DNA-based identification of novel bovine casein gene variants

In cattle, at least 39 variants of the 4 casein proteins (α(S1)-, β-, α(S2)- and κ-casein) have been described to date. Many of these variants are known to affect milk-production traits, cheese-processing properties, and the nutritive value of milk. They also provide valuable information for phylogenetic studies. So far, the majority of studies exploring the genetic variability of bovine caseins considered European taurine cattle breeds and were carried out at the protein level by electrophoretic techniques. This only allows the identification of variants that, due to amino acid exchanges, differ in their electric charge, molecular weight, or isoelectric point. In this study, the open reading frames of the casein genes CSN1S1, CSN2, CSN1S2, and CSN3 of 356 animals belonging to 14 taurine and 3 indicine cattle breeds were sequenced. With this approach, we identified 23 alleles, including 5 new DNA sequence variants, with a predicted effect on the protein sequence. The new variants were only found in indicine breeds and in one local Iranian breed, which has been phenotypically classified as a taurine breed. A multidimensional scaling approach based on available SNP chip data, however, revealed an admixture of taurine and indicine populations in this breed as well as in the local Iranian breed Golpayegani. Specific indicine casein alleles were also identified in a few European taurine breeds, indicating the introgression of indicine breeds into these populations. This study shows the existence of substantial undiscovered genetic variability of bovine casein loci, especially in indicine cattle breeds. The identification of new variants is a valuable tool for phylogenetic studies and investigations into the evolution of the milk protein genes.

DNA-based analysis of protein variants reveals different genetic variability of the paralogous equine ß-lactoglobulin genes LGB1 and LGB2

The genetic variability of milk protein genes may influence the nutritive value or processing and functional properties of the milk. While numerous protein variants are known in ruminants, knowledge about milk protein variability in horses is still limited. Mare's milk is, however, produced for human consumption in many countries. Beta-lactoglobulin belonging to the protein family of lipocalins, which are known as common food- and airborne allergens, is a major whey protein. It is absent from human milk and thus a key agent in provoking cow's milk protein allergy. Mare's milk is, however, usually better tolerated by most affected people. Several functions of β-lactoglobulin have been discussed, but its ultimate physiological role remains unclear. In the current study, the open reading frames of the two equine β-lactoglobulin paralogues LGB1 and LGB2 were re-sequenced in 249 horses belonging to 14 different breeds in order to predict the existence of protein variants at the DNA-level. Thereby, only a single signal peptide variant of LGB1, but 10 different putative protein variants of LGB2 were identified. In horses, both genes are expressed and in such this is a striking previously unknown difference in genetic variability between the two genes. It can be assumed that LGB1 is the ancestral paralogue, which has an essential function causing a high selection pressure. As horses have very low milk fat content this unknown function might well be related to vitamin-uptake. Further studies are, however, needed, to elucidate the properties of the different gene products.

Genetic variability of the equine casein genes.

The casein genes are known to be highly variable in typical dairy species, such as cattle and goat, but the knowledge about equine casein genes is limited. Nevertheless, mare milk production and consumption is gaining importance because of its high nutritive value, use in naturopathy, and hypoallergenic properties with respect to cow milk protein allergies. In the current study, the open reading frames of the 4 casein genes CSN1S1 (αS1-casein), CSN2 (β-casein), CSN1S2 (αS2-casein), and CSN3 (κ-casein) were resequenced in 253 horses of 14 breeds. The analysis revealed 21 nonsynonymous nucleotide exchanges, as well as 11 synonymous nucleotide exchanges, leading to a total of 31 putative protein isoforms predicted at the DNA level, 26 of which considered novel. Although the majority of the alleles need to be confirmed at the transcript and protein level, a preliminary nomenclature was established for the equine casein alleles.

Diagnostic Performance and Additional Value of Elastosonography in Focal Breast Lesions: Statistical Correlation between Size-Dependant Strain Index Measurements, Multimodality-BI-RADS Score, and Histopathology in a Clinical Routine Setting

Objective. To evaluate the diagnostic benefit of real-time elastography (RTE) in clinical routine. Strain indices (SI) for benign and malignant tumors were assessed. Methods. 100 patients with 110 focal breast lesions were retrieved. Patients had mammography (MG), ultrasound (US), and, if necessary, MRI. RTE was conducted after ultrasound. Lesions were assessed with BI-RADS for mammography and ultrasound. Diagnosis was established with histology or follow-up. Results. SI for BI-RADS 2 was 1.71 ± 0.86. Higher SI (2.21 ± 1.96) was observed for BI-RADS 3 lesions. SI of BI-RADS 4 and 5 lesions were significantly higher (16.92 ± 20.89) and (19.54 ± 10.41). 31 malignant tumors exhibited an average SI of 16.13 ± 14.67; SI of benign lesions was 5.29 ± 11.87 (P value <0.0001). ROC analysis threshold was >3.8 for malignant disease. Sensitivity of sonography was 90.3% (specificity 78.5%). RTE showed a sensitivity of 87.1% (specificity 79.7%). Accuracy of all modalities combined was 96.8%. In BI-RADS 3 lesions RTE was able to detect all malignant lesions (sensitivity 100%, specificity 92.9%, and accuracy 93.9%). Conclusions. RTE increased sensitivity and specificity for breast cancer detection when used in combination with ultrasound.

Corpus callosum index and long-term disability in multiple sclerosis patients

Prediction of long-term disability in patients with multiple sclerosis (MS) is essential. Magnetic resonance imaging (MRI) measurement of brain volume may be of predictive value but sophisticated MRI techniques are often inaccessible in clinical practice. The corpus callosum index (CCI) is a normalized measurement that reflects changes of brain volume. We investigated medical records and 533 MRI scans at diagnosis and during clinical follow-up of 169 MS patients (mean age 42 +/- 11 years, 86% relapsing-remitting MS, time since first relapse 11 +/- 9 years). CCI at diagnosis was 0.345 +/- 0.04 and correlated with duration of disease (p = 0.002; r = -0.234) and expanded disability status scale (EDSS) score at diagnosis (r = -0.428; p < 0.001). Linear regression analyses identified age, duration of disease, relapse rate and EDSS at diagnosis as independent predictors for disability after mean of 7.1 years (Nagelkerkes' R:0.56). Annual CCI decrease was 0.01 +/- 0.02 (annual tissue loss: 1.3%). In secondary progressive MS patients, CCI decrease was double compared to that in relapsing-remitting MS patients (p = 0.04). There was a trend of greater CCI decrease in untreated patients compared to those who received disease modifying drugs (p = 0.2). CCI is an easy to use MRI marker for estimating brain atrophy in patients with MS. Brain atrophy as measured with CCI was associated with disability progression but it was not an independent predictor of long-term disability.

Cardiac troponin testing and the simplified Pulmonary Embolism Severity Index. The SWIss Venous ThromboEmbolism Registry (SWIVTER)

A low simplified Pulmonary Embolism Severity Index (sPESI), defined as age ≤80 years and absence of systemic hypotension, tachycardia, hypoxia, cancer, heart failure, and lung disease, identifies low-risk patients with acute pulmonary embolism (PE). It is unknown whether cardiac troponin testing improves the prediction of clinical outcomes if the sPESI is not low. In the prospective Swiss Venous Thromboembolism Registry, 369 patients with acute PE and a troponin test (conventional troponin T or I, highly sensitive troponin T) were enrolled from 18 hospitals. A positive test result was defined as a troponin level above the manufacturers assay threshold. Among the 106 (29%) patients with low sPESI, the rate of mortality or PE recurrence at 30 days was 1.0%. Among the 263 (71%) patients with high sPESI, 177 (67%) were troponin-negative and 86 (33%) troponin-positive; the rate of mortality or PE recurrence at 30 days was 4.6% vs. 12.8% (p=0.015), respectively. Overall, risk assessment with a troponin test (hazard ratio [HR] 3.39, 95% confidence interval [CI] 1.38-8.37; p=0.008) maintained its prognostic value for mortality or PE recurrence when adjusted for sPESI (HR 5.80, 95%CI 0.76-44.10; p=0.09). The combination of sPESI with a troponin test resulted in a greater area under the receiver-operating characteristic curve (HR 0.72, 95% CI 0.63-0.81) than sPESI alone (HR 0.63, 95% CI 0.57-0.68) (p=0.023). In conclusion, although cardiac troponin testing may not be required in patients with a low sPESI, it adds prognostic value for early death and recurrence for patients with a high sPESI.

Novel pressure-to-cornea index in glaucoma

BACKGROUND: Several conversion tables and formulas have been suggested to correct applanation intraocular pressure (IOP) for central corneal thickness (CCT). CCT is also thought to represent an independent glaucoma risk factor. In an attempt to integrate IOP and CCT into a unified risk factor and avoid uncertain correction for tonometric inaccuracy, a new pressure-to-cornea index (PCI) is proposed. METHODS: PCI (IOP/CCT(3)) was defined as the ratio between untreated IOP and CCT(3) in mm (ultrasound pachymetry). PCI distribution in 220 normal controls, 53 patients with normal-tension glaucoma (NTG), 76 with ocular hypertension (OHT), and 89 with primary open-angle glaucoma (POAG) was investigated. PCI's ability to discriminate between glaucoma (NTG+POAG) and non-glaucoma (controls+OHT) was compared with that of three published formulae for correcting IOP for CCT. Receiver operating characteristic (ROC) curves were built. RESULTS: Mean PCI values were: Controls 92.0 (SD 24.8), NTG 129.1 (SD 25.8), OHT 134.0 (SD 26.5), POAG 173.6 (SD 40.9). To minimise IOP bias, eyes within the same 2 mm Hg range between 16 and 29 mm Hg (16-17, 18-19, etc) were separately compared: control and NTG eyes as well as OHT and POAG eyes differed significantly. PCI demonstrated a larger area under the ROC curve (AUC) and significantly higher sensitivity at fixed 80% and 90% specificities compared with each of the correction formulas; optimum PCI cut-off value 133.8. CONCLUSIONS: A PCI range of 120-140 is proposed as the upper limit of "normality", 120 being the cut-off value for eyes with untreated pressures or=22 mm Hg. PCI may reflect individual susceptibility to a given IOP level, and thus represent a glaucoma risk factor. Longitudinal studies are needed to prove its prognostic value.

Intrarenal resistance index for the assessment of early renal function impairment in patients with liver cirrhosis

BACKGROUND: Renovascular vasoconstriction in patients with hepatorenal syndrome can be quantified by the renal arterial resistance index (RI). We investigated the value of RI measurement in detection of renal function impairment in patients with different stages of chronic liver disease. METHODS: Subjects were divided into 4 groups containing 21 patients with liver cirrhosis and ascites, 25 patients with liver cirrhosis without ascites, 35 patients with fatty liver disease and 78 control subjects. All patients underwent abdominal ultrasound examination with renal RI measurement and correlation with laboratory results for renal function. RESULTS: RI was significantly higher in ascitic patients compared to non-ascitic patients (0.74 vs. 0.67, p<0.01) and in non-ascitic patients with liver cirrhosis than in control subjects (0.67 vs. 0.62, p<0.01). 48% (19/40) of patients with liver cirrhosis and normal serum creatinine concentration showed elevated RI levels. There were no significant differences in RI levels between patients with fatty liver disease and controls (0.63 vs. 0.62). CONCLUSIONS: Intrarenal RI measurement is a predictor of renal vasoconstriction and serves to detect early renal function impairment in cirrhotic patients. The diagnosis of elevated RI may be taken into account in the clinical management of these patients.

Relationship between renal resistance index and renal function in liver transplant recipients after cessation of calcineurin inhibitor

End stage renal disease is a major complication after orthotopic liver transplantation (OLT). Vasoconstriction of renal arterial vessels because of calcineurin inhibitor (CNI) treatment plays a pivotal role in the development of renal insufficiency following OLT. Renal resistance can be measured non-invasively by determining the resistance index (RI) of segmental arteries by color-coded duplex ultrasonography, a measure with predictive value for future renal failure. Sixteen OLT patients on long-term CNI therapy were recruited prospectively and randomly assigned either to receive the m-TOR inhibitor sirolimus (SRL) or to continue on CNI treatment, and were followed for one yr. Serum creatinine (crea) declined after conversion to SRL, whereas it tended to increase in patients remaining on CNI (meanDelta crea SRL: -27, -18, -18, -15 micromol/L; meanDelta crea CNI: 4, 5, 8, 11 micromol/L at 1, 3, 6, 12 months, p = 0.02). RI improved after switching to SRL and was lower on SRL than on CNI (meanDeltaRI SRL: -0.04, -0.04, -0.03, -0.03; meanDeltaRI CNI: -0.006, 0.004, -0.007, -0.01 after 1, 3, 6, 12 months, p = 0.016). Individual changes of RI correlated significantly with individual changes of crea (r = 0.54, p < 0.001). Conversion from CNI to SRL can ameliorate renal function accompanied by a reduction of intrarenal RI after OLT.

Auditory event-related potentials, bispectral index, and entropy for the discrimination of different levels of sedation in intensive care unit patients

BACKGROUND: Sedation protocols, including the use of sedation scales and regular sedation stops, help to reduce the length of mechanical ventilation and intensive care unit stay. Because clinical assessment of depth of sedation is labor-intensive, performed only intermittently, and interferes with sedation and sleep, processed electrophysiological signals from the brain have gained interest as surrogates. We hypothesized that auditory event-related potentials (ERPs), Bispectral Index (BIS), and Entropy can discriminate among clinically relevant sedation levels. METHODS: We studied 10 patients after elective thoracic or abdominal surgery with general anesthesia. Electroencephalogram, BIS, state entropy (SE), response entropy (RE), and ERPs were recorded immediately after surgery in the intensive care unit at Richmond Agitation-Sedation Scale (RASS) scores of -5 (very deep sedation), -4 (deep sedation), -3 to -1 (moderate sedation), and 0 (awake) during decreasing target-controlled sedation with propofol and remifentanil. Reference measurements for baseline levels were performed before or several days after the operation. RESULTS: At baseline, RASS -5, RASS -4, RASS -3 to -1, and RASS 0, BIS was 94 [4] (median, IQR), 47 [15], 68 [9], 75 [10], and 88 [6]; SE was 87 [3], 46 [10], 60 [22], 74 [21], and 87 [5]; and RE was 97 [4], 48 [9], 71 [25], 81 [18], and 96 [3], respectively (all P < 0.05, Friedman Test). Both BIS and Entropy had high variabilities. When ERP N100 amplitudes were considered alone, ERPs did not differ significantly among sedation levels. Nevertheless, discriminant ERP analysis including two parameters of principal component analysis revealed a prediction probability PK value of 0.89 for differentiating deep sedation, moderate sedation, and awake state. The corresponding PK for RE, SE, and BIS was 0.88, 0.89, and 0.85, respectively. CONCLUSIONS: Neither ERPs nor BIS or Entropy can replace clinical sedation assessment with standard scoring systems. Discrimination among very deep, deep to moderate, and no sedation after general anesthesia can be provided by ERPs and processed electroencephalograms, with similar P(K)s. The high inter- and intraindividual variability of Entropy and BIS precludes defining a target range of values to predict the sedation level in critically ill patients using these parameters. The variability of ERPs is unknown.

Incremental Value of the CRUSADE, ACUITY, and HAS-BLED Risk Scores for the Prediction of Hemorrhagic Events After Coronary Stent Implantation in Patients Undergoing Long or Short Duration of Dual Antiplatelet Therapy

BACKGROUND Multiple scores have been proposed to stratify bleeding risk, but their value to guide dual antiplatelet therapy duration has never been appraised. We compared the performance of the CRUSADE (Can Rapid Risk Stratification of Unstable Angina Patients Suppress Adverse Outcomes With Early Implementation of the ACC/AHA Guidelines), ACUITY (Acute Catheterization and Urgent Intervention Triage Strategy), and HAS-BLED (Hypertension, Abnormal Renal/Liver Function, Stroke, Bleeding History or Predisposition, Labile INR, Elderly, Drugs/Alcohol Concomitantly) scores in 1946 patients recruited in the Prolonging Dual Antiplatelet Treatment After Grading Stent-Induced Intimal Hyperplasia Study (PRODIGY) and assessed hemorrhagic and ischemic events in the 24- and 6-month dual antiplatelet therapy groups. METHODS AND RESULTS Bleeding score performance was assessed with a Cox regression model and C statistics. Discriminative and reclassification power was assessed with net reclassification improvement and integrated discrimination improvement. The C statistic was similar between the CRUSADE score (area under the curve 0.71) and ACUITY (area under the curve 0.68), and higher than HAS-BLED (area under the curve 0.63). CRUSADE, but not ACUITY, improved reclassification (net reclassification index 0.39, P=0.005) and discrimination (integrated discrimination improvement index 0.0083, P=0.021) of major bleeding compared with HAS-BLED. Major bleeding and transfusions were higher in the 24- versus 6-month dual antiplatelet therapy groups in patients with a CRUSADE score >40 (hazard ratio for bleeding 2.69, P=0.035; hazard ratio for transfusions 4.65, P=0.009) but not in those with CRUSADE score ≤40 (hazard ratio for bleeding 1.50, P=0.25; hazard ratio for transfusions 1.37, P=0.44), with positive interaction (Pint=0.05 and Pint=0.01, respectively). The number of patients with high CRUSADE scores needed to treat for harm for major bleeding and transfusion were 17 and 15, respectively, with 24-month rather than 6-month dual antiplatelet therapy; corresponding figures in the overall population were 67 and 71, respectively. CONCLUSIONS Our analysis suggests that the CRUSADE score predicts major bleeding similarly to ACUITY and better than HAS BLED in an all-comer population with percutaneous coronary intervention and potentially identifies patients at higher risk of hemorrhagic complications when treated with a long-term dual antiplatelet therapy regimen. CLINICAL TRIAL REGISTRATION URL: http://clinicaltrials.gov. Unique identifier: NCT00611286.

Prognostic Value of Secretoneurin in Critically Ill Patients With Infections.

OBJECTIVES Secretoneurin is produced in neuroendocrine cells, and the myocardium and circulating secretoneurin levels provide incremental prognostic information to established risk indices in cardiovascular disease. As myocardial dysfunction contributes to poor outcome in critically ill patients, we wanted to assess the prognostic value of secretoneurin in two cohorts of critically ill patients with infections. DESIGN Two prospective, observational studies. SETTING Twenty-four and twenty-five ICUs in Finland. PATIENTS A total of 232 patients with severe sepsis (cohort #1) and 94 patients with infections and respiratory failure (cohort #2). INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS We measured secretoneurin levels by radioimmunoassay in samples obtained early after ICU admission and compared secretoneurin with other risk indices. In patients with severe sepsis, admission secretoneurin levels (logarithmically transformed) were associated with hospital mortality (odds ratio, 3.17 [95% CI, 1.12-9.00]; p = 0.030) and shock during the hospitalization (odds ratio, 2.17 [1.06-4.46]; p = 0.034) in analyses that adjusted for other risk factors available on ICU admission. Adding secretoneurin levels to age, which was also associated with hospital mortality in the multivariate model, improved the risk prediction as assessed by the category-free net reclassification index: 0.35 (95% CI, 0.06-0.64) (p = 0.02). In contrast, N-terminal pro-B-type natriuretic peptide levels were not associated with mortality in the multivariate model that included secretoneurin measurements, and N-terminal pro-B-type natriuretic peptide did not improve patient classification on top of age. Secretoneurin levels were also associated with hospital mortality after adjusting for other risk factors and improved patient classification in cohort #2. In both cohorts, the optimal cutoff for secretoneurin levels at ICU admission to predict hospital mortality was ≈ 175 pmol/L, and higher levels were associated with mortality also when adjusting for Simplified Acute Physiology Score II and Sequential Organ Failure Assessment scores. CONCLUSIONS Secretoneurin levels provide incremental information to established risk indices for the prediction of mortality and shock in critically ill patients with severe infections.