MicroRNAs as salivary markers for periodontal diseases

The aim of this review is to discuss current findings regarding the roles of miRNAs in periodontal diseases and the potential use of saliva as a diagnostic medium for corresponding miRNA investigations. For periodontal disease, investigations have been restricted to tissue samples and five miRNAs, that is, miR-142-3p, miR-146a, miR-155, miR-203, and miR-223, were repeatedly validated in vivo and in vitro by different validation methods. Particularly noticeable are the small sample sizes, different internal controls, and different case definitions of periodontitis in in vivo studies. Beside of that, the validated miRNAs are associated with inflammation and therefore with various diseases. Furthermore, several studies successfully explored the use of salivary miRNA species for the diagnosis of oral cancer. Different cancer types were investigated and heterogeneous methodology was used; moreover, no overlap of resultswas found. In conclusion, fivemiRNAs have consistently been reported for periodontitis; however, their disease specificity, detectability, and expression in saliva and their importance as noninvasive markers are questionable. In principle, a salivary miRNA diagnostic method seems feasible.However, standardized criteria and protocols for preanalytics, measurements, and analysis should be established to obtain comparable results across different studies

Mehrjährige Versuchsergebnisse zum Einfluss verschiedener Applikationstechniken auf die selektive Gräserkontrolle im Getreide

Die erfolgreiche Bekämpfung von Ackerfuchsschwanz (Alopecurus myosuroides) wird neben der Terminwahl, der Produktwahl und der Aufwandmenge auch von der richtigen Applikationstechnik beeinflusst. Hierzu wurden in den Jahren 2004 bis 2010 Klein- und Großparzellenversuche auf Praxisflächen angelegt. Zur Gräserkontrolle wurde Atlantis WG (Mesosulfuron & Iodosulfuron) mit verschiedenen Wasseraufwandmengen, Düsentypen und zu verschiedenen Tageszeiten ausgebracht.Die Tropfengröße sowie die Wasseraufwandmenge hatten dabei den stärksten Einfluss auf den Bekämpfungserfolg. Die Applikationen mit sehr groben Tropfen (90 % Drift-reduzierende Einstellung) führte zu Wirkungsminderungen. Dieses wird besonders bei Applikationen in den frühen Entwicklungsstadien vom Ackerfuchsschwanz sichtbar. Grobe Tropfen rollen schneller ab (Abrolleffekt). Dagegen führten fein- bis mitteltropfige Applikationen zu sicheren Bekämpfungserfolgen, aber sie können bei hohen Windgeschwindigkeiten und Lufttemperaturen zu Abdrift und Minderwirkung führen. Es wurde kein sichtbarer Unterschied zwischen den Düsentypen Standardflachstrahl, kompakte Injektordüse und Doppelflachstrahldüse festgestellt.Die Herbizidwirkungen wurden dagegen eindeutig durch die Blattfeuchte der Ungräser beeinflusst. Bei feuchten Blattoberflächen besteht die Gefahr des Abrollens von Tropfen. Dieses wurde durch höhere Wasseraufwandmengen (250 l/ha) noch verstärkt. Daher muss bei Früh- und Nachtspritzungen eine Anpassung der Wasseraufwandmengen vorgenommen werden. Das heißt bei Früh- und Nachtapplikationen mit Blattfeuchte sollte die Brüheaufwandmenge auf 100-150 l/ha abgesenkt werden. Es wird empfohlen, die 90 % Drift-reduzierende Einstellung der Düsen nur im sensiblen Randbereich zu fahren und durch Druckanpassung oder Düsenwechsel auf der Restfläche eine hohe Wirkungssicherheit von Atlantis WG zu garantieren.Aus den Versuchsergebnissen ergibt sich eine bevorzugte Düsenempfehlung für die Praxis: Kompakte Injektordüsen vom Typ AIXR, AirMix oder IDK sollten mit einem Kaliber 03 oder 04 bei ungefähr 3 bar Druck verwendet werden, sofern es keine weiteren anwendungsbezogenen Anwendungsbestimmungen gibt.Stichwörter: Abdriftreduzierung, Ackerfuchsschwanzbekämpfung, Düsentechnik, Herbizidwirkung, WasseraufwandmengeMultiannual results on the influence of different application techniques on the efficacy of selective grass control incerealsThe successful control of Alopecurus myosuroides (ALOMY) depends on the application date, the product selection and the dose rate and is also influenced by the correct application technology. Small and large plot trials were conducted at locations with heavy ALOMY infestation in the years 2004 - 2010. Atlantis WG (mesosulfuron & iodosulfuron) with different water dose rates, nozzle types and at different times of day was applied to control ALOMY in winter wheat.The droplet size as well as the water rate had the strongest impact on ALOMY control, while the application speed played a rather subordinated role. The applications with very coarse droplets (90 % drift-reducing potential) lead to efficacy reductions. This becomes particularly visible with applications in the early growth stages of ALOMY. Coarse drops roll off faster. On the other hand, applications with fine or medium droplets tend to result in a better control but they can lead to drift at high wind velocities and evaporation at high air temperatures and therefore cause lower efficacy.There was no visible difference between the types of the tested nozzle tips like standard flat jet, compact injector nozzle and double flat spray nozzle. The herbicidal efficacy was clearly affected by the moisture of the weed leaves. With moist leaf surfaces, the risk of run-off effects exists. This was more expressed with high spray volumes (250 l/ha) and has to be considered at early day or night applications. In situations of moist leaves, the spray volumes can be reduced to 100-150 l/ha. It is recommended to use the 90 % drift reducing nozzles only in the sensitive field bark area and to secure the efficacy of Atlantis WG against ALOMY by adjusting the pressure, the driving speed or by changing the nozzles outside of this sensitive area.From these test results, the following nozzle recommendation is concluded: Compact injector nozzles like AIXR,Air-mix, IDK should be used with a caliber of 03 or 04 at about 3 bar. Hereby the registration related buffer zone distances have to be considered.Keywords: Black grass control, drift reduction, herbicide efficacy, spray nozzle technology, spray volum

Genetic association of objective sleep phenotypes with a functional polymorphism in the neuropeptide S receptor gene

Background: The neuropeptide S receptor (NPSR1) and its ligand neuropeptide S (NPS) have received increased attention in the last few years, as both establish a previously unknown system of neuromodulation. Animal research studies have suggested that NPS may be involved in arousal/wakefulness and may also have a crucial role in sleep regulation. The single nucleotide polymorphism (SNP) rs324981 in NPSR1 has begun to shed light on a function of the NPS-system in human sleep regulation. Due to an amino acid exchange, the T-allele leads to an increased sensitivity of the NPSR1. In the only genomewide association study to date on circadian sleep parameters in humans, an association was found between rs324981 and regular bedtime. However, the sleep parameters in this study were only measured by self-rating. Therefore, our study aimed to replicate these findings using an objective measure of sleep. Methods: The study included n = 393 white subjects (62–79 years) who participated in an actigraphic assessment for determining sleep duration, rest duration, sleep onset, rest onset and sleep onset latency. Genotyping of the SNP rs324981 was performed using the TaqMan OpenArray System. Results: The genotype at rs324981 was not significantly associated with rest onset (bedtime) or sleep onset (p = .146 and p = .199, respectively). However, the SNP showed a significant effect on sleep- and rest duration (p = .007 and p = .003, respectively). Subjects that were homozygous for the minor T-allele had a significantly decreased sleep- and rest duration compared to A-allele carriers. Conclusion: The results of this study indicate that the sleep pattern in humans is influenced by the NPS-system. However, the previously reported association between bedtime and rs324981 could not be confirmed. The current finding of decreased sleep duration in T/T allele carriers is in accordance with studies in rodents reporting similar results after NPS application.:Background; Methods; Results; Conclusion

Validation of an apoptosis assay for extracorporeal photopheresis

Objectives This validation study investigated a flow cytometric apoptosis assay according to good manufacturing practice (GMP). Background Extracorporeal photopheresis (ECP) is a treatment for various immunological diseases and cutaneous T‐cell lymphomas. It is based on the induction of apoptosis by 8‐methoxypsoralene and ultraviolet A light. The quantification of apoptosis is therefore essential for ECP improvements. However, despite numerous publications on apoptosis, validated technical details are lacking. Methods and materials Mononuclear cells were collected by apheresis and treated by ECP or camptothecin. Samples taken before and after ECP were cultured for 24, 48 and 72 h and analysed for apoptosis and viability of T cells and monocytes by flow cytometry with Annexin V and 7‐AAD staining. Accuracy of the assay, intra‐ and inter‐assay precision and the pre‐analytical and analytical stability of the analytes were the investigated parameters. Results Our data indicate that the median intra‐ and inter‐assay precision coefficient of variation for T cells was 3.86% and 4.80%, respectively. Pre‐analytical stability of T cells and monocytes was ensured during short‐term storage for up to 2 h on ice. After staining, analytical stability was limited to 30 min, likely because of ongoing apoptosis and loss of monocytes due to plastic adhesion. Conclusion The results of this validation study show that the assay is GMP‐compliant and that its reliability, accuracy and precision are acceptable. While pre‐analytical stability of the cells was compatible with on‐site procedures, our analytical stability data indicate that this assay is not suited for batch mode analysis of ECP products

Biomarkers for Non-Invasive Stratification of Coronary Artery Disease and Prognostic Impact on Long-Term Survival in Patients with Stable Coronary Heart Disease

Knowledge about cardiac and inflammatory biomarkers in patients with stable coronary artery disease (CAD) is limited. To address this, we analyzed 3072 patients (36% female) with a median follow-up of 10 years in the Leipzig LIFE Heart Study with suspected CAD with coronary angiography. Selected biomarkers included troponin T (hsTNT), N-terminal pro B-type natriuretic peptide (NT-proBNP), copeptin, C-reactive protein (hsCRP), and interleukin-6 (IL-6). Patients were stratified by CAD severity: CAD0 (no sclerosis), CAD1 (non-obstructive, i.e., stenosis < 50%), and CAD2 (one stenosis 50%). Group comparison (GC) included GC1: CAD0 + 1 vs. CAD2; GC2: CAD0 vs. CAD1 + 2. CAD0, CAD1, and CAD2 were apparent in 1271, 631, and 1170 patients, respectively. Adjusted for classical risk factors, hs-cTnT, NT-proBNP, and IL-6 differed significantly in both GC and hsCRP only in GC2. After multivariate analysis, hs-cTnT, NT-proBNP, and IL-6 remained significant in GC1. In GC2, hs-cTnT (p < 0.001) and copeptin (p = 0.014) reached significance. Ten-year survival in groups CAD0, CAD1, and CAD2 was 88.3%, 77.3%, and 72.4%. Incorporation of hs-cTnT, NT-proBNP, copeptin, and IL-6 improved risk prediction (p < 0.001). The studied cardiac and inflammatory biomarkers enable fast and precise non-invasive identification of mortality risk in CAD patients, allowing the tailoring of primary and secondary CAD prevention

The Human Blood Transcriptome in a Large Population Cohort and Its Relation to Aging and Health

Background: The blood transcriptome is expected to provide a detailed picture of an organism’s physiological state with potential outcomes for applications in medical diagnostics and molecular and epidemiological research.We here present the analysis of blood specimens of 3,388 adult individuals, together with phenotype characteristics such as disease history, medication status, lifestyle factors, and body mass index (BMI). The size and heterogeneity of this data challenges analytics in terms of dimension reduction, knowledge mining, feature extraction, and data integration. Methods: Self-organizing maps (SOM)-machine learning was applied to study transcriptional states on a population-wide scale. This method permits a detailed description and visualization of the molecular heterogeneity of transcriptomes and of their association with different phenotypic features. Results: The diversity of transcriptomes is described by personalized SOM-portraits, which specify the samples in terms of modules of co-expressed genes of different functional context. We identified two major blood transcriptome types where type 1 was found more in men, the elderly, and overweight people and it upregulated genes associated with inflammation and increased heme metabolism, while type 2 was predominantly found in women, younger, and normal weight participants and it was associated with activated immune responses, transcriptional, ribosomal, mitochondrial, and telomere-maintenance cell-functions. We find a striking overlap of signatures shared by multiple diseases, aging, and obesity driven by an underlying common pattern, which was associated with the immune response and the increase of inflammatory processes. Conclusions: Machine learning applications for large and heterogeneous omics data provide a holistic view on the diversity of the human blood transcriptome. It provides a tool for comparative analyses of transcriptional signatures and of associated phenotypes in population studies and medical applications

Benchmarking One-Phase Lipid Extractions for Plasma Lipidomics

A key element of successful lipidomics analysis is a sufficient extraction of lipid molecules typically by two-phase systems such as chloroform-based Bligh and Dyer (B&D). However, numerous metabolomics and lipidomics studies today apply easy to use one-phase extractions. In this work, quantitative flow injection analysis high-resolution mass spectrometry was applied to benchmark the lipid recovery of popular one-phase extraction methods for human plasma samples. The following organic solvents were investigated: methanol (MeOH), ethanol (EtOH), 2-propanol (IPA), 1-butanol (BuOH), acetonitrile (ACN) and the solvent mixtures BuOH/MeOH (3:1) and MeOH/ACN (1:1). The recovery of polar lysophospholipids was sufficient for all tested solvents. However, nonpolar lipid classes such as triglycerides (TG) and cholesteryl esters (CE) revealed extraction efficiencies less than 5% due to precipitation in polar solvents EtOH, MeOH, MeOH/ACN, and ACN. Sample pellets also contained a substantial amount of phospholipids, for example, more than 75% of total phosphatidylcholine and sphingomyelin for ACN. The loss of lipids by precipitation was directly related to the polarity of solvents and lipid classes. Although, lipid recovery increased with the volume of organic solvent, recovery in polar MeOH remains incomplete also for less polar lipid classes such as ceramides. Addition of stable isotope-labeled internal standards prior to lipid extraction could compensate for insufficient lipid recovery for polar lipid classes including lysolipids and phospholipids but not for nonpolar CE and TG. In summary, application of one-phase extractions should be limited to polar lipid classes unless sufficient recovery/solubility of nonpolar lipids has been demonstrated. The presented data reveal that appropriate lipid extraction efficiency is fundamental to achieve accurate lipid quantification

Accurate Lipid Quantification of Tissue Homogenates Requires Suitable Sample Concentration, Solvent Composition, and Homogenization Procedure—A Case Study in Murine Liver

Lipidomics aim to quantify lipid species in all kinds of samples, including tissues. To subject a fixed amount of sample to various workflows, tissue homogenates were frequently prepared at defined concentrations in water or by addition of organic solvents. Here, we investigated this first step of tissue lipidomics by quantitative flow injection analysis coupled to Fourier-Transform mass spectrometry (FTMS). The influence of sample concentration, solvent composition, and homogenization procedure on the recovery of lipids was studied in murine liver. Liver homogenates were prepared either by grinding tissue in liquid nitrogen or by bead-based homogenization. Ground samples were dissolved at different concentrations in water, methanol, and water/methanol = 1/1 (v/v). Here, lipid recovery depends on solvent composition and sample concentration. The recovery of nonpolar lipid classes, including triglycerides and cholesteryl ester, was decreased in methanolic homogenates. In contrast, due to superior dispersion of precipitates, bead-based homogenization resulted in efficient lipid recovery independent of the solvent composition. However, lipid distribution within samples, i.e., lipid content of supernatant and pellet following centrifugation, was altered substantially by solvent composition. In conclusion, accurate lipid quantification of tissue homogenates requires evaluation of solvent composition, sample concentration, as well as the homogenization method to guarantee efficient lipid recovery. Due to a potential loss of lipids, removal of precipitates by centrifugation prior to lipid extraction should be avoided

Accurate quantification of lipid species affected by isobaric overlap in Fourier-Transform mass spectrometry

Lipidomics data require consideration of ions with near-identical masses, which comprises amongst others the Type-II isotopic overlap. This overlap occurs in series of lipid species differing only by number of double bonds (DB) mainly due to the natural abundance of 13C-atoms. High-resolution mass spectrometry, such as Fourier-Transform mass spectrometry (FTMS), is capable of resolving Type-II overlap depending on mass resolving power. In this work, we evaluated FTMS quantification accuracy of lipid species affected by Type-II overlap. Spike experiments with lipid species pairs of various lipid classes were analyzed by flow-injection-analysis (FIA)-FTMS. Accuracy of quantification was evaluated without and with Type-II correction (using relative isotope abundance) as well as utilizing the first isotopic peak (M+1). Isobaric peaks, which were sufficiently resolved, were most accurate without Type-II correction. In cases of partially resolved peaks, we observed peak interference causing distortions in mass and intensity, which is a well described phenomenon in FTMS. Concentrations of respective species were more accurate when calculated from M+1. Moreover, some minor species, affected by considerable Type-II overlap, could only be quantified by M+1. Unexpectedly, even completely unresolved peaks were substantially overcorrected by Type-II correction due to peak interference. The described method was validated including intra and inter-day precisions for human serum and fibroblast samples. Taken together, our results show that accurate quantification of lipid species by FTMS requires resolution-depended data analysis