Observational study on associations between resilience indicators based on daily milk yield in first lactation and lifetime profitability.

Resilience is the ability of cows to be minimally affected by disturbances, such as pathogens, heat waves, and changes in feed quality, or to quickly recover. Obvious advantages of resilience are good animal welfare and easy and pleasant management for farmers. Furthermore, economic effects are also expected, but these remain to be determined. The goal of this study was to investigate the association between resilience and lifetime gross margin, using indicators of resilience calculated from fluctuations in daily milk yield using an observational study. Resilience indicators and lifetime gross margin were calculated for 1,325 cows from 21 herds. These cows were not alive anymore and, therefore, had complete lifetime data available for many traits. The resilience indicators were the natural log-transformed variance (LnVar) and the lag-1 autocorrelation (rauto) of daily milk yield deviations from cow-specific lactation curves in parity 1. Good resilience is indicated by low LnVar (small yield response to disturbances) and low rauto (quick yield recovery to baseline). Lifetime gross margin was calculated as the sum of all revenues minus the sum of all costs throughout life. Included revenues were from milk, calf value, and slaughter of the cow. Included costs were from feed, rearing, insemination, management around calving, disease treatments, and destruction in case of death on farm. Feed intake was unknown and, therefore, lifetime feed costs had to be estimated based on milk yield records. The association of each resilience indicator with lifetime gross margin, and also with the underlying revenues and costs, was investigated using analysis of covariance (ANCOVA) models. Mean daily milk yield in first lactation, herd, and year of birth were included as covariates and factors. Natural log-transformed variance had a significantly negative association with lifetime gross margin, which means that cows with stable milk yield (low LnVar, good resilience) in parity 1 generated on average a higher lifetime gross margin than cows that had the same milk yield level but with more fluctuations. The association with lifetime gross margin could be mainly attributed to higher lifetime milk revenues for cows with low LnVar, due to a longer lifespan. Unlike LnVar, rauto was not significantly associated with lifetime gross margin or any of the underlying lifetime costs and revenues. However, it was significantly associated with yearly treatment costs, which is important for ease of management. In conclusion, the importance of resilience for total profit generated by a cow at the end of life was confirmed by the significant association of LnVar with lifetime gross margin, although effects of differences in feed efficiency between resilient and less resilient cows remain to be studied. The economic advantage can be mainly ascribed to benefits of long lifespan.

Rapid turnover of sensor data to genetic evaluation for dairy cows in the cloud.

More and more sensor and automation data are available that enable animal breeders to define novel traits. However, sensor and automation data are often frequently measured differently (e.g., milk yield and different milk components are continuously measured during each milking). These differences are challenging animal breeders to define traits and use the most appropriate analytical models for genetic evaluation and breeding values. Traditionally, the process from raw data to breeding value estimations involves several steps: data curation, trait definition, variance component estimation, genetic evaluation, and validation of the estimated breeding values (EBV). All these steps often take many iterations and several research projects to optimize the final genetic evaluations. To make this entire process-from raw data to validated EBV-more efficient, we combined all these steps in a cloud environment that allows for faster processing and a faster data distribution time. We used real data (including 1,782,373,113 daily milk-yield records of 1,120,550 dairy cows) and a real trait (a resilience trait based on the deviations from expected milk yields) to demonstrate the functioning of this cloud environment. The daily milk-yield records were incorporated into our cloud solution, in which we have set up central binary large object storage. Subsequent steps were all performed in the cloud. The data set was preprocessed in approximately 6 h to obtain the resilience indicator for 352,871 cows in the first 3 lactations. Estimation of genetic parameters (heritabilities and genetic correlations) was performed by splitting the data into 5 subsets in ASReml, and prediction of subsequent EBV was performed on the entire data set using MiXBLUP. Together with the validation of breeding values, this process encompassed 16.5 h. By combining the different steps from preprocessing sensor data to genetic evaluation of new traits in one cloud environment, we generated EBV and validation plots in approximately 1 working day. Moreover, our setup is a flexible design and can be adapted easily to test new, longitudinal sensor-driven traits and compare the performance of these new traits to previous ones.

Validation of resilience indicators by estimating genetic correlations among daughter groups and with yield responses to a heat wave and disturbances at herd level.

Resilient cows are minimally affected in their functioning by disturbances, and if affected, they quickly recover. Previously, the variance and autocorrelation of daily deviations from a lactation curve were proposed as resilience indicators. These traits were heritable and genetically associated with good health and longevity. However, it was unknown if selection for these indicators would lead to desired changes in the phenotype. The first aim of this study was to investigate if forward prediction of the resilience indicators in another environment was possible. Therefore, the resilience indicator records were split into 2 subsets, each containing half of the daughters of each sire, split within sire into cows that calved in early year-seasons and cows that calved in more recent year-seasons. Genetic correlations between the subsets were then estimated for each resilience indicator. The second aim was to estimate genetic correlations between the resilience indicators and traits describing production responses to actual disturbances. The disturbances were a heat wave in July 2015 and yield disturbances at herd level. The latter were selected by decreases in mean yield of all primiparous cows in a herd, indicating that a disturbance occurred. The data set used for calculation of the resilience indicators and the traits describing yield responses contained 62,932,794 daily milk yield records on 199,104 primiparous cows. Genetic correlations (rg) between recent and earlier daughter groups were 1 for both resilience indicators, which suggests that selection will result in changes in the phenotype in the next generation. Furthermore, low variance was genetically correlated with weak response in milk yield to both the heat wave and herd disturbances (rg 0.47 to 0.97). Low autocorrelation was genetically correlated with reduced perturbation length and quick recovery after the heat wave and herd disturbances (0.28 to 0.97). These results suggest that variance and autocorrelation cover different aspects of resilience, and should be combined in a resilience index. In conclusion, genetic selection for the resilience indicators will likely result in favorable changes in the traits themselves, and in response and recovery to actual disturbances, which confirms that they are useful resilience indicators.

Genetic analysis of resilience indicators based on milk yield records in different lactations and at different lactation stages.

Resilience is the ability of cows to cope with disturbances, such as pathogens or heat waves. To breed for improved resilience, it is important to know whether resilience genetically changes throughout life. Therefore, the aim was to perform a genetic analysis on 2 resilience indicators based on data from 3 periods of the first lactation (d 11-110, 111-210, and 211-340) and the first 3 full lactations, and to estimate genetic correlations with health traits. The resilience indicators were the natural log-transformed variance (LnVar) and lag-1 autocorrelation (rauto) of daily deviations in milk yield from an expected lactation curve. Low LnVar and rauto indicate low variability in daily milk yield and quick recovery, and were expected to indicate good resilience. Data of 200,084 first, 155,784 second, and 89,990 third lactations were used. Heritabilities were similar based on different lactation periods (0.12-0.15 for LnVar, 0.05-0.06 for rauto). However, the heritabilities of the resilience indicators based on full first lactation were higher than those based on lactation periods (0.20 for LnVar, 0.08 for rauto), due to lower residual variances. Heritabilities decreased from 0.20 in full lactation 1 to 0.19 in full lactation 3 for LnVar and from 0.08 to 0.06 for rauto. For LnVar, as well as for rauto, the strongest genetic correlation between lactation periods was between period 2 and 3 (0.97 for LnVar, 0.96 for rauto) and the weakest between period 1 and 3 (0.81 for LnVar, 0.65 for rauto). Similarly, for both traits the genetic correlation between full lactations was strongest between lactations 2 and 3 (0.99 for LnVar, 0.95 for rauto) and weakest between lactations 1 and 3 (0.91 for LnVar, 0.71 for rauto). For LnVar, genetic correlations with resilience-related traits, such as udder health, ketosis, and longevity, adjusted for correlations with milk yield, were almost always favorable (-0.59 to 0.02). In most cases these genetic correlations were stronger based on full lactations than on lactation periods. Genetic correlations were similar across full lactations, but the correlation with udder health increased substantially from -0.31 in lactation 1 to -0.51 in lactation 3. For rauto, genetic correlations with resilience-related traits were always favorable in lactation period 1 and in most full lactations, but not in the other lactation periods. However, correlations were weak (-0.27 to 0.15). Therefore, as a resilience indicator for breeding, LnVar is preferred over rauto. A multitrait index based on estimated breeding values for LnVar in lactations 1, 2, and 3 is recommended to improve resilience throughout the lifetime of a cow.

Between-herd variation in resilience and relations to herd performance.

Resilient cows are minimally affected in their functioning by infections and other disturbances, and recover quickly. Herd management is expected to have an effect on disturbances and the resilience of cows, and this effect was investigated in this study. Two resilience indicators were first recorded on individual cows. The effect of herd-year on these resilience indicators was then estimated and corrected for genetic and year-season effects. The 2 resilience indicators were the variance and the lag-1 autocorrelation of daily milk yield deviations from an expected lactation curve. Low variance and autocorrelation indicate that a cow does not fluctuate much around her expected milk yield and is, thus, subject to few disturbances, or little affected by disturbances (resilient). The herd-year estimates of the resilience indicators were estimated for 9,917 herd-year classes based on records of 227,655 primiparous cows from 2,644 herds. The herd-year estimates of the resilience indicators were then related to herd performance variables. Large differences in the herd-year estimates of the 2 resilience indicators (variance and autocorrelation) were observed between herd-years, indicating an effect of management on these traits. Furthermore, herd-year classes with a high variance tended to have a high proportion of cows with a rumen acidosis indication (r = 0.31), high SCS (r = 0.19), low fat content (r = -0.18), long calving interval (r = 0.14), low survival to second lactation (r = -0.13), large herd size (r = 0.12), low lactose content (r = -0.12), and high production (r = 0.10). These correlations support that herds with high variance are not resilient. The correlation between the variance and the proportion of cows with a rumen acidosis indication suggests that feed management may have an important effect on the variance. Herd-year classes with a high autocorrelation tended to have a high proportion of cows with a ketosis indication (r = 0.14) and a high production (r = 0.13), but a low somatic cell score (r = -0.17) and a low proportion of cows with a rumen acidosis indication (r = -0.12). These correlations suggest that high autocorrelation at herd level indicates either good or poor resilience, and is thus a poor resilience indicator. However, the combination of a high variance and a high autocorrelation is expected to indicate many fluctuations with slow recovery. In conclusion, herd management, in particular feed management, seems to affect herd resilience.

Exploration of variance, autocorrelation, and skewness of deviations from lactation curves as resilience indicators for breeding.

The ability of a cow to cope with environmental disturbances, such as pathogens or heat waves, is called resilience. To improve resilience through breeding, we need resilience indicators, which could be based on the fluctuation patterns in milk yield resulting from disturbances. The aim of this study was to explore 3 traits that describe fluctuations in milk yield as indicators for breeding resilient cows: the variance, autocorrelation, and skewness of the deviations from individual lactation curves. We used daily milk yield records of 198,754 first-parity cows, recorded by automatic milking systems. First, we estimated a lactation curve for each cow using 4 different methods: moving average, moving median, quantile regression, and Wilmink curve. We then calculated the log-transformed variance (LnVar), lag-1 autocorrelation (rauto), and skewness (Skew) of the daily deviations from these curves as resilience indicators. A genetic analysis of the resilience indicators was performed, and genetic correlations between resilience indicators and health, longevity, fertility, metabolic, and production traits were estimated. The heritabilities differed between LnVar (0.20 to 0.24), rauto (0.08 to 0.10), and Skew (0.01 to 0.02), and the genetic correlations among the indicators were weak to moderate. For rauto and Skew, genetic correlations with health, longevity, fertility, and metabolic traits were weak or the opposite of what we expected. Therefore, rauto and Skew have limited value as resilience indicators. However, lower LnVar was genetically associated with better udder health (genetic correlations from -0.22 to -0.32), better longevity (-0.28 to -0.34), less ketosis (-0.27 to -0.33), better fertility (-0.06 to -0.17), higher BCS (-0.29 to -0.40), and greater dry matter intake (-0.53 to -0.66) at the same level of milk yield. These correlations support LnVar as an indicator of resilience. Of all 4 curve-fitting methods, LnVar based on quantile regression systematically had the strongest genetic correlations with health, longevity, and fertility traits. Thus, quantile regression is considered the best curve-fitting method. In conclusion, LnVar based on deviations from a quantile regression curve is a promising resilience indicator that can be used to breed cows that are better at coping with disturbances.

Genetic analysis of udder conformation traits derived from automatic milking system recording in dairy cows.

Cartesian teat coordinates measured by automatic milking systems (AMS) provide new opportunities to record udder conformation traits and to study changes in udder conformation genetically and phenotypically within and between parities. The objective of this study was to estimate heritabilities and repeatabilities of AMS-based udder conformation traits within parities, to estimate genetic correlations between parities for AMS-based udder conformation traits, and to estimate genetic correlations between AMS-based udder conformation traits and classifier-based udder conformation traits, longevity, and udder health. Data from 70 herds, including 12,663 first-parity cows, 10,206 second-parity cows, and 7,627 third-parity cows, were analyzed using univariate and bivariate mixed animal models. Heritabilities of the AMS udder conformation traits were large (0.37-0.67) and genetic correlations between the AMS udder conformation traits and classifier-based traits were strong (>0.91). Repeatabilities within parities were large as well (0.89-0.97), indicating that a single record on udder conformation per lactation reflects udder conformation well. Genetic correlations of AMS udder conformation traits between parities were strong (0.88-1.00) and were stronger than the permanent environmental correlations. This shows that udder conformation changes over parities, but this change is mostly due to nongenetic factors. Based on these results, the current herd classification system, where cows are scored on udder conformation once in first parity, is sufficient. The AMS udder conformation traits as defined in this study have limited value as replacement for classifier-based udder conformation traits because they have smaller genetic correlations with functional traits than classifier-based traits. In summary, udder conformation hardly changes genetically between parities and is highly repeatable within parities. Udder conformation traits based on AMS need fine-tuning before they can replace classifier-based traits, and AMS teat coordinates probably contain additional information about udder health that is yet to be explored.

Assessing Restoration Effects on River Hydromorphology Using the Process-based Morphological Quality Index in Eight European River Reaches.

The Morphological Quality Index (MQI) and the Morphological Quality Index for monitoring (MQIm) have been applied to eight case studies across Europe with the objective of analyzing the hydromorphological response to various restoration measures and of comparing the results of the MQI and MQIm as a morphological assessment applied at the reach scale, with a conventional site scale physical-habitat assessment method. For each restored reach, the two indices were applied to the pre-restoration and post-restoration conditions. The restored reach was also compared to an adjacent, degraded reach. Results show that in all cases the restoration measures improved the morphological quality of the reach, but that the degree of improvement depends on many factors, including the initial morphological conditions, the length of the restored portion in relation to the reach length, and on the type of intervention. The comparison with a conventional site scale physical-habitat assessment method shows that the MQI and MQIm are best suited for the evaluation of restoration effects on river hydromorphology at the geomorphologically-relevant scale of the river reach.

The APPLE Tree programme: Active Prevention in People at risk of dementia through Lifestyle, bEhaviour change and Technology to build REsiliEnce-randomised controlled trial.

BACKGROUND: Large-scale trials of multidomain interventions show that modifying lifestyle and psychological risk factors can slow cognitive decline. We aim to determine if a lower intensity, personally tailored secondary dementia prevention programme for older people with subjective or mild objective memory decline, informed by behaviour change theory, reduces cognitive decline over 2 years. METHODS: A multi-site, single-blind randomised controlled trial recruiting 704 older adults at high dementia risk due to mild cognitive impairment (MCI) or subjective cognitive decline (SCD). Participants are randomised using 1:1 allocation ratio to the APPLE Tree intervention versus control arm (dementia prevention information), stratified by site. The intervention explores and implements strategies to promote healthy lifestyle, increase pleasurable activities and social connections and improve long-term condition self-management. Two facilitators trained and supervised by a clinical psychologist deliver ten, 1-h group video call sessions over 6 months (approximately every fortnight), video-call 'tea breaks' (less structured, facilitated social sessions) in intervening weeks and individual goal-setting phone calls every 2 weeks. From 6 to 12 months, participants meet monthly for 'tea breaks', with those not attending receiving monthly goal-setting phone calls. Participants receive a food delivery, pedometer and website access to cognitive training and information about lifestyle modification. Follow-ups for all outcome measures are at 12 and 24 months. The primary outcome is cognition (Neuropsychological Test Battery (NTB) score) at 24 months. Secondary outcomes are quality of life, cost per quality-adjusted life year (QALY) and wellbeing and lifestyle factors the intervention targets (diet, vascular risk, body weight, activity, sleep, anxiety, depression, social networks and loneliness, alcohol intake and smoking). Participants from purposively selected sites participate in qualitative process evaluation interviews, which will be analysed using thematic analytic methods. DISCUSSION: If effective, the intervention design, involving remote delivery and non-clinical facilitators, would facilitate intervention roll-out to older people with memory concerns. TRIAL REGISTRATION: ISRCTN17325135 . Registration date 27 November 2019.

Patients Unmet Needs in Chronic Rhinosinusitis With Nasal Polyps Care: A Patient Advisory Board Statement of EUFOREA.

Background: European patients with chronic rhinosinusitis with nasal polyps (CRSwNP) have had only limited occasions to unite to have their voices heard, hence missing the opportunity to contribute to the improvement of CRSwNP care. Aims: To identify unmet needs in CRSwNP from the perspective of CRSwNP patients from the Patient Advisory Board (PAB) of the European Forum for Research and Education in Allergy and Airways diseases (EUFOREA). Methodology: Semi-structured interviews were conducted individually with 15 European patients with CRSwNP and with a disease history of more than 2 years. Patients shared their burden of the disease and frustrations related to CRSwNP care, experiences with key pillars of current treatment options, shortcomings of the current care pathways and recommendations for improvement of care. A panel of 30 members of the Patient Advisory Board reviewed the interview report and provided further input during 2 virtual meetings. Results: CRSwNP patients indicated the need for greater awareness from society and physicians of the disease burden with impact on social function and well-being. Along with a loss of ability to smell and the continuous presence of secretions in the nose, most patients reported poor sleep quality and psychological impact as the most bothersome symptoms. Patients' frustrations relate primarily to the underestimation of the disease burden, the lack of coordination of care and the limited treatment options available to them. Treatment options with oral corticosteroids and/or sinus surgery both have positive and negative aspects, including the lack of long-lasting efficacy. Better coordination of care, more patient-centered care, greater public awareness, increases in research on the disease mechanisms and better therapeutic options would be warmly welcomed by CRSwNP patients. Conclusions: This statement of the EUFOREA Patient Advisory Board on CRSwNP provides novel insights on the underestimation of the burden of CRSwNP and shortcomings of current care. Multiple recommendations made by the patients can underpin action plans for implementation of better care for CRSwNP among all physicians treating patients with this disabling disease.

Corrigendum: Patients Unmet Needs in Chronic Rhinosinusitis With Nasal Polyps Care: A Patient Advisory Board Statement of EUFOREA.

[This corrects the article DOI: 10.3389/falgy.2021.761388.].

Time of out-of-hospital cardiac arrest is not associated with outcome in a metropolitan area: A multicenter cohort study.

AIM: Whether time of day influences survival after out-of-hospital cardiac arrest (OHCA) remains controversial. We compared outcomes after OHCA between day and night and explored whether characteristics of pre-hospital advanced life support (ALS)-quality varied by time of day. METHODS: We conducted a prospective cohort study of individuals that suffered a non-traumatic OHCA in the city of Vienna between August 2013 and August 2015 and who received resuscitative efforts by EMS. We compared clinical outcomes between day and night, defined as 7:00 pm-7:00 am based on EMS shift time including rates of sustained return of spontaneous circulation (ROSC), 30-day survival and favourable neurologic outcome (cerebral performance category 1 or 2). ALS quality measures included time to first medical contact, time to first shock, total dose of epinephrine, and multiple ALS performance measures. RESULTS: We included 1811 patients (37% female) with a mean age of 67 ± 16 years in our analyses. Rates of ROSC and 30-day survival with favourable neurological outcome did not differ between day or night (30% vs 28%, p =  0.33; 12% vs. 11%, p =  0.51, respectively). These results remained unchanged after multivariate adjustment for ROSC (RR, 1.1; 95% CI, 1.0-1.3, p = 0.19) and 30-day survival with favourable neurological outcome (RR, 1.2; 95% CI, 1.0-1.5, p =  0.10). The quality of ALS did not differ between day and night. CONCLUSIONS: In contrast to previous studies, there was no significant difference in sustained ROSC rates and 30-day survival with favourable neurological outcome after OHCA between day and night in the city of Vienna. This is likely due to nearly identical high bystander CPR rates and identical ALS performance provided by EMS personnel irrespective of time of the day.

Phosphorylation of Helicobacter pylori CagA by c-Abl leads to cell motility.

Helicobacter pylori induces a strong motogenic response in infected gastric epithelial host cells, which is enhanced by translocation of the pathogenic factor cytotoxin-associated gene A (CagA) into host cells via a specialized type IV secretion system. Once injected into the cytosol CagA is rapidly tyrosine phosphorylated by Src family kinases followed by Src inactivation. Hence, it remained unknown why CagA is constantly phosphorylated in sustained H. pylori infections to induce cell migration, whereas other substrates of Src kinases are dephosphorylated. Here, we identify the non-receptor tyrosine kinase c-Abl as a crucial mediator of H. pylori-induced migration and novel CagA kinase in epithelial cells. Upon H. pylori infection c-Abl directly interacts with CagA and localizes in focal adhesion complexes and membrane ruffles, which are highly dynamic cytoskeletal structures necessary for cell motility. Selective inhibition of c-Abl kinase activity by STI571 or shRNA abrogates sustained CagA phosphorylation and epithelial cell migration, indicating a pivotal role of c-Abl in H. pylori infection and pathogenicity. These results implicate c-Abl as a novel molecular target for therapeutic intervention in H. pylori-related gastric diseases.

The effects of commonly prescribed drugs in patients with Alzheimer's disease on the rate of deterioration.

BACKGROUND: Prescribed drugs in patients with Alzheimer's disease may affect the symptomatic progression of their disease, both positively and negatively. AIM: To examine the effects of drugs on the progression of disease in a representative group of patients with Alzheimer's disease. METHODS: Patients with the diagnosis of probable Alzheimer's disease were recruited from the community. The prescribed drugs taken by 224 patients (mean age 82.3 years) were recorded at initial assessment and then correlated in logistic regression analysis with progression of the disease, defined as an increase of one point or more in the Global Deterioration Scale over the next 12-month period. RESULTS: Patients who were taking antipsychotic drugs and sedatives had a significantly higher risk of deterioration than those who were taking none (odds ratios (ORs) 2.74 (95% confidence interval (CI) 1.17 to 6.41) and 2.77 (95% CI 1.14 to 6.73), respectively). Higher risk of deterioration was observed in those who were taking both antipsychotic and sedative drugs together (OR 3.86 (95% CI 1.28 to 11.7). Patients taking drugs licensed for dementia, drugs affecting the renin-angiotensin system and statins had a significantly lower risk of deterioration than those who were not taking any of these drugs (ORs 0.49 (95% CI 0.25 to 0.97), 0.31 (95% CI 0.11 to 0.85) and 0.12 (95% CI 0.03 to 0.52), respectively). CONCLUSION: Our findings have implications for both clinicians and trialists. Most importantly, clinicians should carefully weigh any potential benefits of antipsychotics and benzodiazepines, especially in combination, against the risk of increased decline. Researchers need to be aware of the potential of not only licensed drugs for dementia but also drugs affecting the renin-angiotensin system and statins in reducing progression in clinical trials.

Proteome-based plasma biomarkers for Alzheimer's disease.

Alzheimer's disease is a common and devastating disease for which there is no readily available biomarker to aid diagnosis or to monitor disease progression. Biomarkers have been sought in CSF but no previous study has used two-dimensional gel electrophoresis coupled with mass spectrometry to seek biomarkers in peripheral tissue. We performed a case-control study of plasma using this proteomics approach to identify proteins that differ in the disease state relative to aged controls. For discovery-phase proteomics analysis, 50 people with Alzheimer's dementia were recruited through secondary services and 50 normal elderly controls through primary care. For validation purposes a total of 511 subjects with Alzheimer's disease and other neurodegenerative diseases and normal elderly controls were examined. Image analysis of the protein distribution of the gels alone identifies disease cases with 56% sensitivity and 80% specificity. Mass spectrometric analysis of the changes observed in two-dimensional electrophoresis identified a number of proteins previously implicated in the disease pathology, including complement factor H (CFH) precursor and alpha-2-macroglobulin (alpha-2M). Using semi-quantitative immunoblotting, the elevation of CFH and alpha-2M was shown to be specific for Alzheimer's disease and to correlate with disease severity although alternative assays would be necessary to improve sensitivity and specificity. These findings suggest that blood may be a rich source for biomarkers of Alzheimer's disease and that CFH, together with other proteins such as alpha-2M may be a specific markers of this illness.

Dissipation of potassium and proton gradients inhibits mitochondrial hyperpolarization and cytochrome c release during neural apoptosis.

Exposure of rat hippocampal neurons or human D283 medulloblastoma cells to the apoptosis-inducing kinase inhibitor staurosporine induced rapid cytochrome c release from mitochondria and activation of the executioner caspase-3. Measurements of cellular tetramethylrhodamine ethyl ester fluorescence and subsequent simulation of fluorescence changes based on Nernst calculations of fluorescence in the extracellular, cytoplasmic, and mitochondrial compartments revealed that the release of cytochrome c was preceded by mitochondrial hyperpolarization. Overexpression of the anti-apoptotic protein Bcl-xL, but not pharmacological blockade of outward potassium currents, inhibited staurosporine-induced hyperpolarization and apoptosis. Dissipation of mitochondrial potassium and proton gradients by valinomycin or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone also potently inhibited staurosporine-induced hyperpolarization, cytochrome c release, and caspase activation. This effect was not attributable to changes in cellular ATP levels. Prolonged exposure to valinomycin induced significant matrix swelling, and per se also caused release of cytochrome c from mitochondria. In contrast to staurosporine, however, valinomycin-induced cytochrome c release and cell death were not associated with caspase-3 activation and insensitive to Bcl-xL overexpression. Our data suggest two distinct mechanisms for mitochondrial cytochrome c release: (1) active cytochrome c release associated with early mitochondrial hyperpolarization, leading to neuronal apoptosis, and (2) passive cytochrome c release secondary to mitochondrial depolarization and matrix swelling.

Up-regulation of Bcl-xL in response to subtoxic beta-amyloid: role in neuronal resistance against apoptotic and oxidative injury.

Neuron death in Alzheimer's disease is believed to be triggered by an increased production of amyloidogenic beta-amyloid peptides, involving both increased oxidative stress and activation of a conserved death program. Bcl-xL, an anti-apoptotic protein of the Bcl-2 family, is expressed at high levels in the adult nervous system. Exposure of neuronal cultures to subtoxic concentrations of beta-amyloid peptide 1-40 (1-10microM) or the fragment 25-35 (1-10microM) up-regulated both bcl-xL mRNA and Bcl-xL protein levels, determined by reverse transcriptase-polymerase chain reaction and western blot analysis. Bcl-xL protein was also up-regulated during oxidative stress induced by exposure to hydrogen peroxide (3-100microM) or ferric ions (1-10microM). In contrast, apoptotic stimuli (exposure to staurosporine or serum withdrawal) actually decreased neuronal Bcl-xL expression. To investigate the role of Bcl-xL in cell death relevant to Alzheimer's disease, we stably overexpressed Bcl-xL in human SH-SY5Y neuroblastoma cells. Cells overexpressing Bcl-xL were significantly protected from beta-amyloid neurotoxicity and staurosporine-induced apoptosis compared to vector-transfected controls. In contrast, Bcl-xL overexpression only conferred a mild protection against oxidative injury induced by hydrogen peroxide. We conclude that up-regulation of Bcl-xL expression in response to subtoxic concentrations of beta-amyloid is a stress response that increases the resistance of neurons to beta-amyloid neurotoxicity primarily by inhibiting apoptotic processes.

Use of PCR to screen for promoter elements in genomic DNA library clones.

We report a modified PCR strategy to screen for promoter elements of genes of interest that is based upon consecutive rounds of PCR and appropriate subcloning. Following preliminary identification and sequencing of intron 1 by standardized PCR, the application of a suited cDNA/intron primer combination renders a succeeding PCR-mediated screening of cosmid or P1-derived artificial chromosome (PAC) libraries possible, thus identifying genomic clones comprising the searched promoter elements. We tested our approach in comparison with a commercially available promoter finder kit by searching the promoter elements of the CENP-C gene from the human and mouse genomes. Applying the kit system, we amplified the anticipated promoter from mouse, but failed in isolating human promoter elements. Our approach made use of a 5'-UTR/intron 1 primer combination in the second round of PCR, enabling the identification of positive clones from genomic DNA within a human PAC library possible. Subcloning and final PCR amplification revealed the successful isolation of the human promoter. Therefore, we conclude that our approach might represent a helpful alternative to identify promoter elements, especially when prior art genome walking, STS-based strategies or anchored PCR failed.

Proliferation and differentiation of osteoblasts on Biocement D modified with collagen type I and citric acid.

In this study, the proliferation and differentiation of rat calvarial osteoblasts cultured on either (1) calcium-phosphate bone cement Biocement D, (2) Biocement D with 2.5% (w/w) mineralized collagen type I, or (3) Biocement D with 2.5% (w/w) mineralized collagen type I and 3% (w/w) citric acid were investigated. Incubation of the composites in cell-culture medium resulted in a fast decrease of pH and calcium concentration as well as in an increase of phosphate concentration. Although these effects occurred with all investigated materials, the lowest extent could be observed for the citric-acid-containing composites. As shown by scanning-electron microscopy, osteoblasts adhered to the composite surfaces. Proliferation and differentiation of the cells grown on the composites were found to be reduced compared to cells grown on tissue-culture polystyrene. Cells cultured in the vicinity of the composites but without direct contact also exhibited a reduced rate of proliferation, reduced alkaline phosphatase activity, and reduced mineralization. Simulating the changes in calcium and phosphate concentration occasioned by the composites through exposing cells to EGTA and phosphate gives rise to the same effects of reducing proliferation, ALP activity, and mineralization. No indication for apoptosis in cells exposed to low calcium and high phosphate concentrations was found. The number of necrotic cells, however, increased after incubation with EGTA and phosphate. For assessment of cell-composite interactions and the success of the composites in vivo, as well as for more effective material development, it seems to be important to know how changes in microenvironmental pH and ion composition of the material affect cellular proliferation and differentiation.

The effect of self-presentation on social value orientation.

Social-value orientations in interdependence situations are expected to be influenced by self-presentational concerns because in contrast to proself orientations (individualism and competition), prosocial orientations (cooperation, altruism, equality, and maximin) tend to make a more positive impression on others. In the present study, the influence of self-presentation on social orientation was inhibited by means of a bogus pipeline procedure. Dutch University students in a bogus pipeline condition displayed less prosocial and more proself orientations than students in a condition in which no bogus pipeline was used.