Proteomic analysis of the effect of hemin in breast cancer.

Heme, an iron-containing prosthetic group found in many proteins, carries out diverse biological functions such as electron transfer, oxygen storage and enzymatic reactions. Hemin, the oxidised form of heme, is used to treat porphyria and also to activate heme-oxygenase (HO) which catalyses the rate-limiting step in heme degradation. Our group has previously demonstrated that hemin displays antitumor activity in breast cancer (BC). The aim of this work has been to study the effect of hemin on protein expression modifications in a BC cell line to gain insight into the molecular mechanisms of hemin antitumor activity. For this purpose, we carried out proteome analysis by Mass Spectrometry (MS) which showed that 1309 proteins were significantly increased in hemin-treated cells, including HO-1 and the proteases that regulate HO-1 function, and 921 proteins were significantly decreased. Furthermore, the MS-data analysis showed that hemin regulates the expression of heme- and iron-related proteins, adhesion and cytoskeletal proteins, cancer signal transduction proteins and enzymes involved in lipid metabolism. By biochemical and cellular studies, we further corroborated the most relevant in-silico results. Altogether, these results show the multiple physiological effects that hemin treatment displays in BC and demonstrate its potential as anticancer agent.

Changes in global strain of puborectalis muscle during pregnancy and postpartum.

OBJECTIVE: One of the functional parameters that can be assessed by ultrasound is muscle strain, which represents the extent of deformation of the muscle from its original shape when forces are applied to the tissue under study. The aim of this study was to evaluate the effect of pregnancy and delivery on the puborectalis muscle, by assessing changes in global strain of the muscle during and after pregnancy. METHODS: This was a secondary analysis of a prospective observational study on the association between stress urinary incontinence and levator ani muscle avulsion after delivery. Two-hundred and eighty nulliparous pregnant women underwent four-dimensional transperineal ultrasound assessments at 12 and 36 weeks of gestation, and 6 months postpartum. Tomographic ultrasound images were constructed and the puborectalis muscle was delineated by hand using programming software. After delineation, the length of the midline of the puborectalis muscle was measured at rest and during maximum pelvic floor muscle contraction, and global strain was expressed as percentile difference. Postpartum results were analyzed separately for vaginal, operative (vacuum) vaginal and Cesarean delivery. Differences in global strain of the puborectalis muscle according to mode of delivery were compared using a paired-sample t-test. The effect of partial or complete avulsion of the puborectalis muscle on postpartum strain was evaluated by ANOVA. RESULTS: In total, 254 datasets were analyzed. Global strain of the puborectalis muscle did not change during pregnancy, but after spontaneous or operative vaginal delivery the global strain diminished significantly. No significant change was observed in strain of the puborectalis muscle after Cesarean delivery. Women who suffered complete bilateral avulsion had significantly lower strain compared with women with an intact puborectalis muscle. CONCLUSION: Spontaneous or operative vacuum vaginal birth and complete bilateral avulsion of the puborectalis muscle influence negatively the strain of the puborectalis muscle. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Comparison of translabial three-dimensional ultrasound with magnetic resonance imaging for measurement of levator hiatal biometry at rest.

OBJECTIVES: To compare translabial three-dimensional (3D) ultrasound with magnetic resonance imaging (MRI) for the measurement of levator hiatal biometry at rest in women with pelvic organ prolapse, and to determine the interobserver reliability between two independent observers for ultrasound and MRI measurements. METHODS: Data were derived from a multicenter prospective cohort study in which women scheduled for conventional anterior colporrhaphy underwent translabial 3D ultrasound and MRI prior to surgery. Intraclass correlation coefficients (ICCs) were calculated to estimate interobserver reliability between two independent observers and determine the agreement between ultrasound and MRI measurements. Bland-Altman plots were created to assess the agreement between ultrasound and MRI measurements. RESULTS: Data from 139 women from nine hospitals were included in the study. The interobserver reliability of ultrasound assessment at rest, during Valsalva maneuver and during contraction and of MRI assessment at rest were moderate or good. The agreement between ultrasound and MRI for the measurement of levator hiatal biometry at rest was moderate, with ICCs of 0.52 (95%CI, 0.32-0.66) for levator hiatal area, 0.44 (95%CI, 0.21-0.60) for anteroposterior diameter and 0.44 (95%CI, 0.22-0.60) for transverse diameter. Levator hiatal biometry measurements were statistically significantly larger on MRI than on translabial 3D ultrasound. CONCLUSIONS: The agreement between translabial 3D ultrasound and MRI for measurement of the levator hiatus at rest in women with pelvic organ prolapse was only moderate. The results of translabial 3D ultrasound and MRI should therefore not be used interchangeably in daily practice or in clinical research. Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.

Association between levator hiatal dimensions on ultrasound during first pregnancy and mode of delivery.

OBJECTIVES: To determine the association between levator hiatal dimensions, measured using transperineal ultrasound, in women during their first pregnancy and the subsequent mode of delivery, stratified by the indication for intervention. METHODS: In this prospective observational study, 280 nulliparous women with a singleton pregnancy were invited for transperineal ultrasound examination at 12 and 36 weeks' gestation. Their levator hiatal dimensions were measured at rest, on pelvic floor muscle contraction and on Valsalva maneuver. The subsequent mode of delivery was classified into five categories: spontaneous vaginal delivery, instrumental vaginal delivery owing to fetal distress, instrumental vaginal delivery owing to failure to progress, Cesarean section owing to fetal distress and Cesarean section owing to failure to progress. Levator hiatal dimensions according to mode of delivery were compared by analysis of variance and Tukey's post-hoc test. Since multiple comparison tests were performed, the statistical significance level was corrected using the Bonferroni method. RESULTS: Of the 252 women included, those who delivered by Cesarean section because of failure to progress had a significantly smaller levator hiatal transverse diameter on pelvic floor contraction at 12 weeks' gestation than did women who had a spontaneous vaginal delivery (Tukey's post-hoc test, P < 0.001). There was also a trend towards a smaller hiatal area on pelvic floor contraction at 12 weeks' gestation in women who delivered by Cesarean section because of failure to progress compared to women who had a spontaneous vaginal delivery (Tukey's post-hoc test, P = 0.005). In women who had an instrumental vaginal delivery because of failure to progress there was a trend towards a smaller levator hiatal anteroposterior diameter on pelvic floor contraction at 36 weeks' gestation compared with women who had a spontaneous vaginal delivery (Tukey's post-hoc test, P = 0.033). CONCLUSIONS: Smaller levator hiatal dimensions on pelvic floor contraction during first pregnancy are associated with a subsequent instrumental vaginal delivery or a Cesarean section owing to failure to progress.

Measuring echogenicity and area of the puborectalis muscle: method and reliability.

OBJECTIVES: To develop a semi-automated method to assess puborectalis muscle echogenicity on three-dimensional/four-dimensional (3D/4D) volume transperineal ultrasound images using 4D View and Matlab® software and evaluate its intra- and interobserver reliability. METHOD: The data of 23 women in their first trimester were included. 3D/4D volume datasets were obtained at rest. Two inexperienced observers were trained by an experienced observer to construct tomographic ultrasound images (TUI) from the original data and to delineate all structures. Puborectalis muscle area (PMA) and the mean echogenicity of the puborectalis muscle (MEP) were calculated offline. Intra- and interobserver reliability were determined by intraclass correlation coefficients (ICC) and their 95% CIs. RESULTS: The development of a semi-automated method to calculate puborectalis area and echogenicity is described in detail. PMA and MEP measurements in pregnant women demonstrated almost perfect intraobserver reliability for both inexperienced observers, with ICC values ranging from 0.88 to 0.99. The interobserver reliability showed ICCs of 0.63 for PMA and almost perfect ICC values, of 0.96-0.98, for echogenicity. The majority of intraobserver mismatch between two delineations of PMA occurred near the borders. CONCLUSIONS: Matlab software can be used to provide reliable measurements of the area and echogenicity of the puborectalis muscle. As the latter can be used to assess structural changes in the puborectalis muscle, it appears a promising new tool for studying pelvic floor structural anatomy.

Diagnosing levator avulsions after first delivery by tomographic ultrasound: reliability between observers from different centers.

INTRODUCTION AND HYPOTHESIS: To evaluate the interobserver reliability of diagnosing levator avulsions between observers from different centers using tomographic ultrasound imaging (TUI) in women after their first delivery. METHODS: Transperineal ultrasound volume datasets of 40 women 6 months after their first delivery were analyzed by five observers from four different centers. Levator avulsions were diagnosed using TUI and datasets were assessed as optimal or suboptimal image quality and optimal or suboptimal pelvic floor contraction. Cohen's kappa was used to evaluate the interobserver reliability of diagnosing levator avulsions for the total group, the group with optimal and suboptimal image quality, and the group with optimal and suboptimal pelvic floor contraction. Consensus on the presence or absence of avulsions was scored according to the number of observers who diagnosed an avulsion (0 = consensus on the absence of avulsion, 1-4 = avulsion diagnosed by 1 to 4 observers, 5 = consensus on the presence of avulsion). RESULTS: For the total group, the interobserver reliability varied widely, with kappa values ranging from -0.07 to 0.72. Analyzes in the subgroups showed comparable results. Of the women who potentially have an avulsion (avulsion diagnosed by at least one observer), consensus on the presence of an avulsion was reached in 0.0 to 20.0 %. Of the women who potentially have no avulsion (no avulsion diagnosed by at least one observer), consensus on the absence of an avulsion was reached in 46.7 to 85.7 %. CONCLUSIONS: Diagnosing levator avulsions using TUI in women 6 months after their first delivery is strongly observer-dependent and therefore not generalizable.

PlGF-MMP9-engineered iPS cells supported on a PEG-fibrinogen hydrogel scaffold possess an enhanced capacity to repair damaged myocardium.

Cell-based regenerative therapies are significantly improved by engineering allografts to express factors that increase vascularization and engraftment, such as placental growth factor (PlGF) and matrix metalloproteinase 9 (MMP9). Moreover, the seeding of therapeutic cells onto a suitable scaffold is of utmost importance for tissue regeneration. On these premises, we sought to assess the reparative potential of induced pluripotent stem (iPS) cells bioengineered to secrete PlGF or MMP9 and delivered to infarcted myocardium upon a poly(ethylene glycol)-fibrinogen scaffold. When assessing optimal stiffness of the PEG-fibrinogen (PF) scaffold, we found that the appearance of contracting cells after cardiogenic induction was accelerated on the support designed with an intermediate stiffness. Revascularization and hemodynamic parameters of infarcted mouse heart were significantly improved by injection into the infarct of this optimized PF scaffold seeded with both MiPS (iPS cells engineered to secrete MMP9) and PiPS (iPS cells engineered to secrete PlGF) cells as compared with nonengineered cells or PF alone. Importantly, allograft-derived cells and host myocardium were functionally integrated. Therefore, survival and integration of allografts in the ischemic heart can be significantly improved with the use of therapeutic cells bioengineered to secrete MMP9 and PlGF and encapsulated within an injectable PF hydrogel having an optimized stiffness.

Ultrasound imaging of the pelvic floor: changes in anatomy during and after first pregnancy.

OBJECTIVES: To describe changes in the absolute values of levator hiatal dimensions and in the contractility and distensibility of the levator hiatus during pelvic floor contraction and Valsalva maneuver, using three/four-dimensional (3D/4D) transperineal ultrasound in women during and after their first pregnancy. METHODS: Two-hundred and eighty nulliparous pregnant women underwent ultrasound examination at 12 and 36 weeks' gestation and 6 months postpartum. Hiatal dimensions were measured at rest, on pelvic floor contraction and on Valsalva maneuver. The contractility and distensibility were determined by the difference between hiatal dimensions at rest and those on contraction or Valsalva, respectively. After exclusions, there were 231 datasets from women at rest, 199 for pelvic floor contraction and 230 for Valsalva maneuver. Data at 36 weeks' gestation and 6 months postpartum were compared with data at 12 weeks' gestation. RESULTS: At 36 weeks' gestation, the absolute values of hiatal dimensions and the contractility and distensibility of the levator hiatus were significantly increased compared with those at 12 weeks' gestation. Women who delivered vaginally showed a persistent significant increase in hiatal dimensions on Valsalva, whereas women who delivered by prelabor or first-stage Cesarean section showed no significant changes in hiatal dimensions on Valsalva. After both vaginal and Cesarean section delivery, there was a persistent increase in the distensibility of the hiatus during Valsalva compared with in early pregnancy. CONCLUSION: During first pregnancy, the absolute values of levator hiatal dimensions and the contractility and distensibility of the levator hiatus increase. Regardless of delivery mode, increased distensibility of the levator hiatus during Valsalva persists after childbirth. This increased pelvic floor distensibility may play a role in the development of pelvic floor dysfunction in later life.

Reliability of pelvic floor measurements on three- and four-dimensional ultrasound during and after first pregnancy: implications for training.

OBJECTIVES: To evaluate the reliability of measurements of the levator hiatus and levator-urethra gap (LUG) using three/four-dimensional (3D/4D) transperineal ultrasound in women during their first pregnancy and 6 months postpartum, and to assess the learning process for these measurements. METHODS: An inexperienced observer was taught to perform measurements of the levator hiatus and LUG by an experienced observer. After training, 3D/4D ultrasound volume datasets of 40 women in the first trimester were analyzed by these two observers. Another training session then took place and both observers repeated the analyses of the same volume datasets. Finally, analyses of 40 volume datasets of the women 6 months postpartum were performed by both observers. Intra- and interobserver reliability were determined by intraclass correlation coefficients (ICC) with 95% CIs. RESULTS: For levator hiatal measurements, in the women during their first pregnancy the interobserver reliability was substantial to almost perfect after both the first and second training session (ICC, 0.62-0.83 and 0.71-0.89, respectively, for anteroposterior diameter, transverse diameter and area at rest, on contraction and on Valsalva) and the intraobserver reliability was substantial to almost perfect for both observers. For these measurements performed once the women had delivered, interobserver reliability was moderate to almost perfect. For LUG measurements performed during pregnancy, interobserver reliability was slight to moderate after the first training session (ICC, 0.14-0.54), but improved after the second training session (ICC, 0.38-0.71), and intraobserver reliability was moderate to substantial for the experienced observer and slight to moderate for the inexperienced observer. For these measurements performed when the women had delivered, interobserver reliability was fair to moderate. CONCLUSIONS: The levator hiatus and LUG can be measured reliably using 3D/4D ultrasound in primigravid and primiparous women. The technique to measure dimensions of the levator hiatus requires limited teaching, but LUG measurements are more difficult and require more extensive training.

Associations between subjective overactive bladder symptoms and objective parameters on bladder diary and filling cystometry.

INTRODUCTION AND HYPOTHESIS: A study was conducted to assess associations between different overactive bladder (OAB) symptoms and their outcomes on bladder diary and filling cystometry parameters. METHODS: We performed a retrospective cohort study in database of 6,876 Urinary Distress Inventories, 3,185 bladder diaries and 2,153 filling cystometries from women referred to our urogynecological center between 2003 and 2009. Women were dichotomized into two groups. Group I: those women without symptoms, and those with symptoms that were not bothersome. Group II: women with bothersome symptoms. Data obtained from bladder diaries were: daytime urinary frequency, nocturnal frequency, minimum voided volume, maximum voided volume, average voided volume, and incontinence episodes. From filling cystometries, volumes at first desire to void, normal desire to void, strong desire to void and maximum cystometric capacity, were extracted. Univariate and multiple linear regression analysis were performed to determine associations between OAB symptoms and bladder diary and filling cystometry measurements. RESULTS: After multivariate analysis the objective daytime frequency was most strongly associated with the frequency symptom (ß 0.27, p < 0.05), night time frequency with the nocturia symptom (ß 0.40, p < 0.05) and the number of incontinence episodes with the urge incontinence symptom (ß 0.37, p < 0.05). Both frequency and nocturia symptoms were significantly associated with bladder diary and cystometry filling volumes, and their effect size was the same. The urgency symptom proved to be poorly associated with objective parameters. CONCLUSIONS: In contrast to the frequency and nocturia symptom, the urgency symptom is poorly associated with objective parameters on bladder diary and filling cystometry. Therefore, the current practice of using frequency and incontinence episodes in outcome research of OAB trials is justified.

Relation of risk factors and putative premotor markers for Parkinson's disease.

As both risk and premotor markers are increasingly discussed to play a key role in the pre-diagnostic phase of Parkinson's disease (PD) the aim of this study was to determine the relation between the risk factors hyperechogenicity of the substantia nigra (SN+) and/or positive family history of PD (faPD+) and putative premotor markers for PD. In a cross-sectional analysis of data of the PRIPS cohort, 1,149 volunteers older than 50 years free of PD were included. In addition to the risk factors SN+ and faPD+, olfactory dysfunction was tested using the Sniffin' sticks test and motor examination was performed. History of depression and constipation was evaluated by a semi-structured interview. Of all 1,149 individuals, 880 had none of the risk markers (76.6%), 143 persons (12.4%) had SN+, 84 (7.3%) were classified as faPD+ and 42 (3.7%) persons had both risk factors. Volunteers with SN+ showed olfactory dysfunction and mild motor impairment (p ≤ 0.001) more often. Depression was more prominent in individuals having two risk factors (p = 0.05). An accumulation of premotor markers was seen in the SN+ group with or without concomitant faPD+, but not in persons with faPD+ only. The profile of premotor markers seems to differ in participants having SN+ and/or faPD+, with SN+ showing the overall highest association with most premotor markers, which implies that SN+ might be a strong indicator for a neurodegenerative process.

Valsalva manoeuvre in patients with different Parkinsonian disorders.

The valsalva manoeuvre (VM), used as an autonomic function test, can detect sympathetic and/or parasympathetic autonomic dysfunction. This study investigated the value of VM in patients with different Parkinsonian syndromes (PS). We continuously recorded blood pressure, ECG and respiration among 38 patients with multiple system atrophy (MSA), 32 patients with progressive supranuclear palsy (PSP), 26 patients with idiopathic Parkinson's disease (PD) and in 27 healthy subjects matched in age and sex (Con). VM was performed in addition to metronomic breathing and tilt-table testing. VM could not be analysed in 26% of the ES patients. Valsalva ratio (VR), as a parameter of cardiovagal function, was pathologically decreased in all patient groups. Valsalva ratio (VR) was not able to discriminate parasympathetic dysfunction between patients and controls as well as E/I ratio of metronomic breathing. As a parameter of sympathetic dysfunction during VM, the physiological increase of blood pressure was more often missing during phase IV than phase II especially in PD and MSA patients. Correlation with orthostatic hypotension during tilt-table testing was only moderate. Although VM can demonstrate sympathetic and parasympathetic autonomic dysfunction, we cannot recommend VM as a first line autonomic test in PS patients. Metronomic breathing and tilt-table test seem more capable as parasympathetic resp. and sympathetic function tests to identify cardiovascular abnormalities in PS patients.

Baroreflex sensitivity and power spectral analysis in different extrapyramidal syndromes.

Cardiac autonomic abnormalities have been described in Parkinson's disease and other extrapyramidal syndromes. To investigate baroreflex sensitivity as an important risk marker of cardiovascular mortality in patients with Parkinson's disease and other extrapyramidal syndromes. We recorded continuously blood pressure, ECG and respiration in 35 patients with multiple system atrophy (MSA), 32 patients with progressive supranuclear palsy (PSP), 46 patients with idiopathic Parkinson's disease (PD) and in 27 corresponding healthy subjects (Con). Recordings of 2 min at rest were used to calculate baroreflex and spectral analysis of heart rate and systolic blood pressure. Resting baroreflex sensitivity (BRS) was significantly lower in the MSA and the PSP group but not in the PD group in comparison to the Con group. With increasing Hoehn & Yahr stage, BRS significantly decreased in all patient groups. In spectral analysis, all patient groups had a significantly lower relative low frequency (LF)-band power than the healthy controls. Patients with extrapyramidal disorders frequently demonstrate pathologically decreased BRS values and abnormalities of spectral analysis. This may have fundamental impact on the cardiovascular prognosis of patients with extrapyramidal disease.

Cross-sectional study discloses a positive family history for Parkinson's disease and male gender as epidemiological risk factors for substantia nigra hyperechogenicity.

Hyperechogenicity of the substantia nigra (SN) has been proposed to be a typical finding in Parkinson's disease (PD) and a marker of vulnerability to nigrostriatal dysfunction in healthy subjects. This large cross-sectional study including 1120 subjects older than 50 years without any signs of PD was performed to evaluate the association of SN hyperechogenicity and other proposed epidemiological risk factors for PD. Among all variables assessed only family history of PD and male gender proved to be significantly associated with SN hyperechogenicity, indicating a genetic predisposition for the ultrasound marker.

Pronounced short stature in a girl with tricho-rhino-phalangeal syndrome II (TRPS II, Langer-Giedion syndrome) and growth hormone deficiency.

We report on a 10-year-old girl with tricho-rhino-phalangeal syndrome type II (TRPS II) and pronounced short stature (-4.8 SD). The patient has an interstitial chromosome 8q24.1 deletion of 12-15 Mb. The deletion spans all genes from CSMD3 to at least ANXA13 including the TRPS1 and EXT1 genes, which are responsible for the TRPS II phenotype. In addition to the features of TRPS II, the patient had growth hormone (GH) deficiency with diminished response in three stimulation tests. Therapy with 0.2 mg GH/kg/week led to an increase of growth velocity from 2.5 to 6.6 cm/year. To our knowledge, such a combination of TRPS II and GH deficiency has not yet been described.

Midtrimester screening for microalbuminuria in healthy pregnant women.

To ascertain the effectiveness and feasibility of testing for microalbuminuria and the albumin/creatinine ratio as an early indication of hypertensive disorders of pregnancy, we measured albumin and creatinine excretion in 95 healthy pregnant women between 16 and 20 weeks of pregnancy. Nine women developed hypertensive complications; one of them became pre-eclamptic. There were no statistically significant differences in urine albumin and creatinine concentrations nor in the albumin/creatinine ratio between those women who developed pregnancy-induced hypertension and those who did not. Microalbuminuria testing had a specificity of 0.95 (95% CI: 0.88 - 0.99) and a sensitivity of 0.11 (95% CI: 0.03 - 0.48). The albumin/creatinine ratio had a specificity of 0.98 (95% CI: 0.92 - 1.0) and a sensitivity of 0.22 (95% CI: 0.03 - 0.6). The albumin/creatinine ratio was significantly lower in women who delivered prematurely. We conclude that mid-trimester testing for microalbuminuria and the measurement of the urinary albumin/creatinine ratio are not effective tools for the early recognition of pregnancy-induced hypertension in healthy pregnant women.

Assay for ADP-ribosyl cyclase by reverse-phase high-performance liquid chromatography.

Cyclic ADP-ribose (cADPR), a natural metabolite of beta-NAD(+), is a second messenger for Ca(2+) signaling in T cells. As a tool for purification and identification of ADP-ribosyl cyclase(s) in T cells, a sensitive and specific enzymatic assay using 1,N(6)-etheno-NAD(+) as substrate was developed. A major problem-the sensitivity of 1,N(6)-etheno-cADPR toward the extraction medium perchloric acid-was solved by replacing the perchloric acid extraction procedure of nucleotides by a filtration step. Standard compounds for the HPLC analysis of ADP-ribosyl cyclases and NAD(+)-glycohydrolases, e.g., 1,N(6)-etheno-cADPR, 1,N(6)-etheno-ADPR, and 1,N(6)-etheno-AMP, were produced by ADP-ribosyl cyclase from Aplysia californica and dinucleotide pyrophosphatase. The assay was applied to subcellular fractions prepared from human Jurkat T cells. As a result ADP-ribosyl cyclase and NAD(+)-glycohydrolase activity could be detected and precisely quantified in different subcellular fractions indicating the presence of different isoenzymes in T cells.