Identification of sperm proteins as biomarkers of field fertility in Holstein-Friesian bulls used for artificial insemination.

Despite passing stringent quality control, bulls used in artificial insemination can vary significantly in their fertility, emphasizing the need for reliable markers of sperm quality. This study aimed to identify sperm proteins acting as biomarkers of fertility in 2 different populations of dairy bulls classified based on their field fertility. Semen was collected and cryopreserved from: 54 Holstein bulls located in Ireland, classified according to fertility indexes as low fertility (LF, n = 23), medium fertility (n = 14), or high fertility (HF, n = 17); and 18 Holstein bulls located in Denmark, classified as LF (n = 8) or HF (n = 10). The proteome was measured through liquid chromatography-mass spectrometry and data were analyzed with the R software. Differentially abundant proteins between HF and LF bulls and biomarker proteins were determined through a modified t-test and random forest, respectively, selecting 301 differentially abundant proteins and 34 biomarker proteins. The predictive ability of the 34 biomarkers was evaluated employing support vector machine as the classifier, using their abundance levels in the Irish bulls to train the model and in the Danish bulls for validation. The prediction accuracy was 94.4%, with only one HF bull misclassified, corresponding to the lowest fertility index bull in the HF group. The biomarkers more abundant in sperm of HF bulls enriched axoneme assembly and sperm motility (false discovery rate <0.05), according to functional analysis. In conclusion, a robust model coupled with the application of appropriate bioinformatic tools allowed the identification of functionally relevant sperm proteins predictive of the fertility of Holstein bulls used in artificial insemination.

Sleep macro- and microstructure in breast cancer survivors.

Complaints of sleep disturbance are prevalent among breast cancer (BC) patients and are predictors of quality of life. Still, electrophysiological measures of sleep are missing in patients, which prevents from understanding the pathophysiological consequences of cancer and its past treatments. Using polysomnography, sleep can be investigated in terms of macro- (e.g. awakenings, sleep stages) and micro- (i.e. cortical activity) structure. We aimed to characterize sleep complaints, and macro- and microstructure in 33 BC survivors untreated by chemotherapy and that had finished radiotherapy since at least 6 months (i.e. out of the acute effects of radiotherapy) compared to 21 healthy controls (HC). Compared to HC, BC patients had a larger number of awakenings (p = 0.008); and lower Delta power (p < 0.001), related to sleep deepening and homeostasis; greater both Alpha (p = 0.002) and Beta power (p < 0.001), related to arousal during deep sleep; and lower Theta power (p = 0.004), related to emotion regulation during dream sleep. Here we show that patients have increased cortical activity related to arousal and lower activity related to sleep homeostasis compared to controls. These results give additional insights in sleep pathophysiology of BC survivors and suggest sleep homeostasis disruption in non-advanced stages of BC.

Dietary obesity in mice is associated with lipid deposition and metabolic shifts in the lungs sharing features with the liver.

Obesity is associated with both chronic and acute respiratory illnesses, such as asthma, chronic obstructive pulmonary disease (COPD) or increased susceptibility to infectious diseases. Anatomical but also systemic and local metabolic alterations are proposed contributors to the pathophysiology of lung diseases in the context of obesity. To bring perspective to this discussion, we used NMR to compare the obesity-associated metabolomic profiles of the lung with those of the liver, heart, skeletal muscles, kidneys, brain and serum from male C57Bl/6J mice fed with a high-fat and high-sucrose (HFHSD) diet vs. standard (SD) chow for 14 weeks. Our results showed that the lung was the second most affected organ after the liver, and that the two organs shared reduced one-carbon (1C) metabolism and increased lipid accumulation. Altered 1C metabolism was found in all organs and in the serum, but serine levels were increased only in the lung of HFHSD compared to SD. Lastly, tricarboxylic acid (TCA)-derived metabolites were specifically and oppositely regulated in the serum and kidneys but not in other organs. Collectively, our data highlighted that HFHSD induced specific metabolic changes in all organs, the lung being the second most affected organ, the main alterations affecting metabolite concentrations of the 1C pathway and, to a minor extend, TCA. The absolute metabolite quantification performed in this study reveals some metabolic specificities affecting both the liver and the lung, that may reveal common metabolic determinants to the ongoing pathological process.

Driving performance and EEG fluctuations during on-the-road driving following sleep deprivation.

The present study mainly aimed to assess whether and how sleepiness due to sleep deprivation interacts with Time on Task (ToT) effects both on electroencephalography (EEG) measures and driving performance in real driving conditions. Healthy participants performed a one hour on-the-road monotonous highway driving task while EEG was recorded continuously after one night of normal sleep and after one night of total sleep deprivation. The main outcome parameter in the highway driving test was the Standard Deviation of Lateral Position (SDLP). SDLP and EEG indices (i.e alpha and theta power spectra) increased after sleep deprivation and varied with ToT. The latter was more pronounced after sleep deprivation. Beta power spectra did not differ between conditions but increased with ToT. Changes in SDLP and EEG did not correlate significantly. We conclude that driving performance as well as fatigue and sleepiness fluctuations with ToT were more evident after sleep deprivation as compared to normal sleep.

Ruminococcus gnavus E1 modulates mucin expression and intestinal glycosylation.

AIMS: The molecular cross-talk between commensal bacteria and the gut play an important role in the maintenance of the intestinal homeostasis and general health. Here, we studied the impact of a major Gram-positive anaerobic bacterium of the human gut microbiota, that is, Ruminococcus gnavus on the glycosylation pattern and the production of intestinal mucus by the goblet cells. METHODS AND RESULTS: Our results showed that R. gnavus E1 specifically increases the expression and the glycosylation level of the intestinal glyco-conjugates by goblet cells in the colonic mucosa of mono-associated mice with R. gnavus E1 as well as in human HT29-MTX cells. Such an effect was mediated through induction of the level of mRNA encoding for the major intestinal gel-forming mucin such as MUC2 and various glycosyltransferase enzymes. CONCLUSIONS: This study demonstrates for the first time that R. gnavus E1 possess the ability to modulate the glycosylation profile of the glyco-conjugate molecules and mucus in goblet cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Furthermore, we demonstrated that R. gnavus E1 modified specifically the glycosylation pattern and MUC2 expression by means of a small soluble factor of peptidic nature (<3 kDa) and heat stable in the HT29-MTX cell.

Functioning of the three attentional networks and vigilance in primary insomnia.

BACKGROUND: Results from cognitive measures in primary insomnia (PI) patients are not consistent with the difficulties in performing daily living tasks of which these patients complain about. Lack of sensitivity of the tests and the data concerning some cognitive functions may explain this discordance. The aim of the present investigation was to better characterize cognitive deficits of PI patients in order to further understand their cognitive complaints. We looked at attentional and executive function because of their high involvement in daily living tasks. METHODS: A total of 21 PI patients and 16 good sleepers completed the Attentional Network Test (ANT). We only included untreated PI patients since sleep medication could be a confounding factor when assessing cognition. RESULTS: While PI patients, compared to good sleepers, were found to have a longer overall reaction time (RT) and perform more slowly in the incongruent flanker condition (ie, conflict situation) than in the congruent condition, no group effects were observed for the variables representing the three attentional networks (ie, alerting, orienting, and executive functions). CONCLUSIONS: The present study revealed a conflict resolution deficit in untreated PI patients. This impairment may be linked to the prefrontal alterations reported in neuroimaging studies in these patients. Patients had also an impaired vigilance compared to good sleepers, likely due to the high cognitive load of the ANT. These results would serve to explain the complaints of PI patients about difficulties performing daily living tasks that are demanding and of long duration.

Electroencephalography during on-the-road driving in older untreated insomnia patients and normal sleepers.

Insomniacs report decreased performance in daily routines, which may have detrimental consequences for car driving. We compared changes over time in driving performance (measured as Standard Deviation of Lateral Position - SDLP) and background EEG between 20 untreated insomnia patients (52-70 years old) and 21 normal sleepers (54-73 years old) during a 1h on-the-road driving test after a normal night of sleep, in the morning. SDLP did not differ between groups and increased slightly over time to similar degrees in both groups. EEG alpha and beta power were lower in insomniacs as compared to normal sleepers. Alpha and beta power slightly reduced during driving in normal sleepers but remained at a constant low level in insomniacs. Changes in EEG power and SDLP were not related. It is concluded that on-the-road driving performance does not differ between older insomniacs and older normal sleepers and that changes in spectral EEG measures of cortical arousal and in driving performance are not related.

Effect of dose rate on residual γ-H2AX levels and frequency of micronuclei in X-irradiated mouse lymphocytes.

The biological risks associated with low-dose-rate (LDR) radiation exposures are not yet well defined. To assess the risk related to DNA damage, we compared the yields of two established biodosimetry end points, γ-H2AX and micronuclei (MNi), in peripheral mouse blood lymphocytes after prolonged in vivo exposure to LDR X rays (0.31 cGy/min) vs. acute high-dose-rate (HDR) exposure (1.03 Gy/min). C57BL/6 mice were total-body irradiated with 320 kVP X rays with doses of 0, 1.1, 2.2 and 4.45 Gy. Residual levels of total γ-H2AX fluorescence in lymphocytes isolated 24 h after the start of irradiation were assessed using indirect immunofluorescence methods. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to determine apoptotic cell frequency in lymphocytes sampled at 24 h. Curve fitting analysis suggested that the dose response for γ-H2AX yields after acute exposures could be described by a linear dependence. In contrast, a linear-quadratic dose-response shape was more appropriate for LDR exposure (perhaps reflecting differences in repair time after different LDR doses). Dose-rate sparing effects (P < 0.05) were observed at doses ≤2.2 Gy, such that the acute dose γ-H2AX and TUNEL-positive cell yields were significantly larger than the equivalent LDR yields. At the 4.45 Gy dose there was no difference in γ-H2AX expression between the two dose rates, whereas there was a two- to threefold increase in apoptosis in the LDR samples compared to the equivalent 4.45 Gy acute dose. Micronuclei yields were measured at 24 h and 7 days using the in vitro cytokinesis-blocked micronucleus (CBMN) assay. The results showed that MNi yields increased up to 2.2 Gy with no further increase at 4.45 Gy and with no detectable dose-rate effect across the dose range 24 h or 7 days post exposure. In conclusion, the γ-H2AX biomarker showed higher sensitivity to measure dose-rate effects after low-dose LDR X rays compared to MNi formation; however, confounding factors such as variable repair times post exposure, increased cell killing and cell cycle block likely contributed to the yields of MNi with accumulating doses of ionizing radiation.

The RABiT: high-throughput technology for assessing global DSB repair.

At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a rapid automated biodosimetry tool (RABiT); this is a completely automated, ultra-high-throughput robotically based biodosimetry workstation designed for use following a large-scale radiological event, to perform radiation biodosimetry measurements based on a fingerstick blood sample. High throughput is achieved through purpose built robotics, sample handling in filter-bottomed multi-well plates and innovations in high-speed imaging and analysis. Currently, we are adapting the RABiT technologies for use in laboratory settings, for applications in epidemiological and clinical studies. Our overall goal is to extend the RABiT system to directly measure the kinetics of DNA repair proteins. The design of the kinetic/time-dependent studies is based on repeated, automated sampling of lymphocytes from a central reservoir of cells housed in the RABiT incubator as a function of time after the irradiation challenge. In the present study, we have characterized the DNA repair kinetics of the following repair proteins: γ-H2AX, 53-BP1, ATM kinase, MDC1 at multiple times (0.5, 2, 4, 7 and 24 h) after irradiation with 4 Gy γ rays. In order to provide a consistent dose exposure at time zero, we have developed an automated capillary irradiator to introduce DNA DSBs into fingerstick-size blood samples within the RABiT. To demonstrate the scalability of the laboratory-based RABiT system, we have initiated a population study using γ-H2AX as a biomarker.

Coffin-Siris syndrome is a SWI/SNF complex disorder.

Coffin-Siris syndrome (CSS) is a congenital disorder characterized by intellectual disability, growth deficiency, microcephaly, coarse facial features, and hypoplastic or absent fifth fingernails and/or toenails. We previously reported that five genes are mutated in CSS, all of which encode subunits of the switch/sucrose non-fermenting (SWI/SNF) ATP-dependent chromatin-remodeling complex: SMARCB1, SMARCA4, SMARCE1, ARID1A, and ARID1B. In this study, we examined 49 newly recruited CSS-suspected patients, and re-examined three patients who did not show any mutations (using high-resolution melting analysis) in the previous study, by whole-exome sequencing or targeted resequencing. We found that SMARCB1, SMARCA4, or ARID1B were mutated in 20 patients. By examining available parental samples, we ascertained that 17 occurred de novo. All mutations in SMARCB1 and SMARCA4 were non-truncating (missense or in-frame deletion) whereas those in ARID1B were all truncating (nonsense or frameshift deletion/insertion) in this study as in our previous study. Our data further support that CSS is a SWI/SNF complex disorder.

Serum troponin Ic values in organ donors are related to donor myocardial dysfunction but not to graft dysfunction or rejection in the recipients.

BACKGROUND: Increased plasma cardiac troponin I (cTnI) values in heart donors are associated with donor myocardial dysfunction and increased risk of rejection in the recipients. We investigated the association between cTnI values and myocardial dysfunction in potential heart donors and the relationship between donors' cTnI values and recipients' early myocardial function and 1 year survival and risk of rejection. METHODS: cTnI was measured in 159 consecutive potential heart donors. Myocardial function was estimated by the left ventricular ejection fraction (LVEF) and segmental wall motion abnormalities (SWMA). Results are mean+/-SD (range) or median (interquartile range). RESULTS: cTnI values in potential donors were 2.1+/-5 ng/ml (0-40.4 ng/ml); cTnI values were significantly (P<0.001) higher: 4.2+/-5.9 ng/ml (0-30.6 ng/ml) for potential donors with LVEF <50% versus LVEF >50%: 1.7+/-4.7 ng/ml (0-40.4 ng/ml). cTnI values were significantly lower for donors without SWMA. cTnI values were significantly (P<0.001) lower for the 90 donors whose hearts were harvested: 1.1+/-2.3 ng/ml (0-15.6 ng/ml) versus the not harvested: 3.6+/-6.9 ng/ml (0-40.4 ng/ml). There were 87 recipients followed for 1 year. Donors' cTnI values were not associated with early alteration of LVEF, incidence of rejection or 1 year recipients' survival. CONCLUSION: Increased cTnI values in potential heart donors are statistically associated with myocardial dysfunction and could be helpful for organ selection. In contrast, cTnI values in heart donors were not associated with graft dysfunction or recipient survival after transplantation.

Relationship between procalcitonin values and infection in brain-dead organ donors.

BACKGROUND AND AIMS: An association between the inflammatory reactions estimated by several biomarkers and organ dysfunction has been reported in brain-dead organ donors (BDOD). Procalcitonin (PCT), a biomarker of inflammation due to bacterial infection, is increased among BDOD. However, is not known whether infection changes PCT values in BDOD. MATERIALS AND METHODS: We retrospectively analyzed 82 BDOD including several demographic and clinical parameters, bacterial culture results, antibiotics prescription, and plasma values of PCT measured before organ harvesting. Infection was diagnosed to be either a positive bacterial culture (restricted definition) and/or prescription of antibiotics (extended definition). RESULTS: The median PCT value was 1.5 (interquartile range [IQR], 0.4 to 6.9; range, 0 to 526 ng/mL; n=82). Thirty-eight (46%) and 24 (29%) patients had PCT values>2 ng/mL and >5 ng/mL, respectively. Median PCT values among infected (1.18; IQR, 0.27 to 6.55 ng/mL) versus noninfected (1.57; IQR, 0.53 to 7.15 ng/mL) BDOD (restricted definition) were not different (P=.36). The area under the receiver operating characteristic curve using PCT to predict infection (restricted definition) was 0.52. Specificity of PCT to predict infection was above 80% at PCT values>9 ng/mL. CONCLUSION: Our results confirmed PCT values are increased in BDOD, suggesting that this was not related to an infectious cause (whatever definition was used) unless PCT values are high.

Sequential measurements of troponin Ic values in brain-dead patients considered as potential heart donors.

It was suggested that a single value of normal or increased plasma cardiac troponin T or I (cTnT or cTnI) concentration could contribute to estimate donor myocardial damage and function in brain-dead patients. In patients with acute coronary syndromes, an initial normal value of troponin must be confirmed several hours later but no such recommendations exist for brain-dead patients. We investigated the relationship between two sequential (6 h interval) measurements of plasma cTnI concentrations in brain-dead patients considered as potential heart donors. The first and the second TnIc values were correlated with an adjusted r2 value of 0.92 (p<0.001). Our results suggest therefore that it is not necessary to repeat the measurements, when the value of plasma cTnI concentration is taken into consideration in the algorithm for cardiac harvesting.

Identification of the zinc binding ligands and the catalytic residue in human aspartoacylase, an enzyme involved in Canavan disease.

Canavan disease is an autosomal-recessive neurodegenerative disorder caused by a lack of aspartoacylase, the enzyme that degrades N-acetylaspartate (NAA) into acetate and aspartate. With a view to studying the mechanisms underlying the action of human aspartoacylase (hASP), this enzyme was expressed in a heterologous Escherichia coli system and characterized. The recombinant protein was found to have a molecular weight of 36 kDa and kinetic constants K(m) and k(cat) of 0.20 +/- 0.03 mM and 14.22 +/- 0.48 s(-1), respectively. Sequence alignment showed that this enzyme belongs to the carboxypeptidase metalloprotein family having the conserved motif H(21)xxE(24)(91aa)H(116). We further investigated the active site of hASP by performing modelling studies and site-directed mutagenesis. His21, Glu24 and His116 were identified here for the first time as the residues involved in the zinc-binding process. In addition, mutations involving the Glu178Gln and Glu178Asp residues resulted in the loss of enzyme activity. The finding that wild-type and Glu178Asp have the same K(m) but different k(cat) values confirms the idea that the carboxylate group contributes importantly to the enzymatic activity of aspartoacylase.

Aminoacylase I deficiency: a novel inborn error of metabolism.

This is the first report of a patient with aminoacylase I deficiency. High amounts of N-acetylated amino acids were detected by gas chromatography-mass spectrometry in the urine, including the derivatives of serine, glutamic acid, alanine, methionine, glycine, and smaller amounts of threonine, leucine, valine, and isoleucine. NMR spectroscopy confirmed these findings and, in addition, showed the presence of N-acetylglutamine and N-acetylasparagine. In EBV transformed lymphoblasts, aminoacylase I activity was deficient. Loss of activity was due to decreased amounts of aminoacylase I protein. The amount of mRNA for the aminoacylase I was decreased. DNA sequencing of the encoding ACY1 gene showed a homozygous c.1057 C>T transition, predicting a p.Arg353Cys substitution. Both parents were heterozygous for the mutation. The mutation was also detected in 5/161 controls. To exclude the possibility of a genetic polymorphism, protein expression studies were performed showing that the mutant protein had lost catalytic activity.

Maackiain 3-O-(6'-O-malonyl-beta-D-glucopyranoside) from Oudneya africana, a powerful inhibitor of porcine kidney acylase I.

The inhibitory effect of phenolic extracts of several plants from the Algerian Atlas used traditionally in Arab folk medicine was tested on the porcine kidney acylase I activity. An endemic Saharan plant of the Brassicaceae family, Oudneya africana, has shown a strong inhibitory effect. The active compound was isolated and purified by semi-preparative HPLC and HPLC-photodiode array detection, and structurally determined using 1H, 13C NMR and mass spectroscopy methods. Results indicate that maackiain 3-O-(6'-O-malonyl-beta-D-glucopyranoside) showed a competitive inhibition of porcine kidney acylase I with a Ki value of 11 microM. The malonyl moiety appeared to be a structural key element for the inhibitory activity. This observation indicates interesting structure-activity relationships for the inhibitory action of this compound on the acylase I and its potential role in the toxicity of haloalkene-derived mercapturates and that of the enzyme in detoxication and bioactivation.

Effect of flavonoids from various Mediterranean plants on enzymatic activity of intestinal carboxylesterase.

Flavonol compounds of three Mediterranean plants from the Algerian Atlas used traditionally in Arab folk medicine, Arenaria serpyllifolia, Rhamnus alaternus and Thapsia garganica, were found to inhibit the enzymatic activities of both rat intestine and purified porcine liver carboxylesterase in a concentration-dependent manner. Results indicate that the flavonol compounds from the aerial part of these plants lead to the inactivation of the CE pI = 5.1 with Ki of micromolar range. These results encourage us to perform further biological investigation.

[Analysis of the criteria that contribute to the decision to harvest the heart in brain-dead organ donors]. / Analyse des critères qui participent à la décision de prélèvement cardiaque chez les patients en état de mort encéphalique.

OBJECTIVES: The number of cardiac transplantation procedures does not increase because of the lack of donor hearts despite an increase in the number of brain-dead organ donors. The criteria used to select a donor heart are not formally standardized. The aim of the present study was to analyze the criteria that contribute to the selection of a donor heart. TYPE OF STUDY: Descriptive, retrospective study. PATIENTS AND METHOD: Clinical parameters, the initial causes that lead to brain death, maximum doses of catecholamines, several biochemical markers of myocardial ischaemia/necrosis as well as several echocardiography criteria were extracted from a prospectively collected database. Univariate and multivariate (logistic regression) analyses were performed with the "harvested heart" as dependent variable and the above-cited independent variables. RESULTS: One hundred and eighty consecutive brain-dead patients admitted from 1st October 1998 to 31st December 2000 out of which 112 gave at least one organ were analyzed. Among these 112 patients, 59 (39 males and 20 females) were pre-selected as potential heart donors. Only 44 hearts were harvested. Logistic regression analysis showed that harvesting of the heart was more probable if the donor were a male, had no left ventricle systolic wall motion abnormalities, had low doses of norepinephrine and low serum troponin Ic concentrations. CONCLUSION: After an initial phase of selection, the final decision to harvest a heart is based on several criteria. These results should be an incentive to conceive a score that could allow a more formal decision process for heart harvesting.

Transplantation in Waldenstrom's macroglobulinemia--the French experience.

Published data on transplantation in Waldenstrom's macroglobulinemia (WM) are still limited. We present a retrospective multicentric study of 27 WM patients who underwent 19 autologous (median age, 54 years) and 10 allogeneic (median age, 46 years) transplantations. Median time between diagnosis and transplantation was 36 months; 66% of patients had received three or more treatment lines and 72 % had chemosensitive disease. High-dose therapy (HDT) and autologous transplantation induced a 95% response rate (RR), including 10 major responses. With a median follow-up of 18 months, 12 patients are alive at 10 to 81 months and eight are free of disease progression at 10 to 34 months. The toxic mortality rate (TRM) was 6%. Allogeneic transplantation was preceded by HDT in nine patients and by a nonmyeloablative regimen in one patient. The RR was 80%, including seven major responses. With a median follow-up of 20.5 months, six patients are alive and free of progression at 3 to 76 months. Four patients died, all from toxicity, resulting in a TRM of 40%. HDT followed by autologous transplantation is feasible in WM, even in heavily pretreated patients, with some prolonged responses but a high relapse rate. Conversely, allogeneic transplantation is more toxic, but likely induces a graft-versus-WM effect and may, for some patients, result in long-term disease control.