Base-Paired and Base-Stacked Structures of the Anti-HIV Drug Lamivudine: A Nucleoside DNA-Mimicry with Unprecedented Topology

We have prepared a DNA-mimicry of nucleosides in which the anti-HIV drug lamivudine (beta-L-2',3'-dideoxy-3'-thiacytidine, 3TC) self-assembles into a base-paired and helically base-stacked hexagonal structure. Face-to-face and face-to-tail stacked 3TC=3TC dimers base-paired through two hydrogen bonds between neutral cytosines by either N-H center dot center dot center dot O or N-H center dot center dot center dot N atoms give rise to a right-handed DNA-mimicry of lamivudine with an unusual highly symmetric hexagonal lattice and topology. In addition, a base-paired and base-stacked supramolecular architecture of lamivudine hemihydrochloride hemihydrate was also obtained as a result of our crystal screenings. This structure is formed through partially face-to-face stacked lamivudine pairs held together by protonated and neutral fragments. However, no helical stacking occurs in this structure in which lamivudine also adopts unusual conformations as the C1'-endo and C1'-exo sugar puckers and cytosine orientations intermediate between the anti and syn conformations. As a conclusion drawn from the nucleoside duplex, the hexagonal DNA-mimicry of lamivudine reveals that such double-stranded helices can be assembled without counterions and organic solvents but with higher crystallographic symmetry instead, because only water crystallizes together with lamivudine in this structure.

Lamivudine salts with improved solubilities

To optimize solubility of drugs, current strategies mainly focus on engineering and screening of smart crystal phases. Two salts of the anti-human immunodeficiency virus (HIV) drug lamivudinenamely, lamivudine hydrochloride and lamivudine hydrochloride monohydrate, were prepared in the course of screening the crystallization conditions of lamivudine duplex, an uncommon DNA-mimic, double-stranded helical structure made up of partially protonated drug pairs. Here, water solubilities of lamivudine hydrochloride, lamivudine hydrochloride monohydrate, and lamivudine duplex are reported. The aqueous solubility of this anti-HIV drug was significantly increased in both salts and also in lamivudine duplex in relation to the water solubility of lamivudine form II. In comparison with the lamivudine form II incorporated into therapeutic formulations, the drug solubility was increased at a temperature of 299 +/- 2 K by factors of 1.2, 3.3, and 4.5 in lamivudine hydrochloride, lamivudine hydrochloride monohydrate, and lamivudine duplex, respectively, demonstrating that this solid-state property of lamivudine can be improved by crystal engineering strategies. Solubility profiles were understood on the basis of structural and solventsolute interaction approaches. At last, correlations between solubility and crystal structures allowed for a rational approach to understand how this physicochemical feature could be enhanced by engineering new salts of the drug. (C) 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:21432154, 2012

Aspergillus fumigatus calcineurin interacts with a nucleoside diphosphate kinase

The Ca2+-calcineurin pathway affects virulence and morphogenesis in filamentous fungi. Here, we identified 37 CalA-interacting proteins that interact with the catalytic subunit of calcineurin (CalA) in Aspergillus fumigatus, including the nucleoside diphosphate kinase (SwoH). The in vivo interaction between CalA and SwoH was validated by bimolecular fluorescence complementation. A. fumigatus swoH is an essential gene. Therefore, a temperature-sensitive conditional mutant strain with a point mutation in the active site, SwoH(V83F), was constructed, which demonstrated reduced growth and increased sensitivity to elevated temperatures. The SwoH(V83F) mutation did not cause a loss in virulence in the Galleria mellonella infection model. Taken together these results imply that CalA interacts with SwoH. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Corrosion protection of aluminium alloy by cerium conversion and conducting polymer duplex coatings

The corrosion protection of AA6063 aluminium alloy by cerium conversion, polyaniline conducting polymer and by duplex coatings has been investigated. The electrochemical behaviour was evaluated in aerated 3.5 wt.% NaCl. All coatings tested shifted the corrosion and pitting potentials to more positive values, indicating protection against corrosion. The duplex coatings are significantly more effective than each coating alone: corrosion and pitting potentials were shifted by +183 and +417 mV(SCE), respectively, by duplex coatings in relation to the untreated aluminium alloy. Optical microscopy and scanning electron microscopy are in agreement with the electrochemical results, reinforcing the superior performance of duplex coatings. (C) 2012 Elsevier Ltd. All rights reserved.

Laparoscopic ureteropyeloanastomosis in the treatment of duplex system

Purpose: Duplex system is one of the most common anomalies of upper urinary tract. Anatomical and clinical presentation determine its treatment. Usually, the upper moiety has a poor function and requires resection, but when it is not significantly impaired, preservation is recommended. Laparoscopic reconstruction with upper pole preservation is presented as an alternative treatment. Materials and Methods: Four female patients with duplex system, one presenting with recurrent urinary tract infection and the others with urinary incontinence associated to infrasphincteric ectopic ureter, were treated. Surgical procedure envolved a laparoscopic ureteropyeloanastomosis of the upper pole ureter to the pelvis of the lower moiety, with prior insertion of a double J stent. Results: Surgical time varied from 120 to 150 minutes, with minimal blood loss in all cases. Follow-up varied from 15 to 30 months, with resolution of the clinical symptoms and preservation of the upper moiety function. Conclusion: Laparoscopic ureteropyeloanatomosis is a feasible and safe minimally invasive option in the treatment of duplex system.

Capillary bioreactors based on human purine nucleoside phosphorylase: A new approach for ligands identification and characterization

The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The Km value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255 +/- 29.2 mu M and 133 +/- 114.9 mu M, respectively). A new fourth-generation immucillin derivative (DI4G: IC50 = 40.6 +/- 0.36 nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay. (C) 2011 Elsevier B.V. All rights reserved.

Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2'deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2'deoxy) ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5' hydroxyl group of adenosine and Arg(43*) side chain contributes for the ribosyl radical to adopt an unusual C3'-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl-6 and Br-8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser(90) by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr(91)) is responsible for the lack of negative cooperativity of phosphate binding in this enzyme.

Brazeability of UNS 32101 and UNS S32304 lean duplex stainless steels

CAPES

Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

Background: Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host. Methods: Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated. Results: The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 mu M) and PPADS (50 mu M) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 mu M), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 mu M) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 mu M), TNP-ATP (50 mu M) or the purinergic blockers KN-62 (10 mu M) and Ip5I (10 mu M). Incubating P. berghei infected cells with KN-62 (200 mu M) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 mu M) led to an increase in rings forms (82% +/- 4, n = 11) and a decrease in trophozoite forms (18% +/- 4, n = 11). Conclusions: The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.

Structural and kinetic studies of Schistosoma mansoni adenylate kinases

The human parasite Schistosoma mansoni is totally dependent on the purine salvage pathway in order to supply large quantities of purine precursors for its energy and DNA biosynthetic needs. Adenylate kinase (ADK) is responsible for the conversion of AMP (produced by the adenosine kinase reaction) into ADP, which is subsequently converted into ATP by nucleoside diphosphate kinase (NDPK). ADK and NDPK are the most active enzymes of the pathway, probably reflecting an evolutionary adaptation due to the intense use of the branch in which they participate. However, notwithstanding their importance very little information has been accumulated found regarding these enzymes. In this work two adenylate kinases from S. mansoni were cloned and heterologously expressed in Escherichia coil. The purified products were utilized in activity assays, and displayed kinetic parameters similar to the corresponding human orthologous proteins. The cytosolic S. mansoni ADK was crystallized and its structure solved allowing us to detect a difference in the nucleotide binding site when compared with the human ortholog. (C) 2012 Elsevier B.V. All rights reserved.

Sonothrombolysis for acute ischemic stroke: a systematic review of randomized controlled trials

Object. Sonothrombolysis has recently been considered an emerging modality for the treatment of stroke. The purpose of the present paper was to review randomized clinical studies concerning the effects of sonothrombolysis associated with tissue plasminogen activator (tPA) on acute ischemic stroke. Methods. Systematic searches for literature published between January 1996 and July 2011 were performed for studies regarding sonothrombolysis combined with tPA for acute ischemic stroke. Only randomized controlled trials were included. Data extraction was based on ultrasound variables, patient characteristics, and outcome variables (rate of intracranial hemorrhages and arterial recanalization). Results. Four trials were included in this study; 2 trials evaluated the effect of transcranial Doppler (TCD) ultrasonography on sonothrombolysis, and 2 addressed transcranial color-coded duplex (TCCD) ultrasonography. The frequency of ultrasound waves varied from 1.8 to 2 MHz. The duration of thrombus exposure to ultrasound energy ranged from 60 to 120 minutes. Sample sizes were small, recanalization was evaluated at different time points (60 and 120 minutes), and inclusion criteria were heterogeneous. Sonothrombolysis combined with tPA did not lead to an increase in symptomatic intracranial hemorrhagic complications. Two studies demonstrated that patients treated with ultrasound combined with tPA had statistically significant higher rates of recanalization than patients treated with tPA alone. Conclusions. Despite the heterogeneity and the limitations of the reviewed studies, there is evidence that sonothrombolysis associated with tPA is a safe procedure and results in an increased rate of recanalization in the setting of acute ischemic stroke when wave frequencies and energy intensities of diagnostic ultrasound systems are used. (http://thejns.org/doi/abs/10.3171/2011.10.FOCUS11251)

Brazilian guidelines for the application of transcranial ultrasound as a diagnostic test for the confirmation of brain death

Neurosonological studies, specifically transcranial Doppler (TCD) and transcranial color-coded duplex (TCCD), have high level of specificity and sensitivity and they are used as complementary tests for the diagnosis of brain death (BD). A group of experts, from the Neurosonology Department of the Brazilian Academy of Neurology, created a task force to determine the criteria for the following aspects of diagnosing BD in Brazil: the reliability of TCD methodology; the reliability of TCCD methodology; neurosonology training and skills; the diagnosis of encephalic circulatory arrest; and exam documentation for BD. The results of this meeting are presented in the current paper.

Biowaiver monographs for immediate-release solid oral dosage forms: Stavudine

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate-release (IR) solid oral dosage forms containing stavudine (d4T) are reviewed. According to Biopharmaceutics Classification System (BCS), d4T can be assigned to BCS class I. No problems with BE of IR d4T formulations containing different excipients and produced by different manufacturing methods have been reported and, hence, the risk of bioinequivalence caused by these factors appears to be low. Furthermore, d4T has a wide therapeutic index. It is concluded that a biowaiver is appropriate for IR solid oral dosage forms containing d4T as the single active pharmaceutical ingredient (API) provided that (a) the test product contains only excipients present in the IR d4T drug products that have been approved in a number of countries for the same dosage form, and (b) both test product and its comparator are either very rapidly dissolving or rapidly dissolving with similarity of dissolution profiles demonstrated at pH 1.2, 4.5, and 6.8. (c) 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:1016, 2012

Leishmania Metacyclogenesis Is Promoted in the Absence of Purines

Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that "stressful conditions" will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.

Evaluation of inadequate anti-retroviral treatment in patients with HIV/AIDS

Introduction: Since the emergence of antiretroviral therapy, the survival of patients infected with human immunodeficiency virus has increased. Non-adherence to this therapy is directly related to treatment failure, which allows the emergence of resistant viral strains. Methods: A retrospective descriptive study of the antiretroviral dispensing records of 229 patients from the Center for Health Care, University Hospital, Federal University of Juiz de Fora, Brazil, was conducted between January and December 2009. Results: The study aimed to evaluate patient compliance and determine if there was an association between non-adherence and the therapy. Among these patients, 63.8% were men with an average age of 44.0 +/- 9.9 years. The most used treatment was a combination of 2 nucleoside reverse transcriptase inhibitors with 1 non-nucleoside reverse transcriptase inhibitor (55.5%) or with 2 protease inhibitors (28.8%). It was found that patients taking lopinavir/ritonavir with zidovudine and lamivudine had a greater frequency of inadequate treatment than those taking atazanavir with zidovudine and lamivudine (85% and 83.3%, respectively). Moreover, when the combination of zidovudine/lamivudine was used, the patients were less compliant (chi(2) = 4.468, 1 degree of freedom, p = 0.035). Conclusions: The majority of patients failed to correctly adhere to their treatment; therefore, it is necessary to implement strategies that lead to improved compliance, thus ensuring therapeutic efficacy and increased patient survival.