Multidrug resistance in Helicobacter pylori infection.

Helicobacter pylori (Hp), a well-known human pathogen, causes one of the most common chronic bacterial infections and plays an important role in the emergence of chronic progressive gastric inflammation and a variety of gastrointestinal diseases. The prevalence of Hp infection varies worldwide and is indirectly proportional to socio-economic status, especially during childhood. The response to the eradication therapy significantly depends on the antibiotic resistance specific to each geographical region; thus, currently, given the increasing prevalence of antimicrobial resistance (especially to clarithromycin, metronidazole, and levofloxacin), successful treatment for Hp eradication has become a real challenge and a critical issue. The most incriminated factors associated with multidrug resistance (MDR) in Hp proved to be the overuse or the improper use of antibiotics, poor medication adherence, and bacterial-related factors including efflux pumps, mutations, and biofilms. Up to 30% of first-line therapy fails due to poor patient compliance, high gastric acidity, or high bacteremia levels. Hence, it is of great importance to consider new eradication regimens such as vonoprazan-containing triple therapies, quintuple therapies, high-dose dual therapies, and standard triple therapies with probiotics, requiring further studies and thorough assessment. Strain susceptibility testing is also necessary for an optimal approach.

History of Cobaltabis(dicarbollide) in Potentiometry, No Need for Ionophores to Get an Excellent Selectivity.

This work is a mini-review highlighting the relevance of the θ metallabis(dicarbollide) [3,3'-Co(1,2-C2B9H11)2]- with its peculiar and differentiating characteristics, among them the capacity to generate hydrogen and dihydrogen bonds, to generate micelles and vesicles, to be able to be dissolved in water or benzene, to have a wide range of redox reversible couples and many more, and to use these properties, in this case, for producing potentiometric membrane sensors to monitor amine-containing drugs or other nitrogen-containing molecules. Sensors have been produced with this monoanionic cluster [3,3'-Co(1,2-C2B9H11)2]-. Other monoanionic boron clusters are also discussed, but they are much fewer. It is noteworthy that most of the electrochemical sensor species incorporate an ammonium cation and that this cation is the species to be detected. Alternatively, the detection of the borate anion itself has also been studied, but with significantly fewer examples. The functions of the borate anion in the membrane are different, even as a doping agent for polypyrrole which was the conductive ground on which the PVC membrane was deposited. Apart from these cases related to closo borates, the bulk of the work has been devoted to sensors in which the θ metallabis (dicarbollide) [3,3'-Co(1,2-C2B9H11)2]- is the key element. The metallabis (dicarbollide) anion, [3,3'-Co(1,2-C2B9H11)2]-, has many applications; one of these is as new material used to prepare an ion-pair complex with bioactive protonable nitrogen containing compounds, [YH]x[3,3'-Co(1,2-C2B9H11)2]y as an active part of PVC membrane potentiometric sensors. The developed electrodes have Nernstian responses for target analytes, i.e., antibiotics, amino acids, neurotransmitters, analgesics, for some decades of concentrations, with a short response time, around 5 s, a good stability of membrane over 45 days, and an optimal selectivity, even for optical isomers, to be used also for real sample analysis and environmental, clinical, pharmaceutical and food analysis.

Synergistic effect of serotonin 1A and serotonin 1B/D receptor agonists in the treatment of L-DOPA-induced dyskinesia in 6-hydroxydopamine-lesioned rats.

BACKGROUND: The gold standard for symptomatic relief of Parkinson's disease (PD) is L-DOPA. However, long-term treatment often leads to motor complications such as L-DOPA-induced dyskinesia (LID). While amantadine (Gocovri™) is the only approved therapy for dyskinesia in PD patients on the American market, it is associated with neurological side effects and limited efficacy. Thus, there remains a high unmet need for addressing LID in PD patients worldwide. OBJECTIVE: The objective of this study was to evaluate the efficacy, safety and performance compared to approved treatments of the serotonin receptor 1A (5-HT1A) and 5-HT1B/D agonists buspirone and zolmitriptan in the 6-hydroxydopamine unilaterally lesioned rat model for PD. METHODS: The hemiparkinsonian 6-OHDA-lesioned rats underwent chronic treatment with L-DOPA to induce dyskinesia and were subsequently used for efficacy testing of buspirone, zolmitriptan and comparison with amantadine, measured as abnormal involuntary movement (AIM) scores after L-DOPA challenge. Safety testing was performed in model and naïve animals using forelimb adjusting, rotarod and open field tests. RESULTS: 5-HT1A and 5-HT1B/D agonism effectively reduced AIM scores in a synergistic manner. The drug combination of buspirone and zolmitriptan was safe and did not lead to tolerance development following sub-chronic administration. Head-to-head comparison with amantadine showed superior performance of buspirone and zolmitriptan in the model. CONCLUSIONS: The strong anti-dyskinetic effect found with combined 5-HT1A and 5-HT1B/D agonism renders buspirone and zolmitriptan together a meaningful treatment for LID in PD.

Glutaraldehyde-Polymerized Hemerythrin: Evaluation of Performance as an Oxygen Carrier in Hemorrhage Models.

Hemoglobin-based oxygen carriers (HBOCs) have been proposed and tested for several decades for the treatment of hemorrhage. We have previously proposed replacing hemoglobin (Hb) in HBOC with the oxygen-carrying protein hemerythrin (Hr), from marine worms, showing that Hr-based derivatives can perform at least as well or even better than Hb-based HBOC in a range of in vitro assays involving oxidative and nitrosative stress as well as in top-up animal models, where small amounts of Hr- or Hb-HBOC were injected into rats. Here, these experiments are extended to a hemorrhage experiment, in which Hr polymerized with glutaraldehyde, alone or conjugated with human serum albumin, is administered after a loss of 20-30% blood volume. The performance of these preparations is compared with that of Hb-based HBOC measured under the same conditions. Polymerized Hr is found to decrease the survival rate and can hence cannot be used as an oxygen carrier in transfusions. On the other hand, an Hr-albumin copolymer restores survival rates to 100% and generally yields biochemical and histological parameters similar to those of glutaraldehyde-polymerized bovine hemoglobin, with the exception of an acid-base imbalance. The latter may be solved by employing an allogeneic albumin as opposed to the human albumin employed in the present study.

Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.

Evaluating the involvement of cerebral microvascular endothelial Na+/K+-ATPase and Na+-K+-2Cl- co-transporter in electrolyte fluxes in an in vitro blood-brain barrier model of dehydration.

The blood-brain barrier (BBB) is involved in brain water and salt homeostasis. Blood osmolarity increases during dehydration and water is osmotically extracted from the brain. The loss of water is less than expected from pure osmotic forces, due to brain electrolyte accumulation. Although the underlying molecular mechanisms are unresolved, the current model suggests the luminally expressed Na+-K+-2Cl- co-transporter 1 (NKCC1) as a key component, while the role of the Na+/K+-ATPase remains uninvestigated. To test the involvement of these proteins in brain electrolyte flux under mimicked dehydration, we employed a tight in vitro co-culture BBB model with primary cultures of brain endothelial cells and astrocytes. The Na+/K+-ATPase and the NKCC1 were both functionally dominant in the abluminal membrane. Exposure of the in vitro BBB model to conditions mimicking systemic dehydration, i.e. hyperosmotic conditions, vasopressin, or increased [K+]o illustrated that NKCC1 activity was unaffected by exposure to vasopressin and to hyperosmotic conditions. Hyperosmotic conditions and increased K+ concentrations enhanced the Na+/K+-ATPase activity, here determined to consist of the α1 ß1 and α1 ß3 isozymes. Abluminally expressed endothelial Na+/K+-ATPase, and not NKCC1, may therefore counteract osmotic brain water loss during systemic dehydration by promoting brain Na+ accumulation.

Developmental maturation of activity-induced K+ and pH transients and the associated extracellular space dynamics in the rat hippocampus.

KEY POINTS: Neuronal activity induces fluctuation in extracellular space volume, [K+ ]o and pHo , the management of which influences neuronal function The neighbour astrocytes buffer the K+ and pH and swell during the process, causing shrinkage of the extracellular space In the present study, we report the developmental rise of the homeostatic control of the extracellular space dynamics, for which regulation becomes tighter with maturation and thus is proposed to ensure efficient synaptic transmission in the mature animals The extracellular space dynamics of volume, [K+ ]o and pHo evolve independently with developmental maturation and, although all of them are inextricably tied to neuronal activity, they do not couple directly. ABSTRACT: Neuronal activity in the mammalian central nervous system associates with transient extracellular space (ECS) dynamics involving elevated K+ and pH and shrinkage of the ECS. These ECS properties affect membrane potentials, neurotransmitter concentrations and protein function and are thus anticipated to be under tight regulatory control. It remains unresolved to what extent these ECS dynamics are developmentally regulated as synaptic precision arises and whether they are directly or indirectly coupled. To resolve the development of homeostatic control of [K+ ]o , pH, and ECS and their interaction, we utilized ion-sensitive microelectrodes in electrically stimulated rat hippocampal slices from rats of different developmental stages (postnatal days 3-28). With the employed stimulation paradigm, the stimulus-evoked peak [K+ ]o and pHo transients were stable across age groups, until normalized to neuronal activity (field potential amplitude), in which case the K+ and pH shifted significantly more in the younger animals. By contrast, ECS dynamics increased with age until normalized to the field potential, and thus correlated with neuronal activity. With age, the animals not only managed the peak [K+ ]o better, but also displayed swifter post-stimulus removal of [K+ ]o , in correlation with the increased expression of the α1-3 isoforms of the Na+ /K+ -ATPase, and a swifter return of ECS volume. The different ECS dynamics approached a near-identical temporal pattern in the more mature animals. In conclusion, although these phenomena are inextricably tied to neuronal activity, our data suggest that they do not couple directly.

Cotransporter-mediated water transport underlying cerebrospinal fluid formation.

Cerebrospinal fluid (CSF) production occurs at a rate of 500 ml per day in the adult human. Conventional osmotic forces do not suffice to support such production rate and the molecular mechanisms underlying this fluid production remain elusive. Using ex vivo choroid plexus live imaging and isotope flux in combination with in vivo CSF production determination in mice, we identify a key component in the CSF production machinery. The Na+/K+/2Cl- cotransporter (NKCC1) expressed in the luminal membrane of choroid plexus contributes approximately half of the CSF production, via its unusual outward transport direction and its unique ability to directly couple water transport to ion translocation. We thereby establish the concept of cotransport of water as a missing link in the search for molecular pathways sustaining CSF production and redefine the current model of this pivotal physiological process. Our results provide a rational pharmacological target for pathologies involving disturbed brain fluid dynamics.

The α2ß2 isoform combination dominates the astrocytic Na+ /K+ -ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation.

Synaptic activity results in transient elevations in extracellular K+ , clearance of which is critical for sustained function of the nervous system. The K+ clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na+ /K+ -ATPase. The Na+ /K+ -ATPase consists of an α and a ß subunit, each with several isoforms present in the central nervous system, of which the α2ß2 and α2ß1 isoform combinations are kinetically geared for astrocytic K+ clearance. While transcript analysis data designate α2ß2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and ß isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi-quantification was carried out with polyhistidine-tagged Na+ /K+ -ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both ß1 and ß2 and the semi-quantification of the astrocytic fraction indicated that the astrocytic Na+ /K+ -ATPase is dominated by α2, paired with ß1 or ß2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine-associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2+/G301R mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K+ dynamics.

Managing Brain Extracellular K(+) during Neuronal Activity: The Physiological Role of the Na(+)/K(+)-ATPase Subunit Isoforms.

During neuronal activity in the brain, extracellular K(+) rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K(+) is the Na(+)/K(+)-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na(+)/K(+)-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K(+) from neurons, whereas the neurons themselves become the primary K(+) absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na(+)/K(+)-ATPase are, partly, determined by the accessory ß subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K(+) from the extracellular space in the face of neuronal activity. Understanding the nature, impact and effects of the various Na(+)/K(+)-ATPase isoform combinations in K(+) management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2) and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood). This review addresses aspects of the Na(+)/K(+)-ATPase in the regulation of extracellular K(+) in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease.

Voltammetric and amperometric determination of 2,4-dinitrophenol metabolites.

Methods for determination of 2-amino-4-nitrophenol and 4-amino-2-nitrophenol, metabolites of 2,4-dinitrophenol, were developed using differential pulse (DP) voltammetry and HPLC with amperometric and spectrophotometric detection. The applicability of these methods was tested by the determination of the analytes in model samples of urine after preliminary separation by solid-phase extraction. Voltammetry enabled parallel determination of both analytes, but its application in real matrix was severely limited due to the interference of other compounds present in urine. HPLC allowed the determination in real urine matrix down to micromolar concentrations; amperometric detection proved to be more sensitive and selective than the spectrophotometric one.

Additive tuning of redox potential in metallacarboranes by sequential halogen substitution.

The first artificially made set of electron acceptors is presented that are derived from a unique platform Cs[3,3'-Co(C(2)B(9)H(11))(2)], for which the redox potential of each differs from its predecessor by a fixed amount. The sequence of electron acceptors is made by substituting one, two, or more hydrogen atoms by chlorine atoms, yielding Cs[3,3'-Co(C(2)B(9)H(11-y)Cl(y))(C(2)B(9)H(11-z)Cl(z))]. The higher the number of chlorine substituents, the more prone the platform is to be reduced. The effect is completely additive, so if a single substitution implies a reduction of 0.1 V of the redox potential of the parent complex, then ten substitutions imply a reduction of 1 V.

Cobaltabisdicarbollide anion receptor for enantiomer-selective membrane electrodes.

A potentiometric sensor for enantiomers has been prepared with no chiral additives, only cobaltabisdicarbollide and the protonated enantiomer.

Application of the cobaltabisdicarbollide anion to the development of ion selective PVC membrane electrodes for tuberculosis drug analysis.

The cobaltabisdicarbollide anion is used as a new material able to generate an ion-pair complex used for PVC membrane ion selective electrodes.

Determination of cobalt in pharmaceutical products.

The aim of this paper is to determine the content of cobalt in pharmaceutical products (B(12) vitamin powder, B(12) ampoules, Centrum, Spectrum ABC and Optima Forte) by spectrometric (FAAS, GFAAS and ICP-AES) and electrometric (AdSV) analytical techniques. The samples (approximately 0.5g) were treated with a mixture of 6mL HNO(3) and 1mL H(2)O(2) in the microwave oven. Due to the matrix effects the method of standard addition is preferred. The validity of the methods was tested by recovery studies of standard addition and results were found to be satisfactory.

Analytical contributions to the evaluation of painting authenticity from Princely church of Curtea de Arges.

The present study contains the analyses performed for pigment samples taken from the Princely church of Curtea de Arges, one of the oldest churches in Romania. The results of our investigations have shown the source of these samples, thus being identified the pigments: natural ultramarine, cinnabar, red earth, and calcium carbonate in the painting from the 14th century, the pigments: lead white, zinc white, and Prussian blue in the repainting from the 19th century and the pigments zinc white, titanium dioxide white, bone white, yellow ochre, red ochre, green earth, artificial ultramarine, and mars red in the interventions carried out in the 20th century. The analyses consisted of light microscopy (LM) and microchemical tests, as well as energy dispersive X-ray (EDX) analysis. This system of analyses allows one to precisely determine the authenticity of certain pigments, thus avoiding the dating errors for different interventions carried out on the original mural painting from the Saint Nicholas Princely church of Curtea de Arges.

Differential pulse cathodic stripping voltammetric determination of selenium in pharmaceutical products.

The aim of this paper is to determine selenium from pharmaceutical products by differential pulse cathodic stripping voltammetry. Firstly, were established the optimum parameters for voltammetric determination of selenium (electrolyte, deposition time, pulse duration, pulse amplitude, etc.) and secondly, the content of selenium was determined in five pharmaceutical products. The drug samples were treated with a mixture of 6 ml HNO(3) and 1 ml H(2)O(2) in the microwave oven. Due to the matrix effects the method of addition is preferred. The peak potential is -0.545 V vs. Ag/AgCl, and the calibration curve is linear up to 0.125 ng/ml, but selenium was determined from pharmaceutical products used the calibration curve in the range 8-64 ng/ml, due to the concentration of selenium in these tablets.