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Toxoplasma gondiiis an obligate intracellular parasite whose tachyzoite form causes disease via a lytic growth cycle. Its metabolic and cellular pathways are primarily designed to ensure parasite survival within a host cell. But during its lytic cycle tachyzoites are exposed to the extracellular milieu and prolonged exposure requires activation of stress response pathways that include reprogramming the parasite proteome. Regulation of protein synthesis is therefore important for extracellular survival. We previously reported that in extracellularly stressed parasites that the elongation phase of protein synthesis is negatively regulated by theToxoplasmaoxygen sensing protein, PhyB. PhyB acts by promoting the activity of elongation factor eEF2, which is a GTPase that catalyzes the transfer of the peptidyl-tRNA from the A site to the P site of the ribosome. In the absence of PhyB, eEF2 is hyper-phosphorylated, which inhibits eEF2 from interacting with the ribosome. eEF2 kinases are atypical calcium-dependent kinases and BLAST analyses revealed the parasite kinase, CDPK3, as the most highly homologous to theSaccharomyces cerevisiaeeEF2 kinase,RCK2. In parasites exposed to extracellular stress, loss of CDPK3 leads to decreased eEF2 phosphorylation and enhanced rates of elongation. Furthermore, co-immunoprecipitation studies revealed that CDPK3 and eEF2 interact in stressed parasites. Since CDPK3 and eEF2 normally localize to the plasma membrane and cytosol, respectively, we investigated how the two can interact. We report that under stress conditions that CDPK3 is not N-myristoylated likely leading to its cytoplasmic localization. In summary, we have identified a novel function for CDPK3 as the first protozoan extracellular-stress induced eEF2 kinase.IMPORTANCEHere, we identify the first protozoan kinase that phosphorylate elongation factor 2 and demonstrate that it is part of an integrated stress response.

In the global market era, food product control is very challenging. It is impossible to track and control all production and delivery chains not only for regular customers but also for the State Sanitary Inspections. Certified laboratories currently use accurate food safety and quality inspection methods. However, these methods are very laborious and costly. The present review highlights the need to develop fast, robust, and cost-effective analytical assays to determine food contamination. Application of the molecularly imprinted polymers (MIPs) as selective recognition units for chemosensors' fabrication was herein explored. MIPs enable fast and inexpensive electrochemical and optical transduction, significantly improving detectability, sensitivity, and selectivity. MIPs compromise durability of synthetic materials with a high affinity to target analytes and selectivity of molecular recognition. Imprinted molecular cavities, present in MIPs structure, are complementary to the target analyte molecules in terms of size, shape, and location of recognizing sites. They perfectly mimic natural molecular recognition. The present review article critically covers MIPs' applications in selective assays for a wide range of food products. Moreover, numerous potential applications of MIPs in the food industry, including sample pretreatment before analysis, removal of contaminants, or extraction of high-value ingredients, are discussed.

This is the first study to generate carrot plants for enhanced salinity tolerance using a single-cell in vitro system. Protoplasts of three carrot accessions were exposed to treatment by seven different concentrations of NaCl (10–400 mM). Salt concentrations higher than 50 mM decreased plating efficiency and those of 200–400 mM of NaCl completely arrested mitotic divisions of cultured cells. The protoplast-derived plants from the control and 50–100 mM NaCl treatment were subjected to an 8-week salt stress in greenhouse conditions induced by salinized soil (EC 3 and 6 mS cm−1). 50 mM NaCl stress applied in vitro induced polyploidy among regenerated plants. The regenerants obtained from the 50 and 100 mM NaCl-treated protoplast cultures grown in saline soil had a higher survival rate compared to the regenerants from the control cultures. The salt-stressed plants accumulated anthocyanins in petioles and produced denser hairs on leaves and petioles in comparison to the control plants. Salt stress influenced pollen viability and seed setting of obtained regenerants. The results suggest that salt stress applied in vitro in protoplast cultures creates variation which allows alleviating the negative effects of salt stress on the development and reproduction of the carrot. Salt stress applied to carrot protoplasts generates variability manifested in differences in cellular response and variation in ploidy. The adaptation of carrot regenerants to soil salinity was associated with accumulation of anthocyanins and increased hairiness.

Over the last decade, an increase in the use of locally available, recycled, and waste materials as growing media components have occurred in various regions of the world in extensive green roof technology. For eco-concept reasons, such a strategy appears to be appropriate, but can be problematic due to difficulties in obtaining proper parameters of growing substrate. The growing media should be properly engineered in order to enable the proper functioning of green roofs and provide suitable environment for ideal root growth. The aim of the study was to assess the utility of locally occurring waste materials for growing media composition and estimate plant- and time-dependent changes in the physico-chemical parameters of waste-based substrates in a simulated extensive green roof system during a two-year Sedum acre L. cultivation. Five different substrate compositions were prepared using silica waste, crushed brick, Ca- and Zn-aggregates, melaphyre, tuff, sand, muck soil, urban compost, spent mushroom, and coconut fibres. Optimal water capacity, particle-size distribution, pH and salts concentration were found in all substrates. A higher concentration of macronutrients (N, P, K, Mg) and trace elements (B, Cu, Fe, Mn, Zn, Cd, Ni, Pb, and Cr) was found in waste-based substrates than in the commercial medium. In comparison to the parameters determined before establish the experiment, bulk density of tested growing media decreased, except for the substrates where the source of organic matter was the rapidly mineralising spent mushroom. The organic matter content in substrates after the two-year vegetation increased in relation to the ready-made substrate, with the exception of the composition with spent mushroom. After two years of the experiment, all available macronutrients and trace elements (with the exception of mineral N, K, SO4-S, and B) concentration were higher than in 2014, while pH, salt concentration was lower. In general, plants grown in waste substrates had lower dry matter content and higher biomass. A significantly higher biomass of S. acre L. was found in the first year of the experiment. In the second year of the research, the plants grown in the commercial medium, the substrate with silica waste, and the substrate with spent mushroom produced higher biomass than in the first year. No symptoms of abnormal growth were observed, despite the higher trace element concentrations in plants collected from waste-based substrate. Waste-based growing media can be considered as a valuable root environment for S. acre L. in an extensive green roof system.

Polymer-drug conjugates are currently being more widely investigated for the treatment of hypertension. In view of the above, in the first stage of our work, we used nontoxic &beta, cyclodextrin (&beta, CD) as effective, simple, inexpensive, and safe for the human body initiator for the synthesis of biocompatible and biodegradable functionalized polymers suitable for the medical and pharmaceutical applications. The obtained polymeric products were synthesized through a ring-opening polymerization (ROP) of &epsilon, caprolactone (CL), d,l-, and l,l- lactide (LA and LLA). The chemical structures of synthesized materials were elucidated based on 1H NMR and solid-state carbon-13 cross-polarization/magic angle spinning nuclear magnetic resonance (13C CP/MAS NMR) analysis, while the incorporation of &beta, CD molecule into the polymer chain was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, molecular modeling has been applied to investigate the intrachain rigidities and chain architectures for several representative structures. The obtained and thoroughly characterized branched matrices were then used to generate the first &beta, cyclodextrin/biodegradable polymer/&beta, blocker conjugate through the successful conjugation of pindolol. The conjugates were fabricated by carbodiimide-mediated coupling reaction. The branched biodegradable materials released the drug in vitro in a sustained manner and without &ldquo, burst release&rdquo, and thus have the ability to treat different heart diseases.

Anaphylaxis is defined as severe, life-threatening, systemic or general, immediate reaction of hypersensitivity, with repeatable symptoms caused by the dose of stimulus which is well tolerated by healthy persons. The proper diagnosis, immediate treatment and differential diagnosis are crucial for saving patient's life. However, anaphylaxis is relatively frequently misdiagnosed or confused with other clinical entities. Thus, there is a continuous need for identifying detectable markers improving the proper diagnosis of anaphylaxis. Here we presented currently known markers of anaphylaxis and discussed in more detail the most clinically valuable ones: tryptase, platelet activacting factor (PAF), PAF-acethylhydrolase, histamine and its metabolites.

BACKGROUND The proximity of the internal carotid artery to the pharyngeal wall poses a risk of injury during nasopharyngeal surgery. OBJECTIVES The aim of this study was to assess the distances between the extracranial internal carotid artery (ICA) and the pharyngeal wall. MATERIAL AND METHODS Measurements were taken on certain levels of the pharynx using computed tomography angiography (angio-CT) scans of 97 patients. One-tailed Student's t-test for independent variables and a comparison of expected values for dependent pairs of observations were applied. RESULTS The shortest distance between the ICA and the pharyngeal wall was 1.1 mm. The ICA is closer to the pharyngeal wall at the epiglottis apex level (16.46 ±0.89 mm) than to the Eustachian tube (ET) (19.8 ±0.62 mm) (p < 0.0005). In women, the ICA is closer to the ET (19.44 ±0.78 mm) than in men (20.17 ±0.96 mm) (p = 0.04). In women, the right ICA is closer to the pharyngeal wall than the left ICA at the level of the lower margin of the 2nd cervical corpus vertebra (C2) (right: 17.6 ±1.8 mm; left: 20.7 ±1.7 mm) (p = 0.002) and at the level of the epiglottis apex (right: 15.2 ±1.7 mm; left: 17.4 ±1.4 mm) (p = 0.028). The bifurcation of the common carotid artery (CCA) is higher in men (19.48 ±2.19 mm below the C2) than in women (21.82 ±1.02 mm) (p < 0.001). When the bifurcation is at the level of the epiglottis apex, the ICA is closer to the pharyngeal wall (12.3 ±1.69 mm) than in other cases (16.46 ±0.89 mm) (p = 0.005). In men, the higher the bifurcation is, the closer the ICA is to the pharyngeal wall at the level of the lower margin of the C2 (p = 0.003). CONCLUSIONS The risk of ICA incision during surgery differs between the pharyngeal levels, genders and sides of the neck. The ICA may be much closer to the pharyngeal wall than described in the literature.

The increasing popularity of green roof technology can contribute to urban sustainability and well-being in cities. However, there still is limited knowledge about the effect of substrate, especially composed from waste amendments, on plant species suitable for the extensive green roofs. Currently the urban agglomeration is characterized by a high degree of industrialization and modern agriculture, which contributes to the production of excessive amounts of various waste materials. The usage of locally available waste materials has many potential benefits, including reduced transportation costs, reduced waste disposal and preservation of natural aggregates. However, the waste has to meet many criteria, such as appropriate physical and chemical properties. The main purposes of green roofs in Poland are: to create additional green space in dense urban area and to increase biodiversity, which could be done by using native plants (well adapted to local weather conditions). A 3-year study was carried out in Krakow using a pilot-scale roof system in field containers. Treatments included two substrates—a Si-waste substrate composed of locally available waste materials and a commercial substrate (control)—and three native xerothermic plant taxa: Dianthus carthusianorum, Hieracium pilosella L. and Sedum acre L. The physicochemical properties of substrates, their stability over time and influence on plant nutrient status as well as the visual rating of plants measurements were examined. We observed a tendency to decrease the bulk density and pH value, and a significant increase in the organic matter content in both tested substrates during a subsequent year of the study. The plant nutrient status was similar in terms of macro-elements, regardless of the substrate used. In relation to elevated trace element concentrations in the Si-waste substrate, the amount of metals accumulated in the tissues of all tested taxa was relatively low. We concluded that waste substrate may be a valuable root environment for examined native species which can be successfully used in Europe on green roof plant communities.

To grow and cause disease, intracellular pathogens modulate host cell processes. Identifying these processes as well as the mechanisms used by the pathogens to manipulate them is important for the development of more effective therapeutics. As an example, the intracellular parasite Toxoplasma gondii induces a wide variety of changes to its host cell, including altered membrane trafficking, cytoskeletal reorganization, and differential gene expression. Although several parasite molecules and their host targets have been identified that mediate- these changes, few are known to be required for parasite replication. One exception is the host cell transcription factor, hypoxia-inducible factor-1 (HIF-1), which is required for parasite replication in an oxygen-dependent manner. Toxoplasma activates HIF-1 by stabilizing the HIF-1α subunit, and this is dependent on the signaling from the Activin-Like Kinase (ALK) receptor superfamily. Here, we demonstrate that specific overexpression of the ALK family member, ALK4, increased HIF-1 activity in Toxoplasma-infected cells, and this increase required ALK4 kinase activity. Moreover, Toxoplasma stimulated ALK4 to dimerize with its co-receptor, ActRII, and also increased ALK4 kinase activity, thereby demonstrating that Toxoplasma activates the ALK4 receptor. ALK4 activation of HIF-1 was independent of canonical SMAD signaling but rather was dependent on the non-canonical Rho GTPase and JNK MAP kinase signaling pathways. Finally, Toxoplasma increased rates of ALK4 ubiquitination and turnover. These data provide the first evidence indicating that ALK4 signaling is a target for a microbial pathogen to manipulate its host cell.

Purpose: A case of schizophrenia with coexisting obsessive-compulsive symptoms is reported. Case: The frequency of obsessive-compulsive disorder (OCD) and obsessive-compulsive symptoms (OCS) occurrence among patients suffering from schizophrenia is considerably higher in comparison to general population. The results of some studies show that schizo-obsessive disorder is characterized by higher intensity of negative and depressive symptoms. Patients with comorbid schizophrenia and obsessive-compulsive disorder show greater level of social dysfunction and they exhibit suicidal behaviours more often than patients diagnosed with schizophrenia. We present a 33-year-old female with obsessive-compulsive symptoms with onset in her early teens with no satisfactory response to treatment, in spite of her good intellect and insight into illness and cooperation. Comment: There is some evidence suggesting that patients with “schizo-obsessive disorder” have a worse prognosis compared to the group of patients suffering only from schizophrenia, but the effect of OCD on schizophrenia symptom profile is unclear.

In previous work, we identified xanthine oxidase (XO) as an important enzyme in the interaction between the host and enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC). Many of the biological effects of XO were due to the hydrogen peroxide produced by the enzyme. We wondered, however, if uric acid generated by XO also had biological effects in the gastrointestinal tract. Uric acid triggered inflammatory responses in the gut, including increased submucosal edema and release of extracellular DNA from host cells. While uric acid alone was unable to trigger a chloride secretory response in intestinal monolayers, it did potentiate the secretory response to cyclic AMP agonists. Uric acid crystals were formed in vivo in the lumen of the gut in response to EPEC and STEC infections. While trying to visualize uric acid crystals formed during EPEC and STEC infections, we noticed that uric acid crystals became enmeshed in the neutrophilic extracellular traps (NETs) produced from host cells in response to bacteria in cultured cell systems and in the intestine in vivo . Uric acid levels in the gut lumen increased in response to exogenous DNA, and these increases were enhanced by the actions of DNase I. Interestingly, addition of DNase I reduced the numbers of EPEC bacteria recovered after a 20-h infection and protected against EPEC-induced histologic damage.

By binding to the adaptor protein SKP1 and serving as substrate receptors for the SKP1 Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasma genome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function., Author summary Toxoplasma gondii is a protozoan parasite that can cause devastating and life-threatening disease in immunocompromised patients and in fetuses. Its replication is important to study because parasite growth is responsible for the pathology that develops in toxoplasmosis patients. The parasite replicates by a unique process named endodyogeny in which two daughter parasites develop within the mother cell. Early during this process the parasite creates a structure called the Daughter Cell Scaffold whose function is to mark the site from which daughter parasites will emerge. Here, we report the identification of one of the first proteins recruited to the Daughter Cell Scaffold and its importance in executing its function.

The sustained release of alprenolol, a ß-adrenergic antagonist, could be beneficial for the treatment of various heart diseases while reducing the side effects resulting from its continuous use. The novel and branched copolymers uniquely composed of biodegradable components (lactide and glycolide) have been synthesized using natural and therapeutically-efficient ß-aescin-initiator, and consequently characterized to determine their structures and physicochemical properties. The obtained matrices were not cyto- and genotoxic towards bacterial luminescence, protozoan, and Salmonella typhimurium TA1535. The copolymers release the drug in vitro in a sustained manner and without burst release. The value of the drug released was strongly dependent on the copolymer composition and highly correlated with the hydrolytic matrices’ degradation results.

Background Women have a higher risk of lethal arrhythmias than men in long QT syndrome type 2 (LQTS2), but the mechanisms remain uncertain due to the limited availability of healthy control human tissue. We have previously reported that in female rabbits, estrogen increases arrhythmia risk in drug-induced LQTS2 by upregulating L-type Ca2+ (ICa,L) and sodium-calcium exchange (INCX) currents at the base of the epicardium by a genomic mechanism. This study investigates if the effects of estrogen on rabbit ICa,L and INCX apply to human hearts. Methods Postmortem human left ventricular tissue samples were probed with selective antibodies for regional heterogeneities of ion channel protein expression and compared to rabbit myocardium. Functionally, ICa,L and INCX were measured from female and male cardiomyocytes derived from human induced pluripotent stem cells (iPS-CMs) with the voltage-clamp technique from control and estrogen-treated iPS-CMs. Results In women (n = 12), Cav1.2α (primary subunit of the L-type calcium channel protein 1) and NCX1 (sodium-calcium exchange protein) levels were higher at the base than apex of the epicardium (40 ± 14 and 81 ± 30%, respectively, P

Aim Familial Hypertrophic Cardiomyopathy (HCM) is one the most common heart disorders, with gene mutations in the cardiac sarcomere. Studying HCM with patient-specific induced pluripotent stem cell (iPSC) derived cardiomyocytes (CMs) would benefit the understanding of HCM mechanism, as well as the development of personalized therapeutic strategies. Methods and Results To investigate the molecular mechanism underlying the abnormal cardiomyocyte (CM) functions in HCM, we derived iPSCs from a HCM patient with a single missense mutation (Arginine442Glycine) in the MYH7 gene. CMs were next enriched from HCM and healthy iPSCs, followed with whole transcriptome sequencing and pathway enrichment analysis. A widespread increase of genes responsible for “Cell Proliferation” was observed in HCM iPSC-CMs when compared with control iPSC-CMs. Additionally, HCM iPSC-CMs exhibited disorganized sarcomeres and electrophysiological irregularities. Furthermore, disease phenotypes of HCM iPSC-CMs were attenuated with pharmaceutical treatments. Conclusions Overall this study explored the possible patient-specific and mutation-specific disease mechanism of HCM, and demonstrates the potential of using HCM iPSC-CMs for future development of therapeutic strategies. Additionally, the whole methodology established in this study could be utilized to study mechanisms of other human inherited heart diseases.

The article is a description of the proposal of a critical edition of Zbior mniejszy poezji polskich drobniejszych by Jozef Epifani Minasowicz, a Polish poet of the late Saxon period and the Reign of Stanislaw August Poniatowski. The author describes the problems which have to be faced by a publisher of texts from the past, willing to make readers familiar with the legacy of such a prolific writer. Zbior has over 270 pages and is the smallest volume among the works by Minasowicz, who was an extremely hard-working poet, an interpreter, an editor and a publisher, widely acclaimed by his contemporaries, but almost completely forgotten today. The article presents the most important problems concerning the process of preparing an edition, such as: selecting the type of edition, representativeness of the collection, and rules of edition. A factor worth mentioning is the situation ideal for an editor, in which an author of numerous lengthy volumes of low literary value, and at the same time deserving the attention of contemporary readers, had himself prepared a selection of his works: divided into smaller sections, systematized, and supplemented with explanations. It is only such a selection by the author that allows for preparing a critical edition, and only such an edition is useful in a cognitive sense. The analysis also includes doubts concerning the representativeness of the collection, which the author contradicts against the benefits from describing the character of the 18th century poet.

Host-guest complexes of alpha-, beta-, and gamma-cyclodextrins (α-CD, β-CD, and γ-CD, respectively) with amantadine (1-aminoadamantane, AMA; an antiviral agent) were characterized in aqueous solutions using proton nuclear magnetic resonance (NMR) spectroscopy. Host-guest molecular interactions were manifested by changes in the chemical shifts of AMA protons. NMR Job's plots showed that the stoichiometry of all the studied complexes was 1:1. Two-dimensional T-ROESY experiments demonstrated that the complexes were formed by different degrees of incorporation of the adamantyl group of AMA into the CD cavity. The mode of AMA binding was proposed. The AMA molecule came into the α-CD cavity (the smallest size) or β-CD cavity (the intermediate size) through its wide entrance to become shallowly or deeply accommodated, respectively. In the complex of AMA with γ-CD (the largest cavity size), the adamantyl group was also quite deeply inserted into the CD cavity, but it arrived there through the narrow cavity entrance. It was found that the adamantyl group of AMA was best accommodated by the β-CD cavity. The binding constants Kaa of the studied complexes (in M(-1) ), determined from DOSY NMR, were fairly high; their values in an ascending order were: α-CD (183)γ-CD (306) ≪ β-CD (5150).

Związki o zróżnicowanej budowie, których wspólną cechą jest obecność grupy adamantylowej w cząsteczce, są stosowane w leczeniu poważnych schorzeń neurologicznych, takich jak choroba Parkinsona, choroba Alzheimera, a także w terapii cukrzycy typu 2 oraz jako leki przeciwwirusowe. W przypadku prostych aminowych pochodnych adamantanu grupa adamantylowa bezpośrednio odpowiada za działanie lecznicze substancji. W innych przypadkach pełni funkcję usztywniającą cząsteczkę, zapobiegając niekorzystnym reakcjom wewnątrzcząsteczkowym. Nowe pochodne zawierające grupę adamantylową, tworzone jako potencjalne substancje lecznicze są obecnie badane w aspekcie aktywności przeciwmalarycznej i przeciw wirusom HIV.

The facile self-assembly reactions of 1,3,5-triaza-7-phosphaadamantane-7-oxide (PTA═O) with AgNO3 or Ag2SO4 give rise to the generation of two new distinct silver–organic frameworks [Ag(NO3)(μ3-PTA═O)]n (1) and [Ag2(μ2-SO4)(μ5-PTA═O)(H2O)]n (2), respectively. They have been characterized by IR, 1H and 31P{1H} NMR spectroscopies, electrospray ionization-mass spectrometry (ESI-MS)(±), and elemental and single-crystal X-ray diffraction analyses, the latter featuring infinite three-dimensional (3D) non-interpenetrating networks driven by multiply bridging PTA═O spacers that adopt undocumented N2O- or N3O-coordination modes. The topological analysis of 1 reveals a uninodal 3-connected net with the point (Schlafli) symbol of (103) and the srs topological type, whereas 2 shows a rare trinodal 3,4,5-connected net with the unprecedented topology defined by the point symbol of (5.6.7)(54.6.8)(54.63.83). Compounds 1 and 2 represent the first 3D metal–organic frameworks (MOFs) derived from PTA═O or any cage-like PTA ...

Self-assembly reactions of silver(I) nitrate with 1,3,5-triaza-7-phosphaadamantane (PTA) as a main ligand and linker, and various benzenecarboxylic acids [benzoic (Hba), 4-cyanobenzoic (Hcba), 2-aminobenzoic (Haba), or terephthalic (H2tpa) acid] as ancillary ligand sources, provide the first general synthetic entry toward the controlled N,P-coordination mode of PTA, giving rise to the new series of coordination polymers [Ag(ba)(μ-PTA)]n (1), [Ag(μ-cba)(μ-PTA)]n·5nH2O (2), [Ag(μ-aba)(μ-PTA)]n·3nH2O (3), and [Ag2(μ4-tpa)(μ-PTA)2]n·2nH2O (4). They have been isolated as air- and light-stable crystalline solids, characterized by IR and NMR spectroscopies, and elemental and single crystal X-ray diffraction analyses, which feature the generation of infinite PTA-driven 1D or 2D metal−organic networks composed of various Ag-benzenecarboxylate nodes and PTA linkers. A crucial synthetic and architectural defining role is played by the ancillary benzenecarboxylate ligand, a slight modification of which allows design ...

Although hypercholesterolemia is implicated in the pathophysiology of many renal disorders as well as hypertension, its direct actions in the kidney are not yet clearly understood. In the present study, we evaluated renal responses to administration of cholesterol (8 μg·min−1·100 g body wt−1; bound by polyethylene glycol) into the renal artery of anesthetized male Sprague-Dawley rats. Total renal blood flow (RBF) was measured by a Transonic flow probe, and glomerular filtration rate (GFR) was determined by Inulin clearance. In control rats ( n = 8), cholesterol induced reductions of 10 ± 2% in RBF [baseline (b) 7.6 ± 0.3 μg·min−1·100 g−1], 17 ± 3% in urine flow (b, 10.6 ± 0.9 μg·min−1·100 g−1), 29 ± 3% in sodium excretion (b, 0.96 ± 0.05 μmol·min−1·100 g−1) and 24 ± 2% in nitrite/nitrate excretion (b, 0.22 ± 0.01 nmol·min−1·100 g−1) without an appreciable change in GFR (b, 0.87 ± 0.03 ml·min−1·100 g−1). These renal vasoconstrictor and anti-natriuretic responses to cholesterol were absent in rats pretreated with nitric oxide (NO) synthase inhibitor, nitro-l-arginine methylester (0.5 μg·min−1·100 g−1; n = 6). In rats pretreated with superoxide (O2−) scavenger tempol (50 μg·min−1·100 g−1; n = 6), the cholesterol-induced renal responses remained mostly unchanged, although there was a slight attenuation in anti-natriuretic response. This anti-natriuretic response to cholesterol was abolished in furosemide-pretreated rats (0.3 μg·min−1·100 g−1; n = 6) but remained unchanged in amiloride-pretreated rats (0.2 μg·min−1·100 g−1; n = 5), indicating that Na+/K+/2Cl− cotransport is the dominant mediator of this effect. These data demonstrate that cholesterol-induced acute renal vasoconstrictor and antinatriuretic responses are mediated by a decrease in NO production. These data also indicate that tubular effect of cholesterol on sodium reabsorption is mediated by the furosemide sensitive Na+/K+/2Cl− cotransporter.

We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of alphaENaC using the serine protease subtilisin. Trimeric channels formed with alphaFM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an approximately 10-fold decrease in amiloride-sensitive whole cell conductance as compared with alphaWT (2.2 versus 21.2 microsiemens (microS)). Both alphaWT and alphaFM were activated to similar levels by subtilisin cleavage. Channels formed with alphaFD, a construct that deleted the segment between the two furin sites (Delta175-204), exhibited an intermediate conductance of 13.2 microS. More importantly, alphaFD retained the ability to be activated by subtilisin to 108.8 +/- 20.9 microS, a level not significantly different from that of subtilisin activated alphaWT (125.6 +/- 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of alpha was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with alphaLD, a construct that extended the deleted sequence of alphaFD by 17 amino acids (Delta175-221). Channels with alphaLD were uncleaved, exhibited low baseline activity (4.1 microS), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.

© 2015 Elsevier Inc. Toxoplasma gondii is a protozoan pathogen in the phylum Apicomplexa that resides within an intracellular parasitophorous vacuole (PV) that is selectively permeable to small molecules through unidentified mechanisms. We have identified GRA17 as a Toxoplasma-secreted protein that localizes to the parasitophorous vacuole membrane (PVM) and mediates passive transport of small molecules across the PVM. GRA17 is related to the putative Plasmodium translocon protein EXP2 and conserved across PV-residing Apicomplexa. The PVs of GRA17-deficient parasites have aberrant morphology, reduced permeability to small molecules, and structural instability. GRA17-deficient parasites proliferate slowly and are avirulent in mice. These GRA17-deficient phenotypes are rescued by complementation with Plasmodium EXP2. GRA17 functions synergistically with a related protein, GRA23. Exogenous expression of GRA17 or GRA23 alters the membrane conductance properties of Xenopus oocytes in a manner consistent with a large non-selective pore. Thus, GRA17 and GRA23 provide a molecular basis for PVM permeability and nutrient access.

Although hypoxia has been shown to increase the expression of a variety of proteins involved in iron homeostasis, including transferrin and its receptor, little is known about the effect of low oxygen on formation of isoforms of the major iron transport protein, divalent metal transporter 1, DMT1. Accordingly, we examined the effects of hypoxia on expression and subcellular distribution of the different isoforms of DMT1 in rat PC12 cells. Treatment with low oxygen modestly increased expression of protein and mRNA levels for both the +IRE and −IRE species of DMT1. In contrast, expression of the exon 1A containing species of DMT1 was greatly increased by hypoxia as indicated by Western blot and real-time RT-PCR analysis. Message levels for the 1A isoforms increased approximately 60-fold after exposure of PC12 cells to 1% oxygen for 5 h. The subcellular distribution of exon 1A isoforms of DMT1 remained consistently in the cytoplasmic milieu of the cell after hypoxic exposure, as also did the distribution of +IRE species of DMT1. The −IRE species of DMT1, however, responded to hypoxia by becoming increasingly associated with the regions adjoining the outer cellular membranes, while a portion partially colocalized with an early endosomal marker (EEA). Hypoxia also caused a significant increase in the uptake of manganese in PC12 cells. In summary, these results demonstrate that hypoxia selectively increases expression of exon 1A containing species of DMT1 with lesser increases in either the +IRE or −IRE isoforms the transporter.

Divalent metal transporter 1 (DMT1; also called DCT1, Nramp2, or SLC11A2) has multiple isoforms that localize differently in many cell types. DMT1 +IRE species (encoded by mRNA with an iron-responsive element) are limited to the plasma membrane and cytosolic vesicles. In neural cells, -IRE isoforms of DMT1 (encoded by mRNA lacking an IRE) localize to the nucleus, plasma membrane, and cytosolic vesicles. In considering nuclear compartmentalization of -IRE isoforms, we hypothesized that the newly identified exon 1A in the N-terminus of this transporter might contain a nuclear localization signal. DNA constructs starting with exon 1A and ending with exons encoding alternative isoforms were made and transiently transfected into HEK293T and PC12 cells as well as rat sympathetic neurons. None of the constructs appeared in the nucleus despite the presence of exon 1A. Antibody specific for exon 1A was also used in both immunostaining and Western blots to investigate localization of exon 1A expressed both endogenously and ectopically in cells. Again, nuclear localization of DMT1 containing exon 1A was not observed. Our data suggest that exon 1A is neither sufficient nor necessary for DMT1 to appear in the nucleus.

Recent studies have characterized cardiac ionic currents in cardiocytes derived from human induced pluripotent stem cells. However, direct comparison with human ventricular myocytes is hampered by differences in experimental conditions. Similarly, stem cell derived cardiocytes are different and potentially more variable than native myocytes. We examined commercially prepared cells (iCells) and scale production cells (hIPS-CMs) using electronic expression of IK1 to distinguish ventricular cells, compared with myocytes (VM) from biopsy samples.APs were recorded in VM, iCells and hiPSC-CMs. iCells and hiPSC-CMs had depolarized membrane potentials, slower upstroke velocities, and prolonged AP relative to VM. These differences were minimized by electronic expression of IK1. Capacitances were larger in VM (298.97 ± 27.5 pF (4 hearts, n=44)) than in iCells (73.21 ± 5.4 (n=32), p

Alterations in iron levels are likely to influence the biological actions of Mn in PC12 cells, because both metals are transported via the divalent metal transporter 1 (DMT1; also Nramp2 or DCT1). Studies were performed to determine the effect of the iron chelator desferrioxamine (DfO) on Mn-induced PC12 cell death and neuronal differentiation. Cell death almost doubled when PC12 cells were exposed for 24 hr to both DfO (10 microM) and Mn (0.3 mM) as opposed to Mn alone. DfO also stimulated Mn-induced neuronal differentiation by enhancing the phosphorylation of both ERK1 and 2 and also attenuated the increase in caspase 3-like activity induced by 0.3 mM Mn by approximately 50%, indicating that caspase activation, as reported previously, does not contribute to Mn-induced PC12 cell death. DfO also affected Mn-induced suppression of mitochondrial function as indicated by an additional 16% loss of ATP formation in PC12 cells cotreated with 0.3 mM Mn. Because sequestration of iron by DfO would be expected to lead to increased transport of Mn, studies were performed to determine whether iron inhibited Mn transport in PC12 cells. Iron inhibited 54Mn transport with an IC50 of approximately 20 microM. In addition, coincubation of DfO with Mn in PC12 cells resulted in increased expression of both the iron response element-positive and the iron response element-negative forms of DMT1. Taken together, these results demonstrate that iron status is likely to have a direct effect on the uptake and biological actions of Mn and probably other divalent metals that are transported by DMT1.

The effects of pertussis toxin, an uncoupler of Gi protein from adenylate cyclase, and luzindole, a competitive inhibitor of melatonin receptor binding, were examined for their ability to inhibit melatonin-induced suppression of PC12 cell growth. Both agents inhibited the melatonin response suggesting that melatonin may be acting through one of its Gi coupled cell surface receptors. This is confirmed by Western blots demonstrating the presence of MT1 receptors in PC12 cells. Coupling of the Gi protein to these receptors is demonstrated by failure of melatonin to suppress cell growth in PKA deficient A126-1B2-1 mutant PC12 cells. Similarly, melatonin failed to prevent cell proliferation when cells were incubated in the presence of the PKA inhibitor, Rp-cAMP. Retinoic acid and dexamethasone, agents known to effect PC12 cell growth and/or differentiation, displayed differential effects on the actions of melatonin. In the presence of melatonin and low concentrations of retinoic acid (100 nM), PC12 cell proliferation was stimulated compared to that seen with either agent alone, whereas no increase in cell proliferation was observed when higher concentrations of retinoic acid (100 microM) were used. The effects of dexamethasone on suppression of PC12 cell growth were additive with that of melatonin whereas, 1,25-dihydroxyvitamin D(3) (IC(50)=10 nM), which by itself had no effect on PC12 cell growth, was found to inhibit the melatonin response. This study demonstrates that inhibition of PC12 cell growth, at physiological concentrations of melatonin, is mediated by cAMP-dependent cell surface receptors and this response is altered by other growth factors known to effect PC12 cell proliferation and differentiation.

Calcineurin, a ubiquitous calcium-activated serine phosphatase, plays an important role in the signal transduction. We have previously reported that cyclosporin A (CsA) inhibits the growth and survival of the rat C6 glioma cells due to the inhibition of signaling pathway involving calcineurin and transcription factor nuclear factor of activated T cells (NFAT). In the present study, we show that CsA affects the survival of reactive astrocyte cultures derived from striatal trauma. Exposure of reactive astrocytes to doses of CsA >50 μg/ml for 24–72 h produces morphological changes, including cell body shrinkage and loss of extensions, followed by cell death. This death was accompanied by apoptotic changes in nuclear morphology and DNA fragmentation, as revealed by Hoechst 33258 and positive TUNEL staining. We demonstrated the presence of calcineurin A subunit in reactive astrocytes and corpus callosum (brain structure enriched in astrocytes) and an additional calcineurin-like protein occurring solely in reactive astrocytes. FK506, a calcineurin inhibitor unrelated to CsA, inhibits proliferation of astrocytes and induces death accompanied by apoptotic changes in nuclear morphology and DNA fragmentation. Since calcineurin is a major target for both CsA and FK506, the results suggest that this phosphatase is involved in the regulation of reactive astrocyte survival.

We appreciate the interest in our recent study of the familial hypertrophic cardiomyopathy in induced pluripotent stem cells.1 Questions were raised about our experimental system, based on unpublished findings using simulations from a computer model of human ventricular myocytes. Christ et al. conclude that discrepancies between the iPSC-derived cardiomyocytes and the computer model indicate either a peculiarity in the specific human we studied or a problem with iPSC-derived cardiomyocytes in general. We readily agree that some properties in our HCM cells may be patient specific: this is part of the promise of personalized medicine. However, we propose an alternative interpretation for the discrepancy between iPSC-derived cardiomyocytes and model simulations: the model is not an accurate representation of the cardiomyocytes under study. Nevertheless, computer models can, and should, be used in conjunction with these …

Topotecan is a topoisomerase (topo) I inhibitor with promising activity in preclinical studies. We hypothesized that low-dose intratumoral delivery of topotecan would be highly effective for gliomas. Human glioma cell lines (U87, U138 and U373) displayed different sensitivities to topotecan (IC50 range: 0.037 μM to 0.280 μM) in cell culture. The most resistant of the glioma cell lines (U87) was implanted stereotactically into the brains of nude rats. Twelve days later, at which time tumor diameter measured 2 to 2.5 mm, animals were randomized to three groups: group I, intratumoral topotecan infused via osmotic pump (n = 12); group II, intratumoral saline infusion (n = 7); and group III, no treatment (n = 10). Animals were sacrificed when signs of deterioration developed, or at 60 days. Animals in group I had a mean survival time (MST) of > 55 days (range=40–60); whereas, those in groups II and III had MST of 26.1 (range=21–31) and 26.5 (range = 20–30) days, respectively. The differences in survival between group I and each of the other groups were statistically significant (p < 0.0001; Logrank Mantel-Cox). None of the animals that survived 60 days had histological evidence of residual tumor at sacrifice. Measurement of topotecan levels in normal brain revealed cytotoxic concentrations up to 4.5 mm from the site of infusion. This study demonstrates that intratumoral topotecan delivered via an osmotic pump prolongs survival in the U87 human glioma model.

Human induced pluripotent stem cell derived cardiac myocytes (h-iPSC-CMs) are a major advance in drug safety testing and research into human cardiac electrophysiology. However, these cells have potential limitations when used for quantitative action potential (AP) analysis. These cells are a mixture of atrial and ventricular cell types, which can be distinguished, to some extent, by action potential (AP) morphology. However, the spontaneous activity of h-iPSC-CMs results in parameter variability and anomalous pharmacological responses, leading to cell misidentification. The spontaneous behavior is largely due to the absence of an IK1 inwardly rectifying potassium channel (IK1).We examined the effect of using an in silico interface to electronically express this missing IK1. An in silico interface was developed to express synthetic IK1 in cells under whole cell voltage clamp using a variant of the dynamic clamp approach. Electronic IK1 expression resulted in a stable physiological resting potential, eliminated spontaneous activity, reduced spontaneous early and delayed after depolarizations, and decreased AP variability. Stimulated APs had a rapid upstroke and spike and dome morphology, and the readily recognizable repolarization attributes of ventricular and atrial cells. When examining the late components of outward current, we found that APs classified on the basis of AP morphology as “atrial-like” had a significantly larger sustained outward Kv1.5-like component at +50 mV than “ventricular-like” APs (in pA/pF: 1.76 ± 0.47 (n=6) vs. 0.74 ± 0.07 (n=7), p 0.4). These data indicate that h-IPSC-CM myocytes with synthetic expression of IK1 can easily be differentiated into ventricular-like and atrial-like myocytes by AP morphology, and that atrial-like cells exhibit an atrial-specific current.