Changes in the protonation state of lyophilized proteins can impact structural integrity, chemical stability, and propensity to aggregate upon reconstitution. When a buffer is chosen, the freezing/drying process may result in dramatic changes in the protonation state of the protein due to ionization shift of the buffer. In order to determine whether protonation shifts are occurring, ionizable probes can be added to the formulation. Optical probes (dyes) have shown dramatic ionization changes in lyophilized products, but it is unclear whether the pH indicator is uniform throughout the matrix and whether the change in the pH indicator actually mirrors drug ionization changes. In solid-state NMR (SSNMR) spectroscopy, the chemical shift of the carbonyl carbon in carboxylic acids is very sensitive to the ionization state of the acid. Therefore, SSNMR can be used to measure ionization changes in a lyophilized matrix by employing a small quantity of an isotopically-labeled carboxylic acid species in the formulation. This paper compares the apparent pH of six trehalose-containing lyophilized buffer systems using SSNMR and UV-Vis diffuse reflectance spectroscopy (UVDRS). Both SSNMR and UVDRS results using two different ionization probes (butyric acid and bromocresol purple, respectively) showed little change in apparent acidity compared to the pre-lyophilized solution in a sodium citrate buffer, but a greater change was observed in potassium phosphate, sodium phosphate, and histidine buffers. While the trends between the two methods were similar, there were differences in the numerical values of equivalent pH (pHeq) observed between the two methods. The potential causes contributing to the differences are discussed.
- MeSH
- fosfáty * chemie MeSH
- histidin * chemie MeSH
- koncentrace vodíkových iontů MeSH
- kyselina citronová chemie MeSH
- lyofilizace * metody MeSH
- magnetická rezonanční spektroskopie * metody MeSH
- pufry MeSH
- spektrofotometrie ultrafialová metody MeSH
- trehalosa * chemie MeSH
- Publikační typ
- časopisecké články MeSH
Freezing and lyophilization have been utilized for decades to stabilize pharmaceutical and food products. Freezing a solution that contains dissolved salt and/or organic matter produces pure primary ice crystal grains separated by freeze-concentrated solutions (FCS). The microscopic size of the primary ice crystals depends on the cooling conditions and the concentration of the solutes. It is generally accepted that primary ice crystals size influences the rate of sublimation and also can impact physico-chemical behaviour of the species in the FCS. This article, however, presents a case where the secondary ice formed inside the FCS plays a critical role. We microscoped the structures of ice-cast FCS with an environmental scanning electron microscope and applied the aggregation-sensitive spectroscopic probe methylene blue to determine how the microstructure affects the molecular arrangement. We show that slow cooling at -50 °C produces large salt crystals with a small specific surface, resulting in a high degree of molecular aggregation within the FCS. In contrast, fast liquid nitrogen cooling yields an ultrafine structure of salt crystals having a large specific surface area and, therefore, inducing smaller aggregation. The study highlights a critical role of secondary ice in solute aggregation and introduces methylene blue as a molecular probe to investigate freezing behaviour of aqueous systems with crystalline solute.
- MeSH
- led * MeSH
- lyofilizace MeSH
- methylenová modř * MeSH
- roztoky MeSH
- voda chemie MeSH
- zmrazování MeSH
- Publikační typ
- časopisecké články MeSH
Citrate buffers are commonly utilized in the field of biomolecule stabilization. We investigate their applicability in the frozen state within a range of initial pHs (2.5 to 8.0) and concentrations (0.02 to 0.60 M). Citrate buffer solutions subjected to various cooling and heating temperatures are examined in terms of the freezing-induced acidity changes, revealing that citrate buffers acidify upon cooling. The acidity is assessed with sulfonephthalein molecular probes frozen in the samples. Optical cryomicroscopy combined with differential scanning calorimetry was employed to investigate the causes of the observed acidity changes. The buffers partly crystallize and partly vitrify in the ice matrix; these processes influence the resulting pH and allow designing the optimal storage temperatures in the frozen state. The freezing-induced acidification apparently depends on the buffer concentration; at each pH, we suggest pertinent concentration, at which freezing causes minimal acidification.
Solutions of three Good's buffers (HEPES, MOPS, and MES), both pure and mixed with sodium phosphate buffers (Na-P), are investigated in terms of the freezing-induced acidity changes in their operational pH ranges. The Good's buffers have the tendency to basify upon freezing and, more intensively, at lower pHs. The acidity varies most prominently in MES, where the change may reach the value of two. Importantly, the Good's buffers are shown to mitigate the strong acidification in the Na-P buffer. Diverse concentrations of the Good's buffers are added to cancel out the strong, freezing-induced acidity drop in 50 mM Na-P that markedly contributes to the solution's acidity; the relevant values are 3 mM HEPES, 10 mM MOPS, and 80 mM MES. These buffer blends are therefore proposed to be applied in maintaining approximately the acidity of solutions even after the freezing process and, as such, should limit the stresses for frozen chemicals and biochemicals.
- MeSH
- fosfáty * MeSH
- ionty MeSH
- koncentrace vodíkových iontů MeSH
- pufry MeSH
- zmrazování MeSH
- Publikační typ
- časopisecké články MeSH
Despite their low concentrations in many aquatic environments, evidence exists to suggest that herbicides do affect non-target organisms. Given that burrowing is a primary life-history trait in crayfish, herbicides could potentially have serious negative effects on these ecologically important freshwater macroinvertebrates. In this study, we exposed the red swamp crayfish Procambarus clarkii to terbuthylazine (a triazine) and metazachlor (a chloroacetanilide) at an environmental concentration of 2.0 μg/L for 28 days, and then observed their burrowing behaviour for two days. The metazachlor-exposed males excavated a greater number of burrows than the other tested groups, with comparable depths and volumes relative to individual specimen weight. The relative depth and volume of female burrows were identical in all groups. The natural habit of female crayfish of constructing deeper burrows than males was marginally significant in the control and META groups but was not significant for relative volume. The hypothesized adverse effects of chronic exposure to real environmental concentrations of herbicides were not documented in terms of either relative depth or volume. However, the increased number of burrows in metazachlor-exposed animals may mean that this invasive species will cause greater damage to embankments and river banks. The mechanisms behind these effects require closer study.
- MeSH
- acetamidy MeSH
- herbicidy * toxicita MeSH
- severní raci * MeSH
- triaziny toxicita MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Freshwater ecosystems are negatively impacted by various pollutants, from agricultural, urban and industrial wastewater, with metals being one of the largest concerns. Moreover, freshwater ecosystems are often affected by alien species introductions that can modify habitats and trophic relationships. Accordingly, the threat posed by metals interacts with those by alien species, since the latter can accumulate and transfer these substances across the food web to higher trophic levels. How metals transfer within such communities is little studied. We analysed the concentration of 14 metals/metalloids (Al, As, Cd, Co, Cr, Cu, Fe, Hg, Mg, Mn, Ni, Pb, Se, Zn, hereafter 'metal(s)') of eight fish and three crustacean species co-existing in the Arno River (Central Italy), most of which were alien. To assess the pathway of contaminants within the community, we coupled metal analysis with carbon and nitrogen stable isotope analysis derived from the same specimens. Crustaceans showed higher metal concentration than fish, except for Cd, Hg and Se that were higher in fish. We found evidence of trophic transfer for six metals (Cd, Cr, Hg, Mg, Se, Zn). Additionally, ontogenetic differences and differences among various fish tissues (muscle, liver, and gills) were found in metals concentration. Considerable biomagnification along the trophic chain was found for Hg, while other metals were found to biodilute. Using stable isotopes and Hg as a third diet tracer, we refined the estimations of consumed preys in the diet previously reconstructed with stable isotope mixing models. Alien species reach high biomass and can both survive to and accumulate high pollutants concentrations, potentially posing a risk for their predators and humans. A combined effect of environmental filtering and increased competition may potentially contribute to the disappearance of native species with lower tolerances.
- MeSH
- chemické látky znečišťující vodu * analýza MeSH
- ekosystém MeSH
- lidé MeSH
- monitorování životního prostředí MeSH
- sladká voda MeSH
- těžké kovy * analýza MeSH
- zavlečené druhy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Itálie MeSH
Nonconsumptive predator-driven mortality (NCM), defined as prey mortality due to predation that does not result in prey consumption, is an underestimated component of predator-prey interactions with possible implications for population dynamics and ecosystem functioning. However, the biotic and abiotic factors influencing this mortality component remain largely unexplored, leaving a gap in our understanding of the impacts of environmental change on ecological communities. We investigated the effects of temperature, prey density, and predator diversity and density on NCM in an aquatic food web module composed of dragonfly larvae (Aeshna cyanea) and marbled crayfish (Procambarus fallax f. virginalis) preying on common carp (Cyprinus carpio) fry. We found that NCM increased with prey density and depended on the functional diversity and density of the predator community. Warming significantly reduced NCM only in the dragonfly larvae but the magnitude depended on dragonfly larvae density. Our results indicate that energy transfer across trophic levels is more efficient due to lower NCM in functionally diverse predator communities, at lower resource densities and at higher temperatures. This suggests that environmental changes such as climate warming and reduced resource availability could increase the efficiency of energy transfer in food webs only if functionally diverse predator communities are conserved.
- MeSH
- ekosystém * MeSH
- kapři MeSH
- populační dynamika MeSH
- potravní řetězec * MeSH
- predátorské chování * MeSH
- severní raci MeSH
- sladká voda MeSH
- teplota * MeSH
- vážky MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Freshwater biodiversity is globally threatened by various factors while severe weather events like long-term droughts may be substantially devastating. In order to remain in contact with the water or stay in a sufficiently humid environment at drying localities, the ability to withstand desiccation by dwelling in the hyporheic zone, particularly through vertical burrowing is crucial. We assessed the ability of three European native and five non-native crayfish as models to survive and construct vertical burrows in a humid sandy-clayey substrate under a simulated one-week drought. Three native species (Astacus astacus, A. leptodactylus, and Austropotamobius torrentium) suffered extensive mortalities. Survival of non-native species was substantially higher while all specimens of Cherax destructor and Procambarus clarkii survived. The native species and Pacifastacus leniusculus exhibited no ability to construct vertical burrows. Procambarus fallax f. virginalis and P. clarkii constructed bigger and deeper burrows than C. destructor and Orconectes limosus. In the context of predicted weather fluctuations, the ability to withstand desiccation through constructing vertical burrows into the hyporheic zone under drought conditions might play a significant role in the success of particular crayfish species, as well as a wide range of further hyporheic-dwelling aquatic organisms in general.
- MeSH
- analýza přežití MeSH
- biodiverzita MeSH
- biologické modely MeSH
- období sucha * MeSH
- severní raci klasifikace fyziologie MeSH
- sladká voda MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The spreading of new crayfish species poses a serious risk for freshwater ecosystems; because they are omnivores they influence more than one level in the trophic chain and they represent a significant part of the benthic biomass. Both the environmental change through global warming and the expansion of the pet trade increase the possibilities of their spreading. We investigated the potential of four "warm water" highly invasive crayfish species to overwinter in the temperate zone, so as to predict whether these species pose a risk for European freshwaters. We used 15 specimens of each of the following species: the red swamp crayfish (Procambarus clarkii), the marbled crayfish (Procambarus fallax f. virginalis), the yabby (Cherax destructor), and the redclaw (Cherax quadricarinatus). Specimens were acclimatized and kept for 6.5 months at temperatures simulating the winter temperature regime of European temperate zone lentic ecosystems. We conclude that the red swamp crayfish, marbled crayfish and yabby have the ability to withstand low winter temperatures relevant for lentic habitats in the European temperate zone, making them a serious invasive threat to freshwater ecosystems.
- MeSH
- analýza přežití MeSH
- ekosystém MeSH
- nízká teplota * MeSH
- severní raci fyziologie MeSH
- vodní hospodářství MeSH
- zavlečené druhy * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
