Metal Exposure and Sex Shape the Fatty Acid Profile of Midges and Reduce the Aquatic Subsidy to Terrestrial Food Webs.

Aquatic micropollutants can be transported to terrestrial systems and their consumers by emergent aquatic insects. However, micropollutants, such as metals, may also affect the flux of physiologically important polyunsaturated fatty acids (PUFAs). As certain PUFAs have been linked to physiological fitness and breeding success of terrestrial consumers, reduced fluxes from aquatic systems could affect terrestrial populations and food webs. We chronically exposed larvae of the aquatic insect Chironomus riparius to a range of environmentally relevant sediment contents of cadmium (Cd) or copper (Cu) in a 28-day microcosm study. Since elevated water temperatures can enhance metals' toxic effects, we used two temperature regimes, control and periodically elevated temperatures (heat waves) reflecting an aspect of climate change. Cd and Cu significantly reduced adult emergence by up to 95% and 45%, respectively, while elevated temperatures had negligible effects. Both metal contents were strongly reduced (∼90%) during metamorphosis. Furthermore, the chironomid FA profile was significantly altered during metamorphosis with the factors sex and metal exposure being relevant predictors. Consequently, fluxes of physiologically important PUFAs by emergent adults were reduced by up to ∼80%. Our results suggest that considering fluxes of physiologically important compounds, such as PUFAs, by emergent aquatic insects is important to understand the implications of aquatic micropollutants on aquatic-terrestrial meta-ecosystems.

Insights into the translational and rotational dynamics of cations and anions in protic ionic liquids by means of NMR fast-field-cycling relaxometry.

Understanding the translational and rotational dynamics of cations and anions in hydrogen bonded protic ionic liquids (PIls) is still a challenge. In this study, we determine self-diffusion coefficients and rotational correlation times of both ions in triethylammonium based PILs by means of NMR Fast-Field-Cycling (FFC) relaxometry. Global fits of 1H and 19F nuclear magnetic relaxation dispersion (NMRD) curves allowed proper separation into intra and inter molecular relaxation rates for both NMR sensitive nuclei and thus a reliable description of translational and rotational motion for both ions individually. The diffusion coefficients of the cations are in the order of 6 × 10-11 m2 s-1 at room temperature and about 50 per cent larger than those of the anions. The diffusion coefficients of cations and anions in both PILs were compared with those we derived from applying an universal dispersion power law and those known from pulsed field gradient (PFG) NMR studies. Considering the Nernst-Einstein equation, molar conductivities were calculated from cationic and anionic diffusion coefficients and related to directly measured molar conductivities, allowing the determination of the degree of dissociation. The rotational correlation times τR ranging from 50 ps up to 2 ns as a function of temperature were compared with those obtained from high-field NMR quadrupolar relaxation time measurements addressing explicitly the rotation of the NH vector and giving insights into the acidic proton mobility. The Stokes-Einstein and Stokes-Einstein-Debye relations were applied to relate the diffusion coefficients and rotational correlation times to the macroscopic bulk viscosity. The results were also discussed with respect to the archetypical PIL ethylammonium nitrate.

Facile Synthesis of a Stable Side-on Phosphinyne Complex by Redox Driven Intramolecular Cyclisation.

Alkyne complexes with vicinal substitution by a Lewis acid and a Lewis base at the coordinated alkyne are prospective frustrated Lewis pairs exhibiting a particular mutual distance and, hence, a specific activation potential. In this contribution, investigations on the generation of a WII alkyne complex bearing a phosphine as Lewis base and a carbenium group as Lewis acid are presented. Independently on potential substrates added, an intramolecular cyclisation product was always isolated. A subsequent deprotonation step led to an unprecedented side-on λ5 -phosphinyne complex, which is interpreted as highly zwitterionic according to visible absorption spectroscopy supported by TD-DFT. Low-temperature 31 P NMR and EPR spectroscopic measurements combined with time-dependent IR-spectroscopic monitoring provided insights in the mechanism of the cyclisation reaction. Decomposition of the multicomponent IR spectra by multivariate curve resolution and a kinetic hard-modelling approach allowed the derivation of kinetic parameters. Assignment of the individual IR spectra to potential intermediates was provided by DFT calculations.

UV Sensor Based on Fiber Bragg Grating Covered with Graphene Oxide Embedded in Composite Materials.

Polymer-matrix composites degrade under the influence of UV radiation in the range of the 290-400 nm band. The degradation of polymer-matrix composites exposed to UV radiation is characterized by extensive aging of the epoxy matrix, resulting in deterioration of their mechanical properties. Glass fibers/epoxy resin composites were made by an out-of-autoclave method whereas a fiber optic sensor was placed between different layers of laminates. In our work, we used a fiber Bragg grating sensor covered with graphene oxide and embedded in a polymer matrix composite to monitor UV radiation intensity. Measurements of UV radiation may allow monitoring the aging process of individual components of the polymer composite. In order to estimate the number of microcracks of epoxy resin, microstructure observations were carried out using a scanning electron microscope.

Display glass for low-loss and high-density optical interconnects in electro-optical circuit boards with eight optical layers.

Parallel optical interconnects on-board level requires low propagation loss in wavelength range between 850 and 1550 nm to be compatible with datacom and telecom optical engines. For highest integration density tight waveguide bends and a scalable number of optical layers should be manufacturable for 2D interfaces to optical fiber array connectors and photonic assembly I/O's. We developed a glass waveguide panel process for double-sided processing of commercial available display glass by applying a two-step thermal ion-exchange process for low-loss multi-mode graded-index waveguides. Multiple glass waveguide panels can be embedded between electrical layers. The generic concept enables fabrication of high-density integration (HDI) electro-optical circuit boards (EOCB) with high number of optical and electrical layers. Waveguides with high NA of 0.3 for low bend losses could be achieved in glass with propagation loss of 0.05 dB/cm for all key wavelengths. Four of those glass waveguide panels were embedded in an EOCB demonstrator with size of 280 x 233 mm² providing eight optical layers with 96 channels in an area of 2.8 x 1.5 mm². To the best of our knowledge it's the highest number of layers that has ever been demonstrated for an EOCB.

"I am adhering to HIV treatment so that I can live to support her": A qualitative study of upward intergenerational support in South Africa.

Background: Intergenerational family care, which was upended by the HIV epidemic in sub-Saharan Africa (SSA), may return to a pre-HIV era arrangement as access to antiretroviral therapy (ART) expands and treated adults can once again provide support for older household members. Empirical research has demonstrated positive 'spillover effects' of ART uptake from treated adults to younger generations, yet much less is known about the nature and breadth of such effects to older generations. This study explores the role and lived experiences among adults who take up ART and those of an older generation with whom they live. Methods: We conducted a qualitative study consisting of semi-structured interviews (n = 46) embedded in the Agincourt Health and Demographic Surveillance System (HDSS) in rural South Africa, between July and September 2022. We purposefully sampled two respondent categories: (i) young or middle-aged adults on ART (aged 18-59 years old); and (ii) older adults (aged ≥60 years old) who were affiliated with a young or middle-aged adult on ART. We used thematic content analysis to extract, code, and categorise relevant text by types of upward spillover effects from ART in younger adults to older adults. Quantitative data was extracted from the existing Agincourt HDSS database and matched to qualitative interview data based on Clinic link unique identifiers of study participants. Results: Mean age was 41 years among young or middle-aged adults (n = 29) and 72 years among older adults (n = 17). Among younger adults, time on ART ranged from five months to more than 21 years. Both young or middle-aged adults on ART and older adults reported positive spillover effects for older adults across five main tiers: caregiving, financial support, physical and mental health, living arrangements and household relationships, and stigma and reputation. Spillover challenges included financial costs and caregiving responsibilities following ART initiation of young or middle-aged adults, although these additional caregiving responsibilities were generally not perceived as particularly burdensome. Conclusions: ART is likely to benefit older adults in South Africa whose families are affected by HIV. This study identified a wide range of perceived spillover effects from ART in younger adults to older adults, including improvements to upward intergenerational support. These qualitative findings offer a guide to researchers, policymakers, and donors to capitalise on the broader societal effects of a large-scale health intervention to further support family structures and meet the needs of a growing older population.

The effect of school-entry age on health is understudied in low- and middle-income countries: A scoping review and future directions for research.

Background: Substantive literature has assessed the impact of starting school at younger ages relative to peers on health in high-income countries (HICs), but there is little evidence from low- and middle-income countries (LMICs). Conclusions drawn from HICs may not apply to different education contexts and health threats. This study maps the empirical evidence on the effect of school-entry age on health in LMICs and identifies directions for future research. Methods: We conducted a scoping review between August and September 2022 by systematically searching the health sciences, education, economics, psychology, and general sciences literature and included quantitative and qualitative studies. The exposure of interest was relative age for grade defined as starting or progressing through school at a younger or older age compared to peers who are in the same grade. We extracted key characteristics of included studies and summarized their findings. We categorized results into broad health domains which emerged a posteriori from our analyses of included studies, including neurodevelopment and mental health, sexual and reproductive health, non-communicable diseases, and nutrition. Findings: We identified 8 studies from middle-income countries published between 2017 and 2022. Among those studies, we identified 3 quasi-experimental studies using data from Brazil, Mexico, and Vietnam, and 5 observational studies primarily from Türkiye. Children starting school earlier had an increased risk of being diagnosed with attention deficit hyperactivity disorder, earlier sexual debut and cohabitation, adolescent pregnancy, adolescent marriage, and engaged more frequently in risky behavior compared to children who started school later. Pregnant women who started school younger also had fewer prenatal care visits and experienced more pregnancy complications. Although most studies identified negative health consequences from starting school earlier, the evidence for nutritional outcomes, such as overweight and stunting, was mixed. No studies were identified from low-income countries. Conclusions: Little is known about the health consequences of school-entry age in low-resource settings. Additional research is needed to investigate the impact of relative age for grade, whether and how these effects persist into adulthood, and to inform strategies that can offset potential disadvantages stemming from school-entry cut-off dates.

Intergenerational spillover effects of antiretroviral therapy in sub-Saharan Africa: a scoping review and future directions for research.

BACKGROUND: Antiretroviral therapy (ART) may influence individuals who do not receive the intervention but who are connected in some way to the person who does. Relatively little is known, however, about the size and scope of, what we term, spillover effects of ART. We explored intergenerational spillover effects of ART in sub-Saharan Africa (SSA) and identified several directions for future research. METHODS: We conducted a scoping review between March and April 2022. We systematically searched PubMed, PsycINFO, EconLit, OTseeker, AIDSInfo, Web of Science, CINHAL, Google Scholar and African Index Medicus. We analysed the distribution of included studies over time and summarised their findings. We examined the intergenerational impact of ART provision to working-age adults living with HIV on children ('downward' spillover effects) and older adults ('upward' spillover effects). We categorised types of intergenerational spillover effects according to broad themes which emerged from our analysis of included studies. FINDINGS: We identified 26 studies published between 2005 and 2022 with 16 studies assessing spillover effects from adults to children (downward), and 1 study explicitly assessing spillover effects from working-age adults to older adults (upward). The remaining studies did not fully specify the direction of spillover effects. Most spillover effects of ART to household and family members were beneficial and included improvements in wealth, labour market outcomes, health outcomes and health services utilisation, schooling, and household composition. Both children and older adults benefited from ART availability among adults. Detrimental spillover effects were only reported in three studies and included financial and opportunity costs associated with health services utilisation and food insecurity in the first year after ART. CONCLUSIONS: ART may lead to substantial spillover effects across generations and sectors in SSA. Further research is needed to capitalise on positive spillover effects while mitigating potential negative spillover effects. The returns to investments in large-scale health interventions such as ART may be underestimated without considering these societal benefits.

Enhanced detection of hydrogen sulfide generated in cell culture using an agar trap method.

Lack of reliable methods to accurately measure hydrogen sulfide (H(2)S) produced in vitro has impeded research on the physiology of this gaseous mediator. Current in vitro methods involve measurement of H(2)S in cell culture media following incubation with H(2)S-releasing compounds. However, this method is inaccurate because H(2)S gas has a short life and thus evades detection. To overcome this, we have adapted a method that employs a modified agar layer to instantly trap H(2)S, allowing measurement of H(2)S accumulated with time. The amount of H(2)S trapped in the agar is quantified using an in situ methylene blue assay. We were able to detect H(2)S produced from sodium hydrogen sulfide (NaHS) added at concentrations as low as 10 µM. Following a 24-h incubation of endothelial-like or vascular smooth muscle cells with 50 µM NaHS, we were able to recover twice more H(2)S than conventional methods. When H(2)S-releasing compounds L-cysteine and N-acetylcysteine were added to the cell culture, the amount of H(2)S increased in a concentration-, time-, and cell line-dependent manner. In conclusion, we have developed an improved method to quantify H(2)S generated in vitro. This method could be used to screen compounds to identify potential H(2)S donors and inhibitors for therapeutic use.

Model-based signal tracking in the quantitative analysis of time series of NMR spectra.

Hard modeling of NMR spectra by Gauss-Lorentz peak models is an effective way for dimensionality reduction. In this manner high-dimensional measured data are reduced to low-dimensional information as peak centers, amplitudes or peak widths. For time series of spectra these parameters can be assumed to be smooth functions in time. We suggest to model these time-dependent parameter functions by cubic spline functions, which makes a stable quantitative analysis of NMR series possible even for crossing, highly overlapping peaks. Applications are presented for the batch distillation of methanol and diethylamine, and the reaction of acetic anhydride with 2-propanol.

N-acetylcysteine Provides Cytoprotection in Murine Oligodendrocytes through Heme Oxygenase-1 Activity.

Oligodendrocytic injury by oxidative stress can lead to demyelination, contributing to neurodegeneration. We investigated the mechanisms by which an antioxidant, N-acetylcysteine (NAC), reduces oxidative stress in murine oligodendrocytes. We used normal 158N and mutant 158JP cells with endogenously high reactive oxygen species (ROS) levels. Oxidative stress was induced in 158N cells using hydrogen peroxide (H2O2, 500 µM), and both cells were treated with NAC (50 µM to 500 µM). ROS production, total glutathione (GSH) and cell survival were measured 24 h after treatment. In normal cells, H2O2 treatment resulted in a ~5.5-fold increase in ROS and ~50% cell death. These deleterious effects of oxidative stress were attenuated by NAC, resulting in improved cell survival. Similarly, NAC treatment resulted in decreased ROS levels in 158JP cells. Characterization of mechanisms underlying cytoprotection in both cell lines revealed an increase in GSH levels by NAC, which was partially blocked by an inhibitor of GSH synthesis. Interestingly, we observed heme oxygenase-1 (HO-1), a cytoprotective enzyme, play a critical role in cytoprotection. Inhibition of HO-1 activity abolished the cytoprotective effect of NAC with a corresponding decrease in total antioxidant capacity. Our results indicate that NAC promotes oligodendrocyte survival in oxidative stress-related conditions through multiple pathways.

Reaction rate ambiguities for perturbed spectroscopic data: Theory and implementation.

The analysis of reaction systems and their kinetic modeling is important for both exploratory research and process design. Multivariate curve resolution (MCR) methods are state-of-the-art tools for the analysis of spectral series, but are also affected by an unavoidable solution ambiguity that impacts the obtained concentration profiles, spectra and model parameters. These uncertainties depend on the underlying model and the magnitude of the measurement perturbations. We present a general theoretical approach together with a computational method for the analysis of the solution ambiguity underlying arbitrary kinetic models. The main idea is to determine all those model parameters for which the corresponding pure component factorizations satisfy all given constraints within small error tolerances. This makes it possible to determine bands of concentration profiles and spectra that reflect the underlying ambiguity and circumscribes the potential reliability of MCR solutions. False conclusions on the uniqueness of a solution can be prevented. The procedure can be applied as a post-processing step to MCR methods as MCR-ALS, ReactLab or others. The Matlab program code is freely accessible and includes not only the proposed ambiguity analysis but also an MCR hard-modeling approach. Application studies are presented for two experimental data sets, namely for UV/Vis spectra on the relaxation of a photoexcited state of benzophenone and for Raman spectra on an aldehyde formation process.

Calcium pantothenate modulates gene expression in proliferating human dermal fibroblasts.

Topical application of pantothenate is widely used in clinical practice for wound healing. Previous studies identified a positive effect of pantothenate on migration and proliferation of cultured fibroblasts. However, these studies were mainly descriptive with no molecular data supporting a possible model of its action. In this study, we first established conditions for an in vitro model of pantothenate wound healing and then analysed the molecular effects of pantothenate. To test the functional effect of pantothenate on dermal fibroblasts, cells were cultured and in vitro proliferation tests were performed using a standardized scratch test procedure. For all three donors analysed, a strong stimulatory effect of pantothenate at a concentration of 20 microg/ml on the proliferation of cultivated dermal fibroblasts was observed. To study the molecular mechanisms resulting in the proliferative effect of pantothenate, gene expression was analysed in dermal fibroblasts cultivated with 20 microg/ml of pantothenate compared with untreated cells using the GeneChip Human Exon 1.0 ST Array. A number of significantly regulated genes were identified including genes coding for interleukin (IL)-6, IL-8, Id1, HMOX-1, HspB7, CYP1B1 and MARCH-II. Regulation of these genes was subsequently verified by quantitative real-time polymerase chain reaction analysis. Induction of HMOX-1 expression by pantothenol and pantothenic acid in dermal cells was confirmed on the protein level using immunoblots. Functional studies revealed the enhanced suppression of free radical formation in skin fibroblasts cultured with panthenol. In conclusion, these studies provided new insight in the molecular mechanisms linked to the stimulatory effect of pantothenate and panthenol on the proliferation of dermal fibroblasts.

Nitric oxide and aspirin: a new mediator for an old drug.

Aspirin is known to cause a multitude of pharmacologic actions through inhibition of cyclooxygenase(s) and reduced formation of prostaglandins. Recently, however, novel cytoprotective and antioxidant mechanisms of aspirin have been identified that are independent of cyclooxygenase inhibition. It was shown that aspirin directly stimulates the activity of endothelial nitric oxide (NO) synthase without affecting the expression of endothelial NO synthase. Increased NO formation was found to underlie aspirin-induced sustained protection of endothelial cells from oxidant injury. Downstream targets of NO that mediate tissue protection include the stress proteins heme oxygenase-1 (HO-1) and ferritin. Both HO-1 and ferritin have been identified as targets of, and inducible by, aspirin and, in the case of HO-1, aspirin-triggered lipoxins. It is important to note that these effects are specific to aspirin and not induced by other nonsteroidal antiinflammatory drugs such as diclofenac, indomethacin, or salicylates or by selective cyclooxygenase-2 inhibitors. HO-1 and its antioxidant product bilirubin have been reported to be not only involved in vasoprotection, but to have a similar function in gastric tissue. Stimulation of NO formation through aspirin and ensuing HO-1 induction might therefore help to reduce gastric injury or irritation. Moreover, NO functions as a smooth muscle-relaxing agent and is thus thought to counteract the reduction in gastric blood flow caused by inhibitors of prostaglandin synthesis. It is therefore conceivable that activation of these novel antioxidant pathways contributes, at least in part, to gastric tolerability and the favorable cardiovascular safety profile of aspirin.

Heme oxygenase-1 is a novel target and antioxidant mediator of S-adenosylmethionine.

The sulfur compound and dietary supplement S-adenosylmethionine (SAM) has been reported to have cytoprotective and antioxidant properties. However, the underlying mechanisms remain unresolved. The present study investigates the effect of SAM on the expression of the antioxidant stress proteins heme oxygenase-1 (HO-1) and ferritin in endothelial cells. Induction of the HO-1/ferritin-system leads to protection of tissues against several inflammatory stimuli. SAM increased the protein and mRNA levels of HO-1 in cultured endothelial cells. Induction of HO-1 gene expression was associated with elevated ferritin protein levels and regulated at the transcriptional level via increased promoter activity. HO-1 upregulation by SAM was causally related to a decrease in NADPH-mediated production of oxygen radicals. Our results demonstrate that the HO-1/ferritin-system is a novel target of the antioxidant compound SAM.

The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease.

Vascular diseases such as atherosclerosis, stroke or myocardial infarction are a significant public health problem worldwide. Attempts to prevent vascular diseases often imply modifications and improvement of causative risk factors such as high blood pressure, obesity, an unfavorable profile of blood lipids or insulin resistance. In addition to numerous preventive and therapeutic drug regimens, there has been increased focus on identifying dietary compounds that may contribute to cardiovascular health in recent years. Food-derived bioactive peptides represent one such source of health-enhancing components. They can be released during gastrointestinal digestion or food processing from a multitude of plant and animal proteins, especially milk, soy or fish proteins. Biologically active peptides are considered to promote diverse activities, including opiate-like, mineral binding, immunomodulatory, antimicrobial, antioxidant, antithrombotic, hypocholesterolemic and antihypertensive actions. By modulating and improving physiological functions, bioactive peptides may provide new therapeutic applications for the prevention or treatment of chronic diseases. As components of functional foods or nutraceuticals with certain health claims, bioactive peptides are of commercial interest as well. The current review centers on bioactive peptides with properties relevant to cardiovascular health.

A chemometric study in the area of feasible solution of an acid-base titration of N-methyl-6-oxyquinolone.

Multivariate curve resolution methods aim at recovering the underlying chemical components from spectroscopic data on chemical reaction systems. In most cases the spectra and concentration profiles of the pure components cannot be uniquely determined from the given spectral data. Instead continua of possible factors exist. This fact is known as rotational ambiguity. The sets of all possible pure component factors can be represented in the so-called area of feasible solutions (AFS). This paper presents an AFS study of the pure component reconstruction problem for a series of UV/Vis spectra taken from an acid-base titration of N-methyl-6-oxyquinolone. Additional information on the equilibrium concentration profiles for a varying acid concentration is taken from fluorescence measurements. On this basis chemometric duality arguments lead to the construction of a unique final solution.

Multi-objective optimization for an automated and simultaneous phase and baseline correction of NMR spectral data.

Spectral data preprocessing is an integral and sometimes inevitable part of chemometric analyses. For Nuclear Magnetic Resonance (NMR) spectra a possible first preprocessing step is a phase correction which is applied to the Fourier transformed free induction decay (FID) signal. This preprocessing step can be followed by a separate baseline correction step. Especially if series of high-resolution spectra are considered, then automated and computationally fast preprocessing routines are desirable. A new method is suggested that applies the phase and the baseline corrections simultaneously in an automated form without manual input, which distinguishes this work from other approaches. The underlying multi-objective optimization or Pareto optimization provides improved results compared to consecutively applied correction steps. The optimization process uses an objective function which applies strong penalty constraints and weaker regularization conditions. The new method includes an approach for the detection of zero baseline regions. The baseline correction uses a modified Whittaker smoother. The functionality of the new method is demonstrated for experimental NMR spectra. The results are verified against gravimetric data. The method is compared to alternative preprocessing tools. Additionally, the simultaneous correction method is compared to a consecutive application of the two correction steps.

Antiretroviral therapy coverage associated with increased co-residence between older and working-age adults in Africa.

OBJECTIVES: To determine whether national antiretroviral therapy (ART) coverage is associated with changes in the living arrangements of older adults. DESIGN: Retrospective analysis using 103 nationally representative surveys from 28 African countries between 1991 and 2015. METHODS: The sample consisted of individuals aged at least 60 years. We investigated how three measures of living arrangements of older adults have changed with ART coverage: the number of older individuals living without working-age adults, the number of older individuals living with only dependent children (i.e. 'missing generation' households), and the number of working-age adults per household where an older individual lives. RESULTS: Our sample consisted of 297 331 older adults. An increase in ART coverage of 1% was associated with a 0.7 percentage point reduction (P < 0.001) in the probability of an older adult living without working-age adult and a 0.2 percentage point reduction (P = 0.005) in the probability of an older adult living in a 'missing generation' household. Increases in ART coverage were also associated with more working-age adults in households with at least one older adult. In our study countries, representing 75% (749 million) of the sub-Saharan population, an additional 103 000-358 000 older adults could be living with working-age adults as a result of increased ART coverage (1%). CONCLUSION: The scale-up of ART has likely led to substantial increases in co-residence between older and working-age adults in Africa. Returns to investments in HIV treatment will be too low, if the social benefits from these changes in living arrangements of older adults are not taken into account.

Identification of residues crucially involved in soluble guanylate cyclase activation.

The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the beta(1)-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp(44), Asp(45) and Phe(74) of the beta(1)-subunit were identified as being crucially important for functional enzyme activation. beta(1)Asp(45) may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of beta(1)IIe(145) Tyr is unchanged compared to the native enzyme, suggesting that Tyr(145) does not confer the ability to distinguish between NO and O(2). In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr(145) for ligand discrimination.