and slightly too high percentage error (44%). CONCLUSION: Our results show an extreme inaccuracy of TDCCO by PAC in post-cardiac arrest patients during therapeutic hypothermia. We found a reasonable correlation between BG-SvO2 and PAC-SvO2 and subsequently between FCO calculated with SvO2 obtained either via blood gas or PAC. The decision to start or titrate inotropics should therefore not be guided by TDCCO in this setting.

Na-P(i) cotransporter type I activity causes a transient intracellular alkalinization during ATP depletion in rabbit medullary thick ascending limb cells.

The cellular pathophysiology of renal ischemia-reperfusion injury was investigated in primary cell cultures from rabbit medullary thick ascending limb (MTAL). Metabolic inhibition (MI) was achieved with cyanide and 2-deoxyglucose. Sixty minutes of MI caused a profound but reversible decrease in intracellular concentration of ATP ([ATP]i). Intracellular pH (pHi) first decreased after initiation of MI, followed by a transient alkalinization. When [ATP]i reached its lowest value (<1% of control), the cells slowly acidified to reach a stable pHi of 6.92 after 50 min of MI. In the presence of EIPA (10 micromol/L), the pattern of changes in pHi was unchanged and acidification was not increased, indicating that the Na+/H+ exchangers were inactive during ATP depletion. When inorganic phosphate (P(i)) or Na+ was omitted from the apical solutions during MI, the transient alkalinization was no longer observed and the cytosol slowly acidified. Experiments on Na+-dependent alkalinizations revealed the presence of a Na-P(i) cotransporter in the apical cell membrane. With indirect immunofluorescence, the Na-P(i) cotransporter expressed in these primary cell cultures could be identified as Na-P(i) type I. Although the exact physiological role of Na-P(i) type I still is unresolved, these experiments demonstrate that apical Na-P(i) type I activity is increased at the onset of ATP depletion in MTAL cells.

Rumen-protected methionine fed to dairy cows: bioavailability and effects on plasma amino acid pattern and plasma metabolite and insulin concentrations.

An experiment was performed to compare the bioavailability of D,L-methionine (Met) of two rumen (polymer and fat)-protected Met forms (Smartamine; Rhône-Poulenc, Animal Nutrition, Antony, France and Mepron; Degussa AG, Hanau, Germany, respectively) fed once daily (at 0730 h) in amounts of 50 g per cow for 5 d. Six dairy cows were used in the crossover design. Blood samples were obtained from jugular veins 3 d before and 5 d after the start of Met supplementation at 1000, 1400, and 1800 h, and the three blood samples were pooled for analyses. Smartamine M feeding caused elevations of sulfur-containing amino acids (Met, Cys, and taurine) and reductions of Val and Ile and the total of branched-chain amino acids (Val + Leu + Ile). Mepron M85 feeding caused only a rise of Met concentrations. Concentrations of Met, taurine, and Gln were higher when Smartamine M than when Mepron M85 was fed. Concentrations of nonesterified fatty acids were reduced, and those of insulin were increased only by Mepron M85 feeding. Milk urea concentrations were lower in cows fed Mepron M85 than in controls, but milk yields; concentrations of fat, protein, and lactose; and somatic cell counts did not significantly change during the experiment and were not different between groups. Food intake, body weight, and body condition scores were not affected. In conclusion, only Mepron M85 supplementation influenced nonesterified fatty acids and insulin concentrations. However, the bioavailability of Met from Smartamine M was greater than of Mepron M85 and effects on other plasma-free amino acids were more marked. Whether less Met in the form of Smartamine M as Mepron M is needed to have the same effects on milk yield and composition needs to be investigated with doses used in practice.

Twenty-four-hour patterns of hormones and metabolites in week 9 and 19 of lactation in high-yielding dairy cows fed triglycerides and free fatty acids.

Effects of feeding crystalline triglycerides (TGA), free fatty acids (FFA), and a starch-rich ration (STA) on metabolite and hormone concentrations in blood plasma were studied in high-yielding dairy cows over a 24-h period in week 9 and 19 of lactating. Energy-corrected milk production in the three groups was similar, but was lower in week 19 than in week 9. Energy and protein intakes were greater in week 9 than in week 19, but energy and protein balances in the three groups and in weeks 9 and 19 were similar. Plasma glucose and triglyceride concentrations were lower in week 9 than in week 19. In cows fed FFA, glucose concentrations were highest in week 9. Plasma triglyceride, phospholipid and cholesterol concentrations were highest, whereas beta-hydroxybutyrate concentrations were lowest in FFA-fed cows in weeks 9 and 19. Concentrations of insulin-like growth factor-I in week 19 were lower in cows fed TGA and FFA than in those fed the starch-rich ration. Post-prandial responses were usually greater following morning than afternoon meals. Fructosamine, albumin, urea, growth hormone, thyroxine, and 3,5,3'-triiodothyronine concentrations were similar in weeks 9 and 19 and were not influenced by dietary treatment or feeding times. In conclusion, there were distinct metabolic and endocrine effects of feeding TGA and FFA compared with STA and the concentrations as well as the 24-h changes of various metabolic and endocrine traits in weeks 9 and 19 of lactation were also different.

Variations of 3-methylhistidine in blood of dairy cows.

Blood plasma 3-methylhistidine, in comparison with other blood variables, has been measured in high-yielding dairy cows with relation to energy and protein supply. Circulating 3-methylhistidine markedly increased to 1 wk after calving, then continuously decreased to 5 wk postpartum to lower than during the last 2 wk of pregnancy. In experiments 36 d after calving, circulating 3-methylhistidine did not change during 24 h despite marked variations of food intake. Peak 3-methylhistidine immediately after parturition coincided with relatively low insulin, thyroxine, triiodothyronine, glucose, protein, and urea with elevated concentrations of nonesterified fatty acids and with greatest estimated energy and protein deficiencies. However, during the ensuing period to 12 wk of lactation, circulating 3-methylhistidine was not closely related to these measures nor to creatinine, beta-hydroxybutyrate, and milk production, but relationships to milk protein yield were close. The postparturient increase of 3-methylhistidine may have been a consequence of enhanced breakdown of skeletal muscle and uterine smooth muscle or another pool with a transiently enhanced turnover rate. Variations of plasma 3-methylhistidine were associated only in part with estimated negative energy and protein balances and corresponding endocrine and metabolic adaptations.

Longissimus dorsi muscle diameter, backfat thickness, body condition scores and skinfold values related to metabolic and endocrine traits in lactating dairy cows fed crystalline fat or free fatty acids.

An experiment was performed using 18 dairy cows with > or = 2 lactations from parturition until week 20 of lactation to investigate the effects of feeding rumen-protected crystalline fat (group A) or free fatty acids (group B) compared with a control group not fed supplemental fat or fatty acids (group C). The feeding effects studied were those on milk yield and composition, body weight (BW), body conditions scores (BCS), skinfold values (a measure of subcutaneous fat tissue thickness), backfat thickness and longissimus dorsi muscle diameter (measured by ultrasound), as well as on blood plasma metabolic and endocrine traits. Fat and fatty-acid feeding reduced roughage intake. Net energy and protein balances during the first week of lactation were negative. Milk yield was similar in all groups, but concentrations of milk fat were reduced in group B and of milk protein in groups A and B. BW, BCS, skinfold value, backfat thickness and longissimus dorsi muscle diameter similarly decreased in all groups until weeks 8-12. Concentrations of glucose and fructosamine decreased transiently during early lactation, whereas levels of beta-hydroxybutyrate, urea, total protein and albumin increased reversibly and similarly in all groups. Concentrations of creatinine decreased similarly in all groups until week 4 and those of nonesterified fatty acids until week 12. Triglyceride concentrations increased continuously until week 18, whereas concentrations of phospholipid and cholesterol increased until week 10 and then remained elevated. Triglyceride, phospholipid and cholesterol concentrations were higher in group B than in groups A and C throughout the experiment. Concentrations of insulin, insulin-like growth factor-1, thyroxine and 3,5,3'-triiodothyronine started to increase after the first 6-8 weeks of lactation, while those of growth hormone were transiently low in week 16. None of the hormones exhibited group differences. In this study rumen-protected fat or fatty-acid supplementation failed to improve the energy and protein balances during early lactation and did not affect milk yield, likely due to decreased roughage intake. BCS, skinfold values and backfat ultrasound measurements of subcutaneous adipose tissue mass and of longissimus dorsi muscle diameter indicated mobilization of fat tissue and protein mobilization in early lactation and were mirrored by typical changes of metabolic and endocrine traits.

A simple method for obtaining functionally and morphologically intact primary cultures of the medullary thick ascending limb of Henle's loop (MTAL) from rabbit kidneys.

We describe a simple method for obtaining functionally and morphologically intact primary cultures of cells from the medullary thick ascending limb of rabbit kidneys. After digesting dissected fragments of the inner stripe of the outer medulla with collagenase, a suspension of tubule fragments is obtained, the vast majority of which are medullary thick ascending limb (MTAL) segments. These are identified individually by their morphological appearance and large amounts are collected with a micropipette mounted on a micromanipulator. This ensures maximal homogeneity of the starting material. Monolayers of cells grow out of these MTAL segments after seeding them onto collagen-coated, permeable filter supports. During the week following confluence, the cultures exhibit an apical side-positive transepithelial potential difference. Electron microscopic examination shows a monolayer of polarised cells with characteristics of distal tubular cells. The primary cultures express Tamm-Horsfall protein at their apical surface. Additional evidence for their differentiation and polarisation is the net ammonium influx, which occurs at very high rates across the apical membrane and is much slower across the basolateral membrane, as judged by measurements of intracellular pH. Adenosine 3',5'-cyclic monophosphate (cAMP) production is stimulated by arginine-vasopressin, calcitonin or isoproterenol (all 1 micromol/l). Intracellular calcium signalling is observed after stimulation with 1 micromol/l adenosine, adenosine 5'-triphosphate (ATP) and bradykinin. In addition, we compared these characteristics with those of TALH-SVE cell monolayers, an established immortalised cell line of the same origin.