Boron in marine biogenic carbonates as a proxy for the carbonate system

This thesis has two focus points in the framework of boron in marine biogenic calcium carbonates and its potential as a proxy. Focus point one deals with potential factors, which might influence the boron isotopic composition and boron concentration in marine biogenic calcium carbonates. Since boron in marine biogenic calcium carbonates serves as a proxy for the reconstruction of the carbonate system, the awareness and characterization of potential impacts is of great importance. For that purpose investigations on 1) the effects of temperature and growth rate on the boron concentration and isotopic composition in calcium carbonate were investigated and 2) the potential carbonate system parameters controlling boron incorporation into shells of foraminifers have been determined. The second focus point introduces a new analytical technique which for the first time allows to determine the boron isotopic composition and boron concentration simultaneously in marine biogenic calcium carbonates. This is a great opportunity for applications in the field of paleoceanography and paleoclimatology since the determination of the boron isotopic composition and boron concentration provides two parameters of the carbonate system which are at least required to reconstruct it

On the Functional Analysis of Murine Peroxisomal Testis Specific 1 (Pxt1) Gene

Die Spermatogenese von Säugern ist ein komplexer biologischer Prozess bei dem diploide Spermatogonien in spezialisierte haploide Spermatozoen differenzieren. Die Regulation der männlichen Gametogenese ist verknüpft mit einem hoch organisierten Gen-Netzwerk, welches oft Keimzell-spezifische und/oder Stadien-spezifische Expression zeigt. Kürzlich konnte gezeigt werden, dass Peroxisomen eine essentielle Rolle während der Spermatogenese spielen. Über das murine peroxisomal testis specific 1 (Pxt1) Gen wurde erstmals 2007 berichtet (Grzmil et al., 2007). Zunächst wurde Pxt1 als ein neues, männliches Keimzell-spezifisches Gen beschrieben, welches für ein peroxisomales Protein codiert. In der vorliegenden Arbeit konnten neue Kenntnisse über die physiologischen Funktionen des PXT1 Proteins in der Spermatogenese, und damit auch über Peroxisomen, erhalten werden. Im ersten Teil dieser Arbeit wurde ein Interaktionspartner von PXT1, CCDC33, charakterisiert. Die testikuläre Expression des Ccdc33 Gens ist, ähnlich wie Pxt1, auf männliche Keimzellen beschränkt und beginnt in Pachytän-Spermatozyten. Weiterhin konnte gezeigt werden, dass es mindestens vier verschiedene Splicing-Transkripte des Ccdc33 Gens gibt. Basierend auf in silico Analysen wurden für das CCDC33 Protein zwei putative peroxisomale Zielsignale des Typs 2 (PTS2) vorausgesagt, von denen in dieser Arbeit eines der beiden PTS2 als funktionell charakterisiert werden konnte. Es ist verantwortlich für die Einsortierung des CCDC33-dsRED Fusionsproteins in die Peroxisomen. Die Kolokalisierung von PXT1 und CCDC33 in Vesikel-ähnlichen Strukturen bestätigt die peroxisomale Lokalisierung von CCDC33. Im zweiten Teil dieser Arbeit wurde über die Generierung und die Analyse von transgenen Mäusen berichtet, die PXT1 im Testis überexprimieren. Es konnte gezeigt werden, dass die Infertilität der transgenen Männchen auf eine ausgeprägte Apoptose der Pachytän-Spermatozyten zurück zuführen ist. Ausserdem konnte eine BH3-ähnliche Domäne in der PXT1 Proteinsequenz identifiziert werden, die charakteristisch für proapoptotische BCL-2 Familien-Mitglieder ist. Mit Hilfe von Deletionsanalysen konnte die Funktionalität dieser Domäne in transient transfizierten HeLa und NIH3T3 Zellen bestätigt werden. Zusätzlich konnte eine Interaktion von PXT1 mit dem Apoptose-Regulator BAT3 gezeigt und analysiert werden. In den Säugerzellen konnte die Überexpression von BAT3 die PXT1-induzierte Apoptose unterdrücken und führte zu einer Translokation von PXT1 aus dem Cytosol in den Nukleus. Ausserdem konnte die PXT1-CCDC33 Interaktion mit verschiedenen molekularen Techniken bestätigt werden. Darüber hinaus konnte in zwei PXT1 transgenen Linien gezeigt werden, dass die endogene 3 UTR von Pxt1 nicht in die verzögerte mRNA Translation involviert ist. Vorläufige Experimente zeigten, dass die durch PXT1 Überexpression induzierte Apoptose im Testis durch einen Caspase 9 unabhängigen Signalweg vermittelt wird, möglicherweise durch einen extrinsischen Signalweg. Zusammenfassend liefern die hier erhobenen Daten interessante Einsichten in die Funktion von PXT1 und Peroxisomen im Testis. Dennoch sind weitere Studien, wie zum Beispiel die Analyse von PXT1 knock-out Mäusen, notwendig, um herauszufinden, ob Pxt1 essentiell für die männliche Fertilität ist. Mithilfe der knock-out Mäuse können auch Erkenntnisse über den molekularen Mechanismus der PXT1-induzierten Apoptose gewonnen werden

Investigating the effects of growth rate and temperature on the B/Ca ratio and δ11B during inorganic calcite formation

To deconvolve the effect of growth rate and temperature on the boron partitioning into calcite and its isotope fractionation, seeded calcite precipitation experiments were performed at a constant temperature and various growth rates and at a constant growth rate and various temperatures. We show that boron partitioning increases with increasing growth rate and decreases with increasing temperature. The B isotope fractionation between calcite and B(OH)4− increases with increasing growth rate favoring the lighter B isotope for incorporation into calcite whereas no effect of temperature was observed within the temperature range investigated (12 °C to 32 °C). At the lowest temperature and growth rate δ11B of the calcite almost equals that of B(OH)4− in solution. Applying the surface entrapment model (SEMO) of Watson and Liang (1995) to our data, we demonstrate that the observed effects of temperature and growth rate on B concentration can be explained by processes in the near surface layer of the calcite crystal

Decoupled carbonate chemistry controls on the incorporation of boron into Orbulina universa

In order to fully constrain paleo-carbonate systems, proxies for two out of seven parameters, plus temperature and salinity, are required. The boron isotopic composition (δ11B) of planktonic foraminifera shells is a powerful tool for reconstructing changes in past surface ocean pH. As B(OH)4− is substituted into the biogenic calcite lattice in place of CO32−, and both borate and carbonate ions are more abundant at higher pH, it was suggested early on that B ∕ Ca ratios in biogenic calcite may serve as a proxy for [CO32−]. Although several recent studies have shown that a direct connection of B ∕ Ca to carbonate system parameters may be masked by other environmental factors in the field, there is ample evidence for a mechanistic relationship between B ∕ Ca and carbonate system parameters. Here, we focus on investigating the primary relationship to develop a mechanistic understanding of boron uptake. Differentiating between the effects of pH and [CO32−] is problematic, as they co-vary closely in natural systems, so the major control on boron incorporation remains unclear. To deconvolve the effects of pH and [CO32−] and to investigate their impact on the B ∕ Ca ratio and δ11B, we conducted culture experiments with the planktonic foraminifer Orbulina universa in manipulated culture media: constant pH (8.05), but changing [CO32−] (238, 286 and 534 µmol kg−1 CO32−) and at constant [CO32−] (276 ± 19.5 µmol kg−1) and varying pH (7.7, 7.9 and 8.05). Measurements of the isotopic composition of boron and the B ∕ Ca ratio were performed simultaneously using a femtosecond laser ablation system coupled to a MC-ICP-MS (multiple-collector inductively coupled plasma mass spectrometer). Our results show that, as expected, δ11B is controlled by pH but it is also modulated by [CO32−]. On the other hand, the B ∕ Ca ratio is driven by [HCO3−], independently of pH. This suggests that B ∕ Ca ratios in foraminiferal calcite can possibly be used as a second, independent, proxy for complete paleo-carbonate system reconstructions. This is discussed in light of recent literature demonstrating that the primary relationship between B ∕ Ca and [HCO3−] can be obscured by other environmental parameters

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Simultaneous determination of δ11B and B/Ca ratio in marine biogenic carbonates at nanogram level

In this study we introduce a new in situ technique which allows the determination of the boron isotopic composition and B/Ca ratios simultaneously at the nanogram level using a combination of optical emission spectroscopy and multiple ion counting MC ICP-MS with laser ablation. This technique offers a new application in the paleo-field of oceanography and climatology since small samples like e.g. single foraminiferal shells can be analyzed. The simultaneous determination of the boron isotopic composition and B/Ca ratios provides two independent proxies which allow the reconstruction of the full carbonate system. To test the new technique we performed measurements on the cultured, benthic foraminifer Amphistegina lessonii. Our results yielded an average boron isotopic composition δ11B = 18.0 ± 0.83‰ (SD) with an average internal precision of 0.52‰ (RSE). The boron concentration was 53 ± 7 μg/g (SD). These results agree with the range reported in the literature. The reconstructed mean pH value is in excellent agreement with the measured pH of the seawater in which the foraminifers grew. The analysis of a foraminifer consumed approximately 1200 ng calcium carbonate containing ca. 0.06 ng boron. Compared to bulk analytical methods, this new technique requires less material and reduces the time for sample preparation

The coordination and distribution of B in foraminiferal calcite

The isotopic ratio and concentration of B in foraminiferal calcite appear to reflect the pH and bicarbonate concentration of seawater. The use of B as a chemical proxy tracer has the potential to transform our understanding of the global carbon cycle, and ocean acidification processes. However, discrepancies between the theory underpinning the B proxies, and mineralogical observations of B coordination in biomineral carbonates call the basis of these proxies into question. Here, we use synchrotron X-ray spectromicroscopy to show that B is hosted solely as trigonal BO3 in the calcite test of Amphistegina lessonii, and that B concentration exhibits banding at the micron length scale. In contrast to previous results, our observation of trigonal B agrees with the predictions of the theoretical mechanism behind B palaeoproxies. These data strengthen the use of B for producing palaeo-pH records. The observation of systematic B heterogeneity, however, highlights the complexity of foraminiferal biomineralisation, implying that B incorporation is modulated by biological or crystal growth processes

Objective and Subjective Assessment of the Sound Attenuation Efficiency Obtained by Custom Moulded Earplugs with Various Acoustic Filters – a Preliminary Study

Background: Hearing loss caused by excessive exposure to noise is one of the most common health risks for employees. One solution for noise reduction is the use of hearing protectors, which is a very effective method for protecting hearing from the workplace noise. In order to obtain better attenuation efficiency, custom moulded earplugs can be equipped with a suitable acoustic filter. The effectiveness of the hearing protectors’ attenuation is based on real measurement of hearing thresholds for normal hearing people with and without hearing protectors. However, this is a time consuming process, and the obtained values are characterised by quite large inter-individual variability. The optimal solution is to measure the attenuation characteristics based on the objective method (without the presence of the subject), the results of which will be in accordance with the results of subjective tests. Therefore, the main purpose of the research in this work was to measure the attenuation characteristics of the self-designed custom moulded earplugs with and without acoustic filters through the use of subjective and objective methods, and to compare the results in terms of the research methods. Methods: Measurements of the acoustic attenuation obtained by custom moulded earplugs with designed F1, F2, and F3 acoustic filters (internal diameters dF1 = 1:25 mm, dF2 = 0:85 mm, and dF3 = 0:45 mm), as well as full insert earplugs (without any acoustic filters) were carried out using two methods: objective and subjective. The objective measurements were carried out in an anechoic chamber. The artificial head (High-frequency Head and Torso Simulator Brüel & Kjær Type 5128) was located at a distance of 3 m, directly opposite the loudspeaker. The test signal in the measurements was pink noise – in the frequency range up to 12.5 kHz and the level 85, 90, and 95 dB. The hearing protectors with and without acoustic filters were mounted in the Head and Torso Simulator which was connected with Pulse System Brüel & Kjær. Five normal hearing subjects participated in the subjective measurements. A pink noise signal was used for one-third octave bands: 125, 250, 500, 1000, 2000, 4000, and 8000 Hz. The attenuation value was defined as the difference (in dB) between the hearing threshold of the test signal with a hearing protector and the hearing threshold determined without a hearing protector. Results: The results of the objective method proved that in addition to the significant impact of frequency on the attenuation values, the type of filter used in custom moulded earplugs also had a significant effect. In addition, the results of the objective method showed that in the whole frequency range the highest attenuation values are shown by the full earplugs, achieving slightly above 45 dB for frequency of 8 kHz. The attenuation values obtained from subjective measurements also confirmed that both the frequency and type of filter significantly affect the attenuation values of the tested hearing protectors. Conclusions: The results of this study did not confirm the hypothesis that the measurement method had no significant effect on the attenuation characteristics of self-designed custom moulded earplugs with different types of acoustic filters. The largest differences in attenuation values between the type of measurement methods occur for the low frequency band (250 Hz) and for higher frequencies (4000 Hz mainly). The change of the internal diameter of the F1 filter from 1.25 mm to 0.85 mm (F2 filter) did not significantly affect the attenuation characteristics