Principal Site Research Articles

Mercury transformations in a coastal water column (Gulf of Trieste, northern Adriatic Sea)

The mechanisms of mercury (Hg) microbial transformations in coastal marine water column are poorly understood. In an effort to study seasonal Hg transformation mechanisms and underlying processes in the coastal marine ecosystem of the Gulf of Trieste (northern Adriatic Sea), we have successfully applied the 197Hg radiotracer technique in series of incubation experiments. This area is characterized by continuous Hg input from the Soča/Isonzo River as a consequence of nearly 500 years of activity at the Idrija Mine (W Slovenia). The method proved to be valuable, since it allowed us to follow Hg transformations at concentrations similar to those found in the study site. During our study, Hg methylation in the marine water column could not be detected, suggesting that sediments are the principal methylation site and the source of MeHg to the water column. Conversely, the water column showed a pronounced Hg reduction potential, mostly of photochemical origin, suggesting that the Gulf of Trieste is a source of Hg0 to the atmosphere. However, the high Hg reduction potential observed in autumn was most likely related to a phytoplankton (diatom) autumn bloom, indicated by high Chl a concentrations measured during that period. We hypothesize that the microbial reduction was either linked to the expression of mer genes or, a consequence of non-specific redox reactions. Variations in bacterioplankton community fingerprints (as determined by Denaturing Gradient Gel Electrophoresis) suggested that community structure had little influence on microbial Hg reduction potential. Nevertheless, our results indicate that in the absence of photochemistry, interactions between microbes and organic matter may enhance biogeochemical Hg2 + reduction potential in some coastal marine environments.

Isolation and Culture of Amphibian (Xenopus laevis) Sub-Capsular Liver and Bone Marrow Cells.

The X. laevis sub-capsular liver is thought to be the principal hematopoietic site of Xenopodinae species from early development and, in case of certain species, into adulthood. The Xenopus bone marrow appears to be comprised of precursor cells committed to myeloid lineages, such as macrophage- and granulocyte-progenitor cells. With alarming increases in the contribution of pathogenic infections to the global amphibian declines, now more than ever a better understanding of the mechanisms controlling amphibian immune cell ontogeny and functionality is warranted. Accordingly, here we detail the isolation and culture of the X. laevis hematopoietic cells from the sub-capsular liver and bone marrow. Considering the immunological roles attributed to these amphibian organs, the respective cell isolation protocols described here will be pertinent to garnering further insights into the coordinated regulation of amphibian hematopoiesis and immune defense mechanisms.

M3+O(–Mn4+)2 clusters in doped MnOx catalysts as promoted active sites for the aerobic oxidation of 5-hydroxymethylfurfural

M3+O(–Mn4+)2 clusters in doped MnOx catalysts are principal active sites that make oxygen ‘easy come, easy go’.

Cholesterol-Binding Sites in GIRK Channels: The Devil is in the Details.

In recent years, it has become evident that cholesterol plays a direct role in the modulation of a variety of ion channels. In most cases, cholesterol downregulates channel activity. In contrast, our earlier studies have demonstrated that atrial G protein inwardly rectifying potassium (GIRK) channels are upregulated by cholesterol. Recently, we have shown that hippocampal GIRK currents are also upregulated by cholesterol. A combined computational-experimental approach pointed to putative cholesterol-binding sites in the transmembrane domain of the GIRK2 channel, the primary subunit in hippocampal GIRK channels. In particular, the principal cholesterol-binding site was located in the center of the transmembrane domain in between the inner and outer α-helices of 2 adjacent subunits. Further studies pointed to a similar cholesterol-binding site in GIRK4, a major subunit in atrial GIRK channels. However, a close look at a sequence alignment of the transmembrane helices of the 2 channels reveals surprising differences among the residues that interact with the cholesterol molecule in these 2 channels. Here, we compare the residues that form putative cholesterol-binding sites in GIRK2 and GIRK4 and discuss the similarities and differences among them.

Cerebrospinal fluid and serum MHPG improve Alzheimer's disease versus dementia with Lewy bodies differential diagnosis

IntroductionGiven the challenges concerning the differential diagnosis of dementia, we investigated the possible added value of monoaminergic compounds to the standard cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers. Particularly, regarding the AD versus dementia with Lewy bodies (DLB) comparison, monoamines or their metabolites might have added discriminative value as there is a more severe neuropathological burden in the locus coeruleus of DLB patients, the principal site of noradrenaline synthesis. MethodsWe applied enzyme-linked immunosorbent assay (ELISA) to analyze CSF amyloid β peptide of 42 amino acids, total tau, and tau phosphorylated at threonine 181, in patients with AD, frontotemporal dementia, DLB/Parkinson's disease dementia, and controls. Reversed-phase high-performance liquid chromatography with electrochemical detection was implemented to study monoamine and metabolite levels in CSF and serum. Stepwise forward conditional logistic regression and receiver operating characteristic (ROC) curve analyses were performed to assess the diagnostic accuracy of these newly fitted models containing the most discriminative indicators of disease status. ResultsMost significant differences in CSF and serum were confined to the noradrenergic system. More specifically, CSF 3-methoxy-4-hydroxyphenylglycol (MHPG) levels were higher, whereas serum MHPG levels were lower, in DLB patients compared with all other groups. Addition of CSF and serum MHPG levels to the CSF AD biomarker panel significantly increased diagnostic accuracy between DLB/Parkinson's disease dementia and AD. Interestingly, a model only including CSF and serum MHPG without the classic AD biomarker panel reached similar area under the curve values. DiscussionWe hypothesize that varying degrees of neuronal loss in the locus coeruleus of DLB/Parkinson's disease dementia versus AD patients result in differentially altered MHPG levels, making this metabolite a valuable biomarker.

Clinical Features and Treatment Outcomes among Children with Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis: A 20-Year Study in a Tertiary Referral Hospital.

Aim To determine the probable causative factors, clinical features, and treatment outcomes of Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and SJS-TEN overlap in children. Methods A 20-year database review of all children diagnosed with SJS/TEN/SJS-TEN overlap at the King Chulalongkorn Memorial Hospital, Thailand. Results 36 patients (M : F, 16 : 20) with the mean age of 9.2 ± 4.0 years were identified. There were 20 cases of SJS, 4 cases of SJS-TEN overlap, and 12 cases of TEN. Drugs were the leading cause for the diseases (72.3%); antiepileptics were the most common culprits (36.1%). Cutaneous morphology at presentation was morbilliform rash (83.3%), blister (38.9%), targetoid lesions (25.0%), and purpuric macules (2.8%). Oral mucosa (97.2%) and eye (83.3%) were the 2 most common mucosal involvements. Majority of the cases (77.8%) were treated with systemic corticosteroids, intravenous immunoglobulin, or both. Treatment outcomes between those who received systemic therapy and those who received only supportive care were comparable. Skin and eye were the principal sites of short-term and long-term complications. Conclusions SJS/TEN are not common but are serious diseases which lead to significant morbidities in children. Early withdrawal of suspicious causes and meticulous supportive care are very important. This study found that the systemic therapy was not superior to supportive care because the treatment outcomes for both groups were comparable.

Mitochondria and Reactive Oxygen Species in Aging and Age-Related Diseases.

Aging has been linked to several degenerative processes that, through the accumulation of molecular and cellular damage, can progressively lead to cell dysfunction and organ failure. Human aging is linked with a higher risk for individuals to develop cancer, neurodegenerative, cardiovascular, and metabolic disorders. The understanding of the molecular basis of aging and associated diseases has been one major challenge of scientific research over the last decades. Mitochondria, the center of oxidative metabolism and principal site of reactive oxygen species (ROS) production, are crucial both in health and in pathogenesis of many diseases. Redox signaling is important for the modulation of cell functions and several studies indicate a dual role for ROS in cell physiology. In fact, high concentrations of ROS are pathogenic and can cause severe damage to cell and organelle membranes, DNA, and proteins. On the other hand, moderate amounts of ROS are essential for the maintenance of several biological processes, including gene expression. In this review, we provide an update regarding the key roles of ROS-mitochondria cross talk in different fundamental physiological or pathological situations accompanying aging and highlighting that mitochondrial ROS may be a decisive target in clinical practice.

Hepatotoxicity and the Role of Some Herbal Hepatoprotective Plants in Present Scenario

Liver is the principal site for metabolism and excretion in body. The human liver metabolizes substances by various biochemical pathways including oxidation, reduction, hydration, condensation, hydrolysis, conjugation or isomerization. Disorder of any of the afore mentioned process may lead to liver cell injury, what we call as hepatotoxicity which in turn leads to many diseases. Such diseases are responsible for higher mortality rates worldwide. Hepatotoxicity can be due to medicines, chemicals, dietary disturbances or herb induced liver damage via hepatotoxins. A number of herbal and herbomineral preparations are available in the Ayurveda, the traditional Indian Medicine which have been investigated for their hepatoprotective potential to treat different types of liver disorders. The present review is focused on different herbal plants that have potential to cure the hepatotoxicity.

The impact of cannabis smoke on the performance of pulmonary surfactant under physiologically relevant conditions

The principal site for gaseous exchange within the lung is the alveolar space, which is bathed in a lipid‐protein blend called pulmonary surfactant. This material is the initial contacting site for orally inhaled products and environmental toxins. Using the lung biosimulator, this study investigates the influence of cannabis smoke on the activity of the lung surfactant replacement product, Curosurf. Initially, 50‐mg cannabis material was pyrolysed and the smoke collected. Cannabis smoke profiling was conducted via gas chromatography–mass spectroscopy, with a mean concentration of 1% Δ9‐tetrahydrocannabinol determined. The smoke aliquots were transferred to the lung biosimulator and expansion—contraction cycles were then initiated to mimic tidal breathing. Baseline data confirmed that Curosurf works effectively under physiologically relevant conditions. Exposure to cannabis smoke from 2 independent batches reduced the Langmuir maximum surface pressure values by approximately 20% and increased the compressibility term; interbatch variation was detected. Cannabis smoke impaired the ability of Curosurf to lower the surface tension term. This was ascribed to the penetration of the planar, hydrophobic drug into the two‐dimensional film, and destructive interaction with polar functionalities. The net effect would be increased work of breathing for the individual.

Study of binding interaction of rivaroxaban with bovine serum albumin using multi-spectroscopic and molecular docking approach

BackgroundRivaroxaban is a direct inhibitor of coagulation factor Xa and is used for venous thromboembolic disorders. The rivaroxaban interaction with BSA was studied to understand its PK and PD (pharmacokinetics and pharmacokinetics) properties. Multi-spectroscopic studies were used to study the interaction which included UV spectrophotometric, spectrofluorometric and three dimensional spectrofluorometric studies. Further elucidation of data was done by molecular simulation studies to evaluate the interaction behavior between BSA and rivaroxaban.ResultsRivaroxaban quenched the basic fluorescence of BSA molecule by the process of static quenching since rivaroxaban and BSA form a complex that results in shift of the absorption spectra of BSA molecule. A decline in the values of binding constants was detected with the increase of temperatures (298–308 K) and the binding constants were in range from 1.32 × 105 to 4.3 × 103 L mol−1 indicating the instability of the BSA and rivaroxaban complex at higher temperatures. The data of number of binding sites showed uniformity. The site marker experiments indicated site I (sub-domain IIA) as the principal site for rivaroxaban binding. The thermodynamic study experiments were carried at the temperatures of 298/303/308 K. The ∆G0, ∆H0 and ∆S0 at these temperatures ranged between − 24.67 and − 21.27 kJ mol−1 and the values for ∆H0 and ∆S0 were found to be − 126 kJ mol−1 and ∆S − 340 J mol−1 K−1 The negative value of ∆G0 indicating spontaneous binding between the two molecules. The negative values in ∆H0 and ∆S0 indicated van der Waals interaction and hydrogen bonding were involved during the interaction between rivaroxaban and BSA.ConclusionsThe results of molecular docking were consistent with the results obtained from spectroscopic studies in establishing the principal binding site and type of bonds between rivaroxaban and BSA.

Synthesis, crystal structures, properties and caries prevention efficiency of 2-, 3-, 4-carboxymethylpyridinium hexafluorosilicates

Three pyridinium hexafluorosilicates with the compositions (LH)2[SiF6] (I, II, III, where L=2-, 3-, 4-carboxymethylpyridine) were obtained as crystalline solids upon interaction of hexafluorosilicic acid with isomeric carboxymethylpyridines. All compounds were characterized by elemental analysis, IR, NMR 19F and mass-spectrometry, solubility data, and X-ray crystallography. The binary ionic solids are stabilized by the interplay of intermolecular interactions including strong charge assisted and conventional hydrogen bonds of the NH⋯F, and OH⋯O types along with CH⋯F contacts and π–π stacking interactions. The different supramolecular motifs in I-III are dictated by the different arrangement of the principal binding sites in the isomeric carboxymethylpyridines. The relationship between the salts structure and solubility data is discussed. All hexafluorosilicates reduce the depth and the number of dental caries, and provide efficiency of caries prevention up to 45%.

Mechanistic target of rapamycin is necessary for changes in dendritic spine morphology associated with long-term potentiation

Alterations in the strength of excitatory synapses in the hippocampus is believed to serve a vital function in the storage and recall of new information in the mammalian brain. These alterations involve the regulation of both functional and morphological features of dendritic spines, the principal sites of excitatory synaptic contact. New protein synthesis has been implicated extensively in the functional changes observed following long-term potentiation (LTP), and changes to spine morphology have similarly been documented extensively following synaptic potentiation. However, mechanistic links between de novo translation and the structural changes of potentiated spines are less clear. Here, we assess explicitly the potential contribution of new protein translation under control of the mechanistic target of rapamycin (mTOR) to LTP-associated changes in spine morphology. Utilizing genetic and pharmacological manipulations of mTORC1 function in combination with confocal microscopy in live dissociated hippocampal cultures, we demonstrate that chemically-induced LTP (cLTP) requires do novo protein synthesis and intact mTORC1 signaling. We observed a striking diversity in response properties across morphological classes, with mushroom spines displaying a particular sensitivity to altered mTORC1 signaling across varied levels of synaptic activity. Notably, while pharmacological inhibition of mTORC1 signaling significantly diminished glycine-induced changes in spine morphology, transient genetic upregulation of mTORC1 signaling was insufficient to produce spine enlargements on its own. In contrast, genetic upregulation of mTORC1 signaling promoted rapid expansion in spine head diameter when combined with otherwise sub-threshold synaptic stimulation. These results suggest that synaptic activity-derived signaling pathways act in combination with mTORC1-dependent translational control mechanisms to ultimately regulate changes in spine morphology. As several monogenic neurodevelopmental disorders with links to Autism and Intellectual Disability share a common feature of dysregulated mTORC1 signaling, further understanding of the role of this signaling pathway in regulating synapse function and morphology will be essential in the development of novel therapeutic interventions.

Lithium induces aerobic glycolysis and glutaminolysis in collecting duct principal cells.

Lithium, given to bipolar disorder patients, causes nephrogenic diabetes insipidus (Li-NDI), a urinary-concentrating defect. Li-NDI occurs due to downregulation of principal cell AQP2 expression, which coincides with principal cell proliferation. The metabolic effect of lithium on principal cells, however, is unknown and investigated here. In earlier studies, we showed that the carbonic anhydrase (CA) inhibitor acetazolamide attenuated Li-induced downregulation in mouse-collecting duct (mpkCCD) cells. Of the eight CAs present in mpkCCD cells, siRNA and drug treatments showed that downregulation of CA9 and to some extent CA12 attenuated Li-induced AQP2 downregulation. Moreover, lithium induced cell proliferation and increased the secretion of lactate. Lithium also increased urinary lactate levels in wild-type mice that developed Li-NDI but not in lithium-treated mice lacking ENaC, the principal cell entry site for lithium. Inhibition of aerobic glycolysis with 2-deoxyglucose (2DG) attenuated lithium-induced AQP2 downregulation in mpkCCD cells but did not attenuate Li-NDI in mice. Interestingly, NMR analysis demonstrated that lithium also increased the urinary succinate, fumarate, citrate, and NH4+ levels, which were, in contrast to lactate, not decreased by 2DG. Together, our data reveal that lithium induces aerobic glycolysis and glutaminolysis in principal cells and that inhibition of aerobic glycolysis, but not the glutaminolysis, does not attenuate Li-NDI.

Effects of Cr(VI) on Ca2 +-ATPase activity in the earthworm Eisenia andrei

The effect of Cr(VI) as a soil contaminant on the edaphic worm Eisenia andrei was studied by evaluating the activity of Ca2+-ATPase in the intestinal mucosa. In eukaryotes, Ca2+-ATPase is a key mediator of cell signaling although comparatively little is known about its activity in earthworms. Size and anatomical constraints (i.e. small and complex) led us to develop and optimize a cyto-biochemical method to measure Ca2+-ATPase activity in earthworms. The principal site of enzyme activity was found to be the post clitellar intestinal tract; immunohistochemistry then identified plasma membrane Ca2+-ATPase (PMCA ATPase) in the apical area of the intestinal epithelium. Earthworms exposed for 28days to OECD soil contaminated with 1, 2, and 15mg/Kg Cr(VI) demonstrated about 70% inhibition of Ca2+-ATPase activity at the low Cr (VI) concentration (the half of the Italian law limit for residential areas), rising to approximately 84% inhibition at the highest concentration. Reduced enzyme activity was accompanied by decreased enzyme content and reduced lysosomal membrane stability (LMS), which is a well established early warning biomarker of stress. These data demonstrate the potential utility of Ca2+-ATPase activity as a sensitive parameter with which to detect environmental stress in earthworms.

Medical collecting on the frontiers of natural history: the rise and fall of Haslar Hospital Museum (1827–1855)

Very little is known about the Royal Navy’s Haslar Hospital Museum, located in Gosport, near Portsmouth, and yet the now defunct institution was one of the nineteenth century’s principal sites of medical, natural history and ethnographic investigation. Here, imperial specimens were collected, studied and used as tools in the education of generations of Naval surgeons, servicemen and scientific explorers, most notably Thomas Henry Huxley. Although principally a Naval and medical institution, Haslar Hospital Museum both assisted and challenged Britain’s best-known scientific collections, in particular the Royal Botanic Gardens, Kew and the British Museum. This paper presents the first history of the museum’s growth and decline, between 1827 and 1855. Particular attention is paid to the agency of Naval surgeons in developing new imperial knowledge, and so to the museum’s success in carving out a privileged space for object-based science at the intersection of medicine and natural history.

Comparison of different models for predicting soil bulk density. Case study – Slovakian agricultural soils

Abstract Soil bulk density is one of the main direct indicators of soil health, and is an important aspect of models for determining agroecosystem services potential. By way of applying multi-regression methods, we have created a distributed prediction of soil bulk density used subsequently for topsoil carbon stock estimation. The soil data used for this study were from the Slovakian partial monitoring system-soil database. In our work, two models of soil bulk density in an equilibrium state, with different combinations of input parameters (soil particle size distribution and soil organic carbon content in %), have been created, and subsequently validated using a data set from 15 principal sampling sites of Slovakian partial monitoring system-soil, that were different from those used to generate the bulk density equations. We have made a comparison of measured bulk density data and data calculated by the pedotransfer equations against soil bulk density calculated according to equations recommended by Joint Research Centre Sustainable Resources for Europe. The differences between measured soil bulk density and the model values vary from −0.144 to 0.135 g cm−3 in the verification data set. Furthermore, all models based on pedotransfer functions give moderately lower values. The soil bulk density model was then applied to generate a first approximation of soil bulk density map for Slovakia using texture information from 17 523 sampling sites, and was subsequently utilised for topsoil organic carbon estimation.

Comparative study of the formation of brominated disinfection byproducts in UV/persulfate and UV/H2O2 oxidation processes in the presence of bromide.

The objective of this research was to compare the transformation of Br- and formation of brominated byproducts in UV/persulfate (PS) and UV/H2O2 processes. It was revealed that Br- was efficiently transformed to free bromine which reacted with humic acid (HA) or dihydroxybenzoic acid resulting in the formation of brominated byproducts such as bromoacetic acids (BAAs) in UV/PS system. In contrast, no free bromine and brominated byproducts could be detected in UV/H2O2 system, although the oxidization of Br- was evident. We presumed that the oxidation of Br- by hydroxyl radicals led to the formation of bromine radicals. However, the bromine radical species could be immediately reduced back to Br- by H2O2 before coupling to each other to form free bromine, which explains the undetection of free bromine and BAAs in UV/H2O2. In addition to free bromine, we found that the phenolic functionalities in HA molecules, which served as the principal reactive sites for free chlorine attack, could be in situ generated when HA was exposed to free radicals. This study demonstrates that UV/H2O2 is more suitable than UV/PS for the treatment of environmental matrices containing Br-. Graphical abstract Graphical abstract.

The Essence That Is Belonging Together: Identity, Embodiment, and Skillful Coping During the 2015 Greek Referendum

In the forward of Heidegger’s Identity and Difference, Joan Stambaugh proposes that in his statement “identity is belonging together,” the emphasis ought to be placed on the word belonging. By emphasizing belonging rather than together, one is better able to jettison thinking that centers identity as a fundamental trait of Being, the traditional view of identity held by metaphysics. This article will explicate this idea by drawing from a series of unstructured interviews conducted with young people in Athens during the 2015 July 5th Greek Referendum. This article argues that with attention devoted to the practice of skillful coping, the essence of identity, rather than a reflective cognitive undertaking, correlates to what Heidegger refers to as the event of appropriation. When finding the self affected by the world, what becomes revealed is an intimate identification with the world and things within it as it unfolds. With mobile media technologies, we are privileged to witness a particular way of being affected by the world, one where material and immaterial locations (such as social media arenas) converge together and whose perceptual distinctions become inseparable. Subsequently, we can also witness how the act of skillful coping, the motivated relationship between the body and the object, stands as the principal site where engulfment and investment become unconcealed.

K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site.

Polymodal K2P (KCNK) thermo- and mechanosensitive TREK1 potassium channels, generate ‘leak’ currents that regulate neuronal excitability, respond to lipids, temperature, and mechanical stretch, and influence pain, temperature perception, and anesthetic responses1–3. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore forming regions per subunit4–6. Contrasting other potassium channels, K2Ps use a selectivity filter ‘C-type’ gate7–10 as the principal gating site. Despite recent advances3,11,12, K2Ps suffer from a poor pharmacologic profile limiting mechanistic and biological studies. Here, we describe a new small molecule TREK activator class that directly stimulates the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1(TREK-1) alone with two selective K2P2.1(TREK-1) and K2P10.1(TREK-2) activators, an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402, define a cryptic binding pocket unlike other ion channel small molecule binding sites and, together with functional studies, identify a cation-π interaction that controls selectivity. Together, our data unveil a previously unknown, druggable K2P site that stabilizes the C-type gate ‘leak mode’ and provide direct evidence for K2P selectivity filter gating.

Inhalation therapy for equine lower respiratory tract disease

The delivery of drugs to horses via the inhalation route is being increasingly adopted as a therapeutic approach in equine practice; predominantly used to treat equine lower airway disease, including,...