Flow cytometry and FISH to measure the average length of telomeres (flow FISH)

Telomeres have emerged as crucial cellular elements in aging and various diseases including cancer. To measure the average length of telomere repeats in cells, we describe our protocols that use fluorescent in situ hybridization (FISH) with labeled peptide nucleic acid (PNA) probes specific for telomere repeats in combination with fluorescence measurements by flow cytometry (flow FISH). Flow FISH analysis can be performed using commercially available flow cytometers, and has the unique advantage over other methods for measuring telomere length of providing multi-parameter information on the length of telomere repeats in thousands of individual cells. The accuracy and reproducibility of the measurements is augmented by the automation of most pipetting (aspiration and dispensing) steps, and by including an internal standard (control cells) with a known telomere length in every tube. The basic protocol for the analysis of nucleated blood cells from 22 different individuals takes about 12 h spread over 2-3 days.

[Fixed-combination of losartan/hydrochlorothiazide 100 mg/25 mg. Tolerability and efficacy on blood pressure measured in the practice and for 24 hours]

BACKGROUND: A high proportion of patients with essential hypertension need a combination therapy to reach the therapeutic goal. In the present study, the tolerability and efficacy of a fixed, once daily combination of the AT1 blocker Losartan (100 mg) and the diuretic hydrochlorothiazide (HCTZ) (25 mg) for patients in the real-life situation was investigated. Special consideration was given to the results of ambulatory 24-hourblood pressure (ABP) measurements. METHODS: The open label, prospective non-interventional surveillance study took place from October 2005 to June 2006. A total of 1139 patients over 18 years in age were included whose blood pressures could not be adequately treated with HCTZ alone and for whom an individual dose titration for Losartan and HCTZ had already been performed. RESULTS: The average age (+/- standard deviation) of the patients was 61.2 +/- 11.6 years; 55.8% were men. Comorbidities were common. Specifically, left ventricular hypertrophy was present in 3.1% of the patients, coronary heart disease in 30.1%, chronic heart failure in 11.8% and status post myocardial infarction in 10.5%, respectively. In addition to the Losartan/HCTZ treatment, 61.0% of the patients received a second antihypertensive medicine. After an average treatment duration of 50.4 +/- 17.2 days, the base line systolic blood pressure of 160.8 +/- 16.3 mmHg decreased by 24.0 +/- 17.0 mmHg (-14.4%) and the diastolic blood pressure of 94.4 +/- 9.9 mmHg decreased by 11.8 +/- 10.2 mmHg (-11.8%). For the ABP measurements, the overall average systolic and diastolic blood pressures fell by 16.9 +/- 14.2 mmHg and 8.8 +/-10.3 mmHg, the day average by 17.3 +/- 14.8 mmHg and 9.0 +/- 10.2 mmHg and the night average by 15.1 +/- 17.6 mmHg and 7.8 +/- 11.7 mmHg, respectively. In twelve of the 1139 patients (1.1%), a total of 15 adverse events occurred. A causal connection with the medication was suspected in only in one case (one patient with three). CONCLUSION: The combination of Losartan/HCTZ 100/25 mg, as the exclusive therapy or in addition to other antihypertensive medicines, was for patients, many of whom who had comorbidities, in the real-life situation well tolerated and effective. The efficacy was demonstrated also during the night through ABP.

Estimating the mean and standard deviation of concentration distributions using censored samples

Environmental data sets of pollutant concentrations in air, water, and soil frequently include unquantified sample values reported only as being below the analytical method detection limit. These values, referred to as censored values, should be considered in the estimation of distribution parameters as each represents some value of pollutant concentration between zero and the detection limit. Most of the currently accepted methods for estimating the population parameters of environmental data sets containing censored values rely upon the assumption of an underlying normal (or transformed normal) distribution. This assumption can result in unacceptable levels of error in parameter estimation due to the unbounded left tail of the normal distribution. With the beta distribution, which is bounded by the same range of a distribution of concentrations, $\rm\lbrack0\le x\le1\rbrack,$ parameter estimation errors resulting from improper distribution bounds are avoided. This work developed a method that uses the beta distribution to estimate population parameters from censored environmental data sets and evaluated its performance in comparison to currently accepted methods that rely upon an underlying normal (or transformed normal) distribution. Data sets were generated assuming typical values encountered in environmental pollutant evaluation for mean, standard deviation, and number of variates. For each set of model values, data sets were generated assuming that the data was distributed either normally, lognormally, or according to a beta distribution. For varying levels of censoring, two established methods of parameter estimation, regression on normal ordered statistics, and regression on lognormal ordered statistics, were used to estimate the known mean and standard deviation of each data set. The method developed for this study, employing a beta distribution assumption, was also used to estimate parameters and the relative accuracy of all three methods were compared. For data sets of all three distribution types, and for censoring levels up to 50%, the performance of the new method equaled, if not exceeded, the performance of the two established methods. Because of its robustness in parameter estimation regardless of distribution type or censoring level, the method employing the beta distribution should be considered for full development in estimating parameters for censored environmental data sets. ^

(Tables 1-15) Morphometric characteristics of the genus Hinia (Nassariidae, Gastropoda) in the Tertiary of northwest Germany

The genus Hinia is divided in 4 subgenera; other subgenera are not represented in the area studied. It was possible to find criteria for a better discrimination of the highly variable species H. (Hinia) schlotheimi and H. (Hinia) turbinella. The species "fuchsi" has been placed in the synonymy of H. (Hinia) turbinella. The species H. (Hinia) schlotheimi (BEYRICH) and H. (Telasco) schroederi (KAUTSKY) have been united under the name H. (Hinia) schlotheimi. The easily distinguishable species H. (Tritonella) tenuistriata and H. (Hinia) sulcata belong to two different genera. H. (Tritonella) cimbrica andersoni of the Viol- and Katzheide-Beds (Reinbek-stage) is separable from the population found in the Hemmoor-stage, it turned out to be a valuable guide subspecies for the Reinbek-stage. The species H. (Tritonella) serraticosta, H. (Tritonella) catulli, H. (Hinia) holsatica, and H. (Telasco) syltensis are all similar in respect to shape and ornamentation. Criteria have been found for a better discrimination of these species. The species contabulata, effusa and seminodifera described by SPEYER (1864), turned out to be contogenetic stages of H. (Tritonella) pygmaea. H. (Tritonella) cavata, previously described from the Tertiary of the North sea area, was proven to be absent from the area investigated. The forms described under that name, belong to H. (Tritonella) woodwardi.

Constructing a stacked benthic d18O record

Composite stacks were constructed by superimposing 6 to 13 benthic foraminiferal d18O records covering the period 0-850 ka. An initial timescale for each core was established using radioisotopic age control points and assuming constant sedimentation rates between these points. The average of these records is our 13-core "untuned" stack. Next, we matched the 41 kyr component of each record individually to variations in Earth's obliquity. Four of the 13 records produced timescales that were inconsistent with one or more of the known radioisotopic ages. The nine remaining cores were averaged to create a "minimally tuned" stack. Six of the minimally tuned cores were assembled into a "tropical" stack. For each stack we estimated the uncertainty envelope from the standard deviation of the constituents. Spectral analysis of the three stacks indicates that benthic d18O is dominated by a 100 kyr oscillation that has a narrow spectral peak. The contribution of precession to the total variance is small when compared to prior results from planktic stacks.

(Table 1) Heat flow measurements in the Black Sea

New data show that the thermal field is definitely related to the geologic structure at depth and to other geophysical fields. Low heat-flow values along reliably established subsurface faults suggest absence of a heated zone of the earth's crust and upper mantle in these regions.

Tab. 1+2: C14-dates cf the older and younger emergence periods

In the neighbourhood of Oobloyah Bay various phenomena ean be eneountered whieh point to a ularge-seale uplift of shorelines, i.e. to an emergence of 200 m. Delta terraces, deltaic fan terraces and glacio-marine sands are regarded by the author as being the most reliable evidence of this. The marine limit documented by glacio-marine sand is to be found at ~170 m a.s.l. Hints of ancient shorelines located at a higher level exist only in the shape of badly preserved raised beaches. They were classified as less reliable records of past sea-levels, due to the lack of marine fossils and/or drift wood, and furthermore because those forms had been strongly influenced by periglacial processes. Deltaic deposits are of more importance in this context. The glacio-marine deltaic sands of several terrace levels contain terrestrial plant remnants which delivered C14dates. Using these dates und the relative elevations of terraces the emergenee of the area investigated could be recorded. This occured in a series of phases (and steps) which were summarized into two periods: an early period of emergenee which took place from at least 25 300 years B.P. to later than 17 340 years B.P. and a later one from at least 12 870 years B.P. up to the present day. The emergence seems to represent a discontinuous but regular sequence of relative sea level movements without intermittent submergence. Since the deltaic fans of the early emergence period were accumulated by sediments through glacio-fluvial channels of an adjacent glacier body the appropriate location of this glacial stage for one of the glaciers delivering meltwater (Nukapingwa Glacier) could be reconstructed. This stage of the glacier appears to belong to a retreating phase of the Mid-Wisconsin (?). The later period of emergence resulted in six rather glacio-marine delta terrace generations at the mouths of the main rivers with glaciofluvial regimen debouching into the Oobloyah Bay. A connection of this emergence with the glacial history of the field area is discussed. If one may rely on the age determinations of land derived plant fossils and their application for the climatic history of the area investigated, it must be concluded that the Heidelberg Valley, to a large extent, was alreaely deglaciated 25 000 years ago. The existence of a "Cockburn"-Phase in the sense of a major readvanee in Late Wisconsin times appears to be doubtful, or has been developed rather weakly.

Average fluorescence and dissolved iron and Fe-binding ligand characteristics during POLARSTERN expedition ANT-XXIV/3 in 2008

Organic complexation of dissolved iron (dFe) was investigated in the Atlantic sector of the Southern Ocean in order to understand the distribution of Fe over the whole water column. The total concentration of dissolved organic ligands ([Lt]) measured by voltammetry ranged between 0.54 and 1.84 nEq of M Fe whereas the conditional binding strength (K') ranged between 10**21.4 and 10**22.8. For the first time, trends in Fe-organic complexation were observed in an ocean basin by examining the ratio ([Lt]/[dFe]), defined as the organic ligand concentration divided by the dissolved Fe concentration. The [Lt]/[dFe] ratio indicates the saturation state of the natural ligands with Fe; a ratio near 1 means saturation of the ligands leading to precipitation of Fe. Reversely, high ratios mean Fe depletion and show a high potential for Fe solubilisation. In surface waters where phytoplankton is present low dissolved Fe and high variable ligand concentrations were found. Here the [Lt]/[dFe] ratio was on average 4.4. It was especially high (5.6-26.7) in the HNLC (High Nutrient, Low Chlorophyll) regions, where Fe was depleted. The [Lt]/[dFe] ratio decreased with depth due to increasing dissolved Fe concentrations and became constant below 450 m, indicating a steady state between ligand and Fe. Relatively low [Lt]/[dFe] ratios (between 1.1 and 2.7) existed in deep water north of the Southern Boundary, facilitating Fe precipitation. The [Lt]/[dFe] ratio increased southwards from the Southern Boundary on the Zero Meridian and from east to west in the Weddell Gyre due to changes both in ligand characteristics and in dissolved iron concentration. High [Lt]/[dFe] ratio expresses Fe depletion versus ligand production in the surface. The decrease with depth reflects the increase of [dFe] which favours scavenging and (co-) precipitation, whereas a horizontal increase in the deep waters results from an increasing distance from Fe sources. This increase in the [Lt]/[dFe] ratio at depth shows the very resistant nature of the dissolved organic ligands.