Publications by authors named "Julio Martin"

59 Publications

Scaffold-Hopping Strategy on a Series of Proteasome Inhibitors Led to a Preclinical Candidate for the Treatment of Visceral Leishmaniasis.

J Med Chem 2021 Apr 27. Epub 2021 Apr 27.

Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry, University of Dundee, Dundee DD1 5EH, U.K.

There is an urgent need for new treatments for visceral leishmaniasis (VL), a parasitic infection which impacts heavily large areas of East Africa, Asia, and South America. We previously reported on the discovery of GSK3494245/DDD01305143 () as a preclinical candidate for VL and, herein, we report on the medicinal chemistry program that led to its identification. A hit from a phenotypic screen was optimized to give a compound with efficacy, which was hampered by poor solubility and genotoxicity. The work on the original scaffold failed to lead to developable compounds, so an extensive scaffold-hopping exercise involving medicinal chemistry design, profiling, and subsequent synthesis was utilized, leading to the preclinical candidate. The compound was shown to act via proteasome inhibition, and we report on the modeling of different scaffolds into a cryo-EM structure and the impact this has on our understanding of the series' structure-activity relationships.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00047DOI Listing
April 2021

Multiple unbiased approaches identify oxidosqualene cyclase as the molecular target of a promising anti-leishmanial.

Cell Chem Biol 2021 Feb 26. Epub 2021 Feb 26.

Division of Biological Chemistry and Drug Discovery, Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Electronic address:

Phenotypic screening identified a benzothiophene compound with activity against Leishmania donovani, the causative agent of visceral leishmaniasis. Using multiple orthogonal approaches, oxidosqualene cyclase (OSC), a key enzyme of sterol biosynthesis, was identified as the target of this racemic compound and its enantiomers. Whole genome sequencing and screening of a genome-wide overexpression library confirmed that OSC gene amplification is associated with resistance to compound 1. Introduction of an ectopic copy of the OSC gene into wild-type cells reduced susceptibility to these compounds confirming the role of this enzyme in resistance. Biochemical analyses demonstrated the accumulation of the substrate of OSC and depletion of its product in compound (S)-1-treated-promastigotes and cell-free membrane preparations, respectively. Thermal proteome profiling confirmed that compound (S)-1 binds directly to OSC. Finally, modeling and docking studies identified key interactions between compound (S)-1 and the LdOSC active site. Strategies to improve the potency for this promising anti-leishmanial are proposed.
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http://dx.doi.org/10.1016/j.chembiol.2021.02.008DOI Listing
February 2021

The Upper-Arm Basilic-Cephalic Loop: A Valueable Alternative for Below-Knee Arterial Reconstruction.

Vasc Endovascular Surg 2021 May 22;55(4):348-354. Epub 2021 Jan 22.

Department of Vascular and Endovascular Surgery, University Hospital Salzburg, 31507Paracelsus Medical University, Salzburg, Austria.

Introduction: Despite advances of endovascular interventions, bypass surgery remains the gold standard for treatment of long and complex arterial occlusions in the lower limb. Autologous vein is regarded superior to other options. As the graft of first choice, the great saphenous vein (GSV) is often not available due to previous bypass, stripping or poor quality. Other options like arm veins (AV) are important alternatives. As forearm portions of AVs are often unusable, a graft created from the upper arm basilic and cephalic veins provides a valuable alternative.

Patients And Methods: We analyzed consecutive patients treated at an academic tertiary referral center between 01/1998 and 07/2018 using arm veins as the main peripheral bypass graft. Study endpoints were primary patency, secondary patency, limb salvage and survival.

Results: In the observed time period 2702 bypass procedures were performed at our institution for below-knee arterial reconstructions. Vein grafts used included the ipsilateral GSV (iGSV; n = 1937/71.7%), contralateral GSV (cGSV; 192/7.1%), small saphenous vein (SSV; 133/4.9%), prosthetic conduits (61/2.3%) and different configurations of AV (379/14%). In the majority of patients receiving AV grafts a complete continuous cephalic or basilic vein (CAV) was used (n = 292/77%). If it was not possible to use major parts of these 2 veins, either spliced arm vein grafts (SAV) (42/11%) or an upper arm basilic-cephalic loop graft (45/12%) were used. Median follow-up was 27 (interquartile range: 8-50) months. After 3 years secondary patency (CAV: 85%; SAV: 62%; loop: 66%; p = 0.125) and limb salvage rates (CAV: 79%, SAV: 68%; loop: 79%; p = 0.346) were similar between the 3 bypass options.

Conclusion: The encouraging results of alternative AV configurations highlight their value in case the basilic or cephalic veins are not useable in continuity. Especially for infragenual redo-bypass procedures, these techniques should be considered to offer patients durable revascularization options.
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http://dx.doi.org/10.1177/1538574420980610DOI Listing
May 2021

Combined Preimplantation Genetic Testing for Autosomal Dominant Polycystic Kidney Disease: Consequences for Embryos Available for Transfer.

Genes (Basel) 2020 06 24;11(6). Epub 2020 Jun 24.

Igenomix, 46980 Valencia, Spain.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and presents with genetic and clinical heterogeneity. ADPKD can also manifest extra-renally, and seminal cysts have been associated with male infertility in some cases. ADPKD-linked male infertility, along with female age, have been proposed as factors that may influence the clinical outcomes of preimplantation genetic testing (PGT) for monogenic disorders (PGT-M). Large PGT for aneuploidy assessment (PGT-A) studies link embryo aneuploidy to increasing female age; other studies suggest that embryo aneuploidy is also linked to severe male-factor infertility. We aimed to assess the number of aneuploid embryos and the number of cycles with transferable embryos in ADPKD patients after combined-PGT. The combined-PGT protocol, involving PGT-M by PCR and PGT-A by next-generation sequencing, was performed in single trophectoderm biopsies from 289 embryos in 83 PGT cycles. Transferable embryos were obtained in 69.9% of cycles. The number of aneuploid embryos and cycles with transferable embryos did not differ when the male or female had the ADPKD mutation. However, a significantly higher proportion of aneuploid embryos was found in the advanced maternal age (AMA) group, but not in the male factor (MF) group, when compared to non-AMA and non-MF groups, respectively. Additionally, no significant differences in the percentage of cycles with transferable embryos were found in any of the groups. Our results indicate that AMA couples among ADPKD patients have an increased risk of aneuploid embryos, but ADPKD-linked male infertility does not promote an increased aneuploidy rate.
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http://dx.doi.org/10.3390/genes11060692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349812PMC
June 2020

The Q Site of Cytochrome is a Promiscuous Drug Target in and .

ACS Infect Dis 2020 03 30;6(3):515-528. Epub 2020 Jan 30.

Division of Biological Chemistry and Drug Discovery, Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.

Available treatments for Chagas' disease and visceral leishmaniasis are inadequate, and there is a pressing need for new therapeutics. Drug discovery efforts for both diseases principally rely upon phenotypic screening. However, the optimization of phenotypically active compounds is hindered by a lack of information regarding their molecular target(s). To combat this issue we initiate target deconvolution studies at an early stage. Here, we describe comprehensive genetic and biochemical studies to determine the targets of three unrelated phenotypically active compounds. All three structurally diverse compounds target the Q active-site of cytochrome , part of the cytochrome complex of the electron transport chain. Our studies go on to identify the Q site as a promiscuous drug target in and with a propensity to rapidly mutate. Strategies to rapidly identify compounds acting via this mechanism are discussed to ensure that drug discovery portfolios are not overwhelmed with inhibitors of a single target.
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http://dx.doi.org/10.1021/acsinfecdis.9b00426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076694PMC
March 2020

Towards discovery of new leishmanicidal scaffolds able to inhibit GSK-3.

J Enzyme Inhib Med Chem 2020 Dec;35(1):199-210

Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain.

Previous reports have validated the glycogen synthase kinase-3 (GSK-3) as a druggable target against the human protozoan parasite . This prompted us to search for new leishmanicidal scaffolds as inhibitors of this enzyme from our in-house library of human GSK-3β inhibitors, as well as from the Leishbox collection of leishmanicidal compounds developed by GlaxoSmithKline. As a result, new leishmanicidal inhibitors acting on GSK-3 at micromolar concentrations were found. These inhibitors belong to six different chemical classes (thiadiazolidindione, halomethylketone, maleimide, benzoimidazole, -phenylpyrimidine-2-amine and oxadiazole). In addition, the binding mode of the most active compounds into GSK-3 was approached using computational tools. On the whole, we have uncovered new chemical scaffolds with an appealing prospective in the development and use of GSK-3 inhibitors against this infectious protozoan.
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http://dx.doi.org/10.1080/14756366.2019.1693704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882465PMC
December 2020

Identification of Small Molecules Disrupting the Ubiquitin Proteasome System in Malaria.

ACS Infect Dis 2019 12 7;5(12):2105-2117. Epub 2019 Nov 7.

Tres Cantos Medicines Development Campus, Diseases of the Developing World . GlaxoSmithKline , Severo Ochoa 2 , Tres Cantos , 28760 Madrid , Spain.

The ubiquitin proteasome system (UPS) is one of the main proteolytic pathways in eukaryotic cells, playing an essential role in key cellular processes such as cell cycling and signal transduction. Changes in some of the components of this pathway have been implicated in various conditions, including cancer and infectious diseases such as malaria. The success of therapies based on proteasome inhibitors has been shown in human clinical trials. In addition to its proven tractability, the essentiality of the UPS underlines its potential as a source of targets to identify new antimalarial treatments. Two assays, previously developed to quantify the parasite protein ubiquitylation levels in a high throughput format, have been used to identify compounds that inhibit parasite growth by targeting UPS. Among the positive hits, specific inhibitors of the proteasome have been identified and characterized. Hits identified using this approach may be used as starting points for development of new antimalarial drugs. They may also be used as tools to further understand proteasome function and to identify new targets in UPS.
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http://dx.doi.org/10.1021/acsinfecdis.9b00216DOI Listing
December 2019

Novel chemical starting points for drug discovery in leishmaniasis and Chagas disease.

Int J Parasitol Drugs Drug Resist 2019 08 22;10:58-68. Epub 2019 May 22.

Calibr at Scripps Research, La Jolla, CA, USA. Electronic address:

Visceral leishmaniasis (VL) and Chagas disease (CD) are caused by kinetoplastid parasites that affect millions of people worldwide and impart a heavy burden against human health. Due to the partial efficacy and toxicity-related limitations of the existing treatments, there is an urgent need to develop novel therapies with superior efficacy and safety profiles to successfully treat these diseases. Herein we report the application of whole-cell phenotypic assays to screen a set of 150,000 compounds against Leishmania donovani, a causative agent of VL, and Trypanosoma cruzi, the causative agent of CD, with the objective of finding new starting points to develop novel drugs to effectively treat and control these diseases. The screening campaign, conducted with the purpose of global open access, identified twelve novel chemotypes with low to sub-micromolar activity against T. cruzi and/or L. donovani. We disclose these hit structures and associated activity with the goal to contribute to the drug discovery community by providing unique chemical tools to probe kinetoplastid biology and as hit-to-lead candidates for drug discovery.
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http://dx.doi.org/10.1016/j.ijpddr.2019.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545338PMC
August 2019

The differential diagnoses of uterine leiomyomas and leiomyosarcomas using DNA and RNA sequencing.

Am J Obstet Gynecol 2019 10 20;221(4):320.e1-320.e23. Epub 2019 May 20.

Igenomix Foundation-Instituto de Investigación Sanitaria INCLIVA, Valencia, Spain; Department of Pediatrics, Obstetrics and Gynecology, School of Medicine, University of Valencia, Valencia, Spain; Department of Obstetrics and Gynecology, School of Medicine, Stanford University, Stanford, CA.

Background: Although uterine leiomyomas and leiomyosarcomas are considered biologically unrelated tumors, they share morphologic and histologic characteristics that complicate their differential diagnosis. The long-term therapeutic option for leiomyoma is laparoscopic myomectomy with morcellation, particularly for patients who wish to preserve their fertility. However, because of the potential dissemination of undiagnosed or hidden leiomyosarcoma from morcellation, there is a need to develop a preoperative assessment of malignancy risk.

Objective: Through an integrated comparative genomic and transcriptomic analysis, we aim to identify differential genetic targets in leiomyomas vs leiomyosarcomas using next-generation sequencing as the first step toward preoperative differential diagnosis.

Study Design: Targeted sequencing of DNA and RNA coding regions for solid tumor-associated genes was performed on formalin-fixed paraffin-embedded samples from 13 leiomyomas and 13 leiomyosarcoma cases. DNA sequencing was used to identify copy number variations, single-nucleotide variants, and small insertions/deletions. RNA sequencing was used to identify gene fusions, splice variants, and/or differential gene expression profiles.

Results: In leiomyosarcomas, tumor mutation burden was higher in terms of copy number variations, single nucleotide variants, small insertions/deletions, and gene fusions compared with leiomyomas. For copy number variations, 20 genes were affected by deletions in leiomyosarcomas, compared with 6 observed losses in leiomyomas. Gains (duplications) were identified in 19 genes in leiomyosarcomas, but only 3 genes in leiomyomas. The most common mutations (single-nucleotide variants and insertions/deletions) for leiomyosarcomas were identified in 105 genes of all analyzed leiomyosarcomas; 82 genes were affected in leiomyomas. Of note, 1 tumor previously diagnosed as leiomyosarcoma was established as inflammatory myofibroblastic tumor along this study with a novel ALK-TNS1 fusion. Finally, a differential transcriptomic profile was observed for 11 of 55 genes analyzed in leiomyosarcomas; 8.5% of initially diagnosed leiomyosarcomas showed high-confidence, novel gene fusions that were associated with these tumors.

Conclusion: Through integrated comparative genomic and transcriptomic analyses, we identified novel differential genetic targets that potentially differentiate leiomyosarcomas and leiomyomas. This provides a new insight into the differential diagnosis of these myometrial tumors.
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http://dx.doi.org/10.1016/j.ajog.2019.05.018DOI Listing
October 2019

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Proc Natl Acad Sci U S A 2019 05 8;116(19):9318-9323. Epub 2019 Apr 8.

Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.

Visceral leishmaniasis (VL), caused by the protozoan parasites and , is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant and isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the β5 subunit of the proteasome. High-resolution cryo-EM structures of apo and compound 8-bound 20S proteasome reveal a previously undiscovered inhibitor site that lies between the β4 and β5 proteasome subunits. This induced pocket exploits β4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.
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http://dx.doi.org/10.1073/pnas.1820175116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511062PMC
May 2019

Relative Contributions of and Bacteriophage to Bacterial Cell Death under Various Environmental Conditions.

mBio 2018 08 7;9(4). Epub 2018 Aug 7.

School of the Environment, Florida Agricultural and Mechanical University, Tallahassee, Florida, USA

The role of protists and bacteriophages in bacterial predation in the microbial food web has been well studied. There is mounting evidence that and like organisms (BALOs) also contribute to bacterial mortality and, in some cases, more so than bacteriophages. A full understanding of the ecologic function of the microbial food web requires recognition of all major predators and the magnitude of each predator's contribution. Here we investigated the contribution of , one of the BALOs, and bacteriophages when incubated with their common prey, , in a seawater microcosm. We observed that was the greatest responder to the prey, increasing 18-fold with a simultaneous 4.4-log-unit reduction of at 40 h, whereas the bacteriophage population showed no significant increase. In subsequent experiments to formulate a medium that would support the predatory activities and replication of both predators, low-nutrient media favored the predation and replication of the , whereas higher-nutrient media enhanced phage growth. The greatest prey reduction and replication of both and phage were observed in media with moderate nutrient levels. Additional experiments show that the predatory activities of both predators were influenced by environmental conditions, specifically, temperature and salinity. The two predators combined exerted greater control on , a synergism that may be exploited for practical applications to reduce bacterial populations. These findings suggest that along with bacteriophage and protists, has the potential to have a prominent role in bacterial mortality and cycling of nutrients, two vital ecologic functions. Although much has been reported about the marine microbial food web and the role of micropredators, specifically viruses and protists, the contribution of -like predators has largely been ignored, posing a major gap in understanding food web processes. A complete scenario of the microbial food web cannot be developed until the roles of all major micropredators and the magnitude of their contributions to bacterial mortality, structuring of microbial communities, and cycling of nutrients are assessed. Here we show compelling evidence that , a predatory bacterium, is a significant contributor to bacterial death and, in some cases, may rival viruses as agents of bacterial mortality. These results advance current understanding of the microbial loop and top-down control on the bacterial community.
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http://dx.doi.org/10.1128/mBio.01202-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6083911PMC
August 2018

Importance of secondary screening with clinical isolates for anti-leishmania drug discovery.

Sci Rep 2018 08 6;8(1):11765. Epub 2018 Aug 6.

Diseases of the developing world, GlaxoSmithKline, Tres cantos, Spain.

The growing drug resistance (DR) raises major concerns for the control of visceral leishmaniasis (VL), a neglected disease lethal in 95 percent of the cases if left untreated. Resistance has rendered antimonials (SSG) obsolete in the Indian Sub-Continent (ISC) and the first miltefosine-resistant Leishmania donovani were isolated. New chemotherapeutic options are needed and novel compounds are being identified by high-throughput screening (HTS). HTS is generally performed with old laboratory strains such as LdBOB and we aimed here to validate the activity of selected compounds against recent clinical isolates. In this academic/industrial collaboration, 130 compounds from the GSK "Leishbox" were screened against one SSG-sensitive and one SSG-resistant strain of L. donovani recently isolated from ISC patients, using an intracellular assay of L. donovani-infected THP1-derived macrophages. We showed that only 45% of the compounds were active in both clinical isolates and LdBOB. There were also different compound efficiencies linked to the SSG susceptibility background of the strains. In addition, our results suggested that the differential susceptibility profiles were chemical series-dependent. In conclusion, we demonstrate the potential value of including clinical isolates (as well as resistant strains) in the HTS progression cascade.
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http://dx.doi.org/10.1038/s41598-018-30040-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6078976PMC
August 2018

Metabolic Clustering Analysis as a Strategy for Compound Selection in the Drug Discovery Pipeline for Leishmaniasis.

ACS Chem Biol 2018 05 24;13(5):1361-1369. Epub 2018 Apr 24.

Centre for Metabolomics and Bioanalysis (CEMBIO) , Facultad de Farmacia, Universidad CEU San Pablo , Campus Montepríncipe, Boadilla del Monte , 28668 Madrid , Spain.

A lack of viable hits, increasing resistance, and limited knowledge on mode of action is hindering drug discovery for many diseases. To optimize prioritization and accelerate the discovery process, a strategy to cluster compounds based on more than chemical structure is required. We show the power of metabolomics in comparing effects on metabolism of 28 different candidate treatments for Leishmaniasis (25 from the GSK Leishmania box, two analogues of Leishmania box series, and amphotericin B as a gold standard treatment), tested in the axenic amastigote form of Leishmania donovani. Capillary electrophoresis-mass spectrometry was applied to identify the metabolic profile of Leishmania donovani, and principal components analysis was used to cluster compounds on potential mode of action, offering a medium throughput screening approach in drug selection/prioritization. The comprehensive and sensitive nature of the data has also made detailed effects of each compound obtainable, providing a resource to assist in further mechanistic studies and prioritization of these compounds for the development of new antileishmanial drugs.
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http://dx.doi.org/10.1021/acschembio.8b00204DOI Listing
May 2018

Identifying inhibitors of the Leishmania inositol phosphorylceramide synthase with antiprotozoal activity using a yeast-based assay and ultra-high throughput screening platform.

Sci Rep 2018 03 2;8(1):3938. Epub 2018 Mar 2.

Department of Biosciences, Durham University, Stockton Road, Durham, DH1 3LE, UK.

Leishmaniasis is a Neglected Tropical Disease caused by the insect-vector borne protozoan parasite, Leishmania species. Infection affects millions of the world's poorest, however vaccines are absent and drug therapy limited. Recently, public-private partnerships have developed to identify new modes of controlling leishmaniasis. Drug discovery is a significant part of these efforts and here we describe the development and utilization of a novel assay to identify antiprotozoal inhibitors of the Leishmania enzyme, inositol phosphorylceramide (IPC) synthase. IPC synthase is a membrane-bound protein with multiple transmembrane domains, meaning that a conventional in vitro assay using purified protein in solution is highly challenging. Therefore, we utilized Saccharomyces cerevisiae as a vehicle to facilitate ultra-high throughput screening of 1.8 million compounds. Antileishmanial benzazepanes were identified and shown to inhibit the enzyme at nanomolar concentrations. Further chemistry produced a benzazepane that demonstrated potent and specific inhibition of IPC synthase in the Leishmania cell.
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http://dx.doi.org/10.1038/s41598-018-22063-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834442PMC
March 2018

Complex Interplay between Sphingolipid and Sterol Metabolism Revealed by Perturbations to the Leishmania Metabolome Caused by Miltefosine.

Antimicrob Agents Chemother 2018 05 26;62(5). Epub 2018 Apr 26.

Centre for Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad CEU San Pablo, Campus Montepríncipe, Boadilla del Monte, Madrid, Spain

With the World Health Organization reporting over 30,000 deaths and 200,000 to 400,000 new cases annually, visceral leishmaniasis is a serious disease affecting some of the world's poorest people. As drug resistance continues to rise, there is a huge unmet need to improve treatment. Miltefosine remains one of the main treatments for leishmaniasis, yet its mode of action (MoA) is still unknown. Understanding the MoA of this drug and parasite response to treatment could help pave the way for new and more successful treatments for leishmaniasis. A novel method has been devised to study the metabolome and lipidome of axenic amastigotes treated with miltefosine. Miltefosine caused a dramatic decrease in many membrane phospholipids (PLs), in addition to amino acid pools, while sphingolipids (SLs) and sterols increased. promastigotes devoid of SL biosynthesis through loss of the serine palmitoyl transferase gene (ΔLCB2) were 3-fold less sensitive to miltefosine than wild-type (WT) parasites. Changes in the metabolome and lipidome of miltefosine-treated mirrored those of A lack of SLs in the ΔLCB2 mutant was matched by substantial alterations in sterol content. Together, these data indicate that SLs and ergosterol are important for miltefosine sensitivity and, perhaps, MoA.
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http://dx.doi.org/10.1128/AAC.02095-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923112PMC
May 2018

Efforts Aimed To Reduce Attrition in Antimalarial Drug Discovery: A Systematic Evaluation of the Current Antimalarial Targets Portfolio.

ACS Infect Dis 2018 04 24;4(4):568-576. Epub 2018 Jan 24.

Tres Cantos Medicines Development Campus, DDW , GlaxoSmithKline , Severo Ochoa, 2 , 28760 Tres Cantos , Madrid , Spain.

Malaria remains a major global health problem. In 2015 alone, more than 200 million cases of malaria were reported, and more than 400,000 deaths occurred. Since 2010, emerging resistance to current front-line ACTs (artemisinin combination therapies) has been detected in endemic countries. Therefore, there is an urgency for new therapies based on novel modes of action, able to relieve symptoms as fast as the artemisinins and/or block malaria transmission. During the past few years, the antimalarial community has focused their efforts on phenotypic screening as a pragmatic approach to identify new hits. Optimization efforts on several chemical series have been successful, and clinical candidates have been identified. In addition, recent advances in genetics and proteomics have led to the target deconvolution of phenotypic clinical candidates. New mechanisms of action will also be critical to overcome resistance and reduce attrition. Therefore, a complementary strategy focused on identifying well-validated targets to start hit identification programs is essential to reinforce the clinical pipeline. Leveraging published data, we have assessed the status quo of the current antimalarial target portfolio with a focus on the blood stage clinical disease. From an extensive list of reported Plasmodium targets, we have defined triage criteria. These criteria consider genetic, pharmacological, and chemical validation, as well as tractability/doability, and safety implications. These criteria have provided a quantitative score that has led us to prioritize those targets with the highest probability to deliver successful and differentiated new drugs.
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http://dx.doi.org/10.1021/acsinfecdis.7b00211DOI Listing
April 2018

Remotely delivering real-time parent training to the home: An initial randomized trial of Internet-delivered parent-child interaction therapy (I-PCIT).

J Consult Clin Psychol 2017 Sep 26;85(9):909-917. Epub 2017 Jun 26.

Judge Baker Children's Center, Harvard Medical School.

Objective: Remote technologies are increasingly being leveraged to expand the reach of supported care, but applications to early child-behavior problems have been limited. This is the first controlled trial examining video-teleconferencing to remotely deliver behavioral parent training to the home setting with a live therapist.

Method: Racially/ethnically diverse children ages 3-5 years with disruptive behavior disorders, and their caregiver(s), using webcams and parent-worn Bluetooth earpieces, participated in a randomized trial comparing Internet-delivered parent-child interaction therapy (I-PCIT) versus standard clinic-based PCIT (N = 40). Major assessments were conducted at baseline, midtreatment, posttreatment, and 6-month follow-up. Linear regressions and hierarchical linear modeling using maximum-likelihood estimation were used to analyze treatment satisfaction, diagnoses, symptoms, functioning, and burden to parents across conditions.

Results: Intent-to-treat analyses found 70% and 55% of children treated with I-PCIT and clinic-based PCIT, respectively, showed "treatment response" after treatment, and 55% and 40% of children treated with I-PCIT and clinic-based PCIT, respectively, continued to show "treatment response" at 6-month follow-up. Both treatments had significant effects on children's symptoms and burden to parents, and many effects were very large in magnitude. Most outcomes were comparable across conditions, except that the rate of posttreatment "excellent response" was significantly higher in I-PCIT than in clinic-based PCIT, and I-PCIT was associated with significantly fewer parent-perceived barriers to treatment than clinic-based PCIT. Both treatments were associated with positive engagement, treatment retention, and very high treatment satisfaction.

Conclusion: Findings build on the small but growing literature supporting the promising role of new technologies for expanding the delivery of behavioral parent training. (PsycINFO Database Record
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http://dx.doi.org/10.1037/ccp0000230DOI Listing
September 2017

Unravelling the rate of action of hits in the Leishmania donovani box using standard drugs amphotericin B and miltefosine.

PLoS Negl Trop Dis 2017 May 25;11(5):e0005629. Epub 2017 May 25.

Kinetoplastids DPU, Diseases of the Developing World (DDW), GlaxoSmithKline, Tres Cantos, Madrid, Spain.

In recent years, the neglected diseases drug discovery community has elected phenotypic screening as the key approach for the identification of novel hit compounds. However, when this approach is applied, important questions related to the mode of action for these compounds remain unanswered. One of such questions is related to the rate of action, a useful piece of information when facing the challenge of prioritising the most promising hit compounds. In the present work, compounds of the "Leishmania donovani box" were evaluated using a rate of action assay adapted from a replicative intracellular high content assay recently developed. The potency of each compound was determined every 24 hours up to 96 hours, and standard drugs amphotericin B and miltefosine were used as references to group these compounds according to their rate of action. Independently of this biological assessment, compounds were also clustered according to their minimal chemical scaffold. Comparison of the results showed a complete correlation between the chemical scaffold and the biological group for the vast majority of compounds, demonstrating how the assay was able to bring information on the rate of action for each chemical series, a property directly linked to the mode of action. Overall, the assay here described permitted us to evaluate the rate of action of the "Leishmania donovani box" using two of the currently available drugs as references and, also, to propose a number of fast-acting chemical scaffolds present in the box as starting points for future drug discovery projects to the wider scientific community. The results here presented validate the use of this assay for the determination of the rate of action early in the discovery process, to assist in the prioritisation of hit compounds.
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http://dx.doi.org/10.1371/journal.pntd.0005629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5462473PMC
May 2017

A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani.

Antimicrob Agents Chemother 2016 06 23;60(6):3524-32. Epub 2016 May 23.

Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identified in vitro did not translate their activities when tested in in vivo models, highlighting the need to develop more predictive in vitro assays. In the present work, we describe the development of a robust replicative, high-content, in vitro intracellular L. donovani assay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypic in vitro infection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation. In vitro and in vivo results for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay.
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http://dx.doi.org/10.1128/AAC.01781-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879429PMC
June 2016

Compound profiling and 3D-QSAR studies of hydrazone derivatives with activity against intracellular Trypanosoma cruzi.

Bioorg Med Chem 2016 Apr 23;24(8):1608-18. Epub 2016 Feb 23.

Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal de Pernambuco, 50740-520 Recife, PE, Brazil. Electronic address:

Chagas disease is a tropical disease caused by the parasite Trypanosoma cruzi, which is endemic in Central and South America. Few treatments are available with effectiveness limited to the early (acute) stage of disease, significant toxicity and widespread drug resistance. In this work we report the outcome of a HTS-ready assay chemical library screen to identify novel, nontoxic, small-molecule inhibitors of T. cruzi. We have selected 50 compounds that possess hydrazone as a common group. The compounds were screened using recombinant T. cruzi (Tulahuen strain) expressing beta-galactosidase. A 3D quantitative structure-activity relationship (QSAR) analysis was performed using descriptors calculated from comparative molecular field analysis (CoMFA). Our findings show that of the fifty selected hydrazones, compounds LpQM-19, 28 and 31 displayed the highest activity against T. cruzi, leading to a selectivity index (SI) of 20-fold. The 3D-QSAR analysis indicates that a particular electrostatic arrangement, where electron-deficient atoms are aligned along the molecule main axis positively correlates with compound biological activity. These results provide new candidate molecules for the development of treatments against Chagas disease.
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http://dx.doi.org/10.1016/j.bmc.2016.02.027DOI Listing
April 2016

Comprehensive carrier genetic test using next-generation deoxyribonucleic acid sequencing in infertile couples wishing to conceive through assisted reproductive technology.

Fertil Steril 2015 Nov 3;104(5):1286-93. Epub 2015 Sep 3.

IGenomix, Parc Cientific Univeristat Valencia, Valencia, Spain; Instituto Valenciano de Infertilidad Valencia and Fundación Instituto Valenciano de Infertilidad, Valencia, Spain; Department of Obstetrics and Gynecology, Valencia University, Instituto Universitario IVI/INCLIVA, Valencia, Spain.

Objective: To develop an expanded pan-ethnic preconception carrier genetic screening test for use in assisted reproductive technology (ART) patients and donors.

Design: Retrospective analysis of results obtained from 2,570 analyses.

Setting: Reproductive genetic laboratory.

Patient(s): The 2,570 samples comprised 1,170 individuals from the gamete donor programs; 1,124 individuals corresponding to the partner of the patient receiving the donated gamete; and 276 individuals from 138 couples seeking ART using their own gametes.

Intervention(s): None.

Main Outcome Measure(s): Next-generation sequencing of 549 recessive and X-linked genes involved in severe childhood phenotypes reinforced with five complementary tests covering high prevalent mutations not detected by next-generation sequencing.

Result(s): Preclinical validation included 48 DNA samples carrying known mutations for 27 genes, resulting in a sensitivity of 99%. In the clinical dataset, 2,161 samples (84%) tested positive, with an average carrier burden of 2.3 per sample. Five percent of the couples using their own gametes were found to have pathogenic variants conferring high risk for six different diseases. These high-risk couples and patients received genetic counseling and recommendations for preimplantation genetic diagnosis. For patients receiving gamete donation, we applied a genetic testing and blinded matching system to avoid high-risk combinations regardless of their carrier burden. For female donors, 1.94% were positive for X-linked conditions; they received genetic counselling and were discarded.

Conclusion(s): We have developed a comprehensive carrier genetic screening test that, combined with our matching system and genetic counseling, constitutes a powerful tool to avoid more than 600 mendelian diseases in the offspring of patients undergoing ART.
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http://dx.doi.org/10.1016/j.fertnstert.2015.07.1166DOI Listing
November 2015

Psychological Intervention in Primary Care After Earthquakes in Lorca, Spain.

Prim Care Companion CNS Disord 2015 26;17(1). Epub 2015 Feb 26.

Mental Health Branch of Murcia Health Service, El Palmar (Mssrs Martin and Garriga) and La Viña Health Centre, Lorca (Ms Egea), Murcia, Spain.

Objective: After the earthquakes that occurred in Lorca, Spain, on May 11, 2011, the regional mental health management employed 2 clinical psychologists for 6 months to provide care to people referred by primary care physicians. The objective was to address the expected increased demand for treatment of mental disorders, notably posttraumatic stress disorder (PTSD) and adjustment disorders.

Method: Referred individuals were evaluated and treated according to a clinical protocol designed ad hoc from June 12, 2011, to November 30, 2011. The protocol provided a stepped intervention guided by clinical and psychometric assessment using "normalization" for those with no psychiatric diagnosis, brief group treatment for mild to moderate PTSD or adjustment disorders, individual treatment for more severe PTSD, and referral to the local mental health center for other mental health disorders. Standard adult and child scales to assess posttraumatic, depression, and anxiety symptoms and resilience were used at initial assessment to guide treatment allocation and repeated to assess outcome status. Psychologists also provided a clinical assessment of symptom resolution at the end of the study.

Results: Rates of symptom resolution and improvements on all scales (PTSD, depression, anxiety, and resilience) demonstrated clinically and statistically significant improvement in all treatment groups (P = .000). Dropout was low. Medications were prescribed frequently to adults; no child received medication as a result of the earthquakes. No case of mental disorder related to the earthquakes was referred to the local mental health center during the 6 months of psychologist intervention.

Conclusion: The structured intervention resulted in a high resolution of cases and low dropout, allowing treatment of a larger number of people with optimal frequency (weekly), devoting more time to the most severe cases and less to those moderately or mildly affected.
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http://dx.doi.org/10.4088/PCC.14m01691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468883PMC
July 2015

Core muscle activity in a series of balance exercises with different stability conditions.

Gait Posture 2015 Jul 27;42(2):186-92. Epub 2015 May 27.

Research Group in Sport and Health, Department of Physical Education and Sports, University of Valencia, Valencia, Spain. Electronic address:

Literature that provides progression models based on core muscle activity and postural manipulations is scarce. The purpose of this study was to investigate the core muscle activity in a series of balance exercises with different stability levels and additional elastic resistance. A descriptive study of electromyography (EMG) was performed with forty-four healthy subjects that completed 12 exercises in a random order. Exercises were performed unipedally or bipedally with or without elastic tubing as resistance on various unstable (uncontrolled multiaxial and uniaxial movement) and stable surfaces. Surface EMG on the lumbar multífidus spinae (LM), thoracic multífidus spinae (TM), lumbar erector spinae (LE), thoracic erector spinae (TE) and gluteus maximus (GM), on the dominant side of the body were collected to quantify the amount of muscle activity and were expressed as a % of the maximum voluntary isometric contraction (MVIC). Significant differences (p<.001) were found between exercises. The three unipedal standing exercises with additional elastic resistance generated the greatest EMG values, ranging from 19% MVIC to 30% MVIC. Postural manipulations with additional elastic resistance and/or unstable devices increase core muscle activity. An adequate exercise progression based on global core EMG could start with seated positions, progressing to bipedal standing stance (i.e., from either multiaxial or stable surface to uniaxial surface). Following this, unipedal standing positions may be performed (i.e., from either multiaxial or stable surface to uniaxial surface) and finally, elastic resistance must be added in order to increase EMG levels (i.e., from stable surface progressing to any of the used unstable surfaces).
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http://dx.doi.org/10.1016/j.gaitpost.2015.05.008DOI Listing
July 2015

Development of two novel high-throughput assays to quantify ubiquitylated proteins in cell lysates: application to screening of new anti-malarials.

Malar J 2015 May 14;14:200. Epub 2015 May 14.

Ubiquitylation and Cancer Molecular Biology, Inbiomed, Mikeletegi 81, 20009, San Sebastian, Spain.

Background: The ubiquitin proteasome system (UPS) is one of the main proteolytical pathways in eukaryotic cells and plays an essential role in key cellular processes such as cell cycle, stress response, signal transduction, and transcriptional regulation. Many components of this pathway have been implicated in diverse pathologies including cancer, neurodegeneration and infectious diseases, such as malaria. The success of proteasome inhibitors in clinical trials underlines the potential of the UPS in drug discovery.

Methods: Plasmodium falciparum, the malaria causative pathogen, has been used to develop two assays that allow the quantification of the parasite protein ubiquitylation levels in a high-throughput format that can be used to find new UPS inhibitors.

Results: In both assays tandem ubiquitin binding entities (TUBEs), also known as ubiquitin traps, have been used to capture ubiquitylated proteins from cell lysates. The primary assay is based on AlphaLISA technology, and the orthogonal secondary assay relies on a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) system. A panel of well-known proteasome inhibitors has been used to validate both technologies. An excellent correlation was obtained between these biochemical assays and the standard whole cell assay that measures parasite growth inhibition.

Conclusions: The two assays presented can be used in a high-throughput format to find new UPS inhibitors for P. falciparum and could help to identify new targets within this system. This methodology is also applicable to other cellular contexts or pathologies.
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http://dx.doi.org/10.1186/s12936-015-0708-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4440562PMC
May 2015

New compound sets identified from high throughput phenotypic screening against three kinetoplastid parasites: an open resource.

Sci Rep 2015 Mar 5;5:8771. Epub 2015 Mar 5.

Molecular Discovery Research, Tres Cantos Medicines Development Campus, GlaxoSmithKline, Tres Cantos, Spain.

Using whole-cell phenotypic assays, the GlaxoSmithKline high-throughput screening (HTS) diversity set of 1.8 million compounds was screened against the three kinetoplastids most relevant to human disease, i.e. Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. Secondary confirmatory and orthogonal intracellular anti-parasiticidal assays were conducted, and the potential for non-specific cytotoxicity determined. Hit compounds were chemically clustered and triaged for desirable physicochemical properties. The hypothetical biological target space covered by these diversity sets was investigated through bioinformatics methodologies. Consequently, three anti-kinetoplastid chemical boxes of ~200 compounds each were assembled. Functional analyses of these compounds suggest a wide array of potential modes of action against kinetoplastid kinases, proteases and cytochromes as well as potential host-pathogen targets. This is the first published parallel high throughput screening of a pharma compound collection against kinetoplastids. The compound sets are provided as an open resource for future lead discovery programs, and to address important research questions.
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http://dx.doi.org/10.1038/srep08771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4350103PMC
March 2015

Exercise intensity progression for exercises performed on unstable and stable platforms based on ankle muscle activation.

Gait Posture 2014 18;39(1):404-9. Epub 2013 Aug 18.

Laboratory of Physical Activity and Health, Research Group in Sport and Health, Department of Physical Education and Sports, University of Valencia, Valencia, Spain.

Ankle sprains are a common sports injury. The literature focuses on the application of neuromuscular training for the improvement of balance, injury prevention and rehabilitation. However, there is a dearth of knowledge about the appropriate prescription of exercises using unstable platforms and surfaces. The purpose of this study was to devise an ankle rehabilitation or training program with exercise progression based on the extent of muscle activation, employing platforms with different levels of stability and additional resistance. A descriptive study of electromyography (EMG) during ankle exercises was performed with a convenience sample of healthy subjects. Forty-four subjects completed 12 exercises performed in a random order. Exercises were performed unipedally or bipedally with or without elastic tubing as resistance on various unstable (uncontrolled multiaxial and uniaxial movement) and stable surfaces. Surface EMG from the tibialis anterior (TA), peroneus longus (PL) and soleus (SOL) were collected to quantify the amount of muscle activity. Significant differences were found between exercise conditions for PL (p<.001), TA (p=.011), and SOL (p<.001). The greatest EMG activity for all muscles occurred with an upright unipedal stance on a soft stability surface with resistance. The least EMG activity for the TA and SOL were in a seated position and for the PL in an erect bipedal position without resistance. Based on the level of ankle muscle activation, exercises for the ankle should progress from bilateral exercises on exercise balls (lowest intensity), to a unipedal position on a soft surface in combination with elastic tubing (highest intensity) in order to achieve progressively greater ankle muscle activation.
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http://dx.doi.org/10.1016/j.gaitpost.2013.08.006DOI Listing
July 2014

The impact of next-generation sequencing technology on preimplantation genetic diagnosis and screening.

Fertil Steril 2013 Mar;99(4):1054-61.e3

Iviomics, Valencia, Spain.

Largely because of efforts required to complete the Human Genome Project, DNA sequencing has undergone a steady transformation with still-ongoing developments of high-throughput sequencing machines for which the cost per reaction is falling drastically. Similarly, the fast-changing landscape of reproductive technologies has been improved by genetic approaches. Preimplantation genetic diagnosis and screening were established more than two decades ago for selecting genetically normal embryos to avoid inherited diseases and to give the highest potential to achieve stable pregnancies. Most recent additions to the IVF practices (blastocyst/trophectoderm biopsy, embryo vitrification) and adoption of new genetics tools such as array comparative genome hybridization have allowed setting up more precise and efficient programs for clinical embryo diagnosis. Nevertheless, there is always room for improvements. Remarkably, a recent explosion in the release of advanced sequencing benchtop platforms, together with a certain maturity of bioinformatics tools, has set the target goal of sequencing individual cells for embryo diagnosis to be a realistically feasible scenario for the near future. Next-generation sequencing technology should provide the opportunity to simultaneously analyze single-gene disorders and perform an extensive comprehensive chromosome screening/diagnosis by concurrently sequencing, counting, and accurately assembling millions of DNA reads.
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http://dx.doi.org/10.1016/j.fertnstert.2013.02.001DOI Listing
March 2013

Mining the potential of label-free biosensors for seven-transmembrane receptor drug discovery.

Prog Mol Biol Transl Sci 2013 ;115:123-42

GlaxoSmithKline, Medicines Research Centre, Stevenage, United Kingdom.

Label-free is a broad term used to describe a number of cutting-edge biosensor technologies that have attracted considerable attention in the area of drug discovery for seven-transmembrane G protein-coupled receptors (GPCRs). Label-free biosensors resolve receptor-mediated responses noninvasively in real time and living cells and do so with high textural information and broad signaling-pathway coverage. They should facilitate studies of the receptor's integrated signal transduction biology intractable to classical assays with single pathway focus. Label-free occupies a privilege niche with respect to mechanistic studies in human native cells-healthy or disease-relevant-and the probing of context-dependent pharmacology in relation to whole biological system efficacy. It is expected that implementation of label-free approaches into the drug discovery process will improve clinical predictability of drug candidates at early stages of discovery research by their exquisite capability to sense whole cellular responses akin to tissue bioassays. Here, we present an overview of promises and challenges this rapidly evolving technology offers to drug screening and we also discuss the prospect of advancing drug discovery.
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http://dx.doi.org/10.1016/B978-0-12-394587-7.00003-8DOI Listing
June 2014

Self-correction in tripronucleated human embryos.

Fertil Steril 2011 Oct;96(4):951-6

Universitary Institute Instituto Valenciano de Infertilidad Valencia, Valencia, Spain.

Objective: To explore the occurrence of ploidy and parental self-correction in tripronuclear (TPN) human embryos.

Design: Experimental.

Setting: Research facility.

Patient(s): Thirty-two TPN embryos resulting from intracytoplasmic sperm injection (ICSI-TPN) and 18 TPN embryos resulting from conventional IVF (IVF-TPN).

Intervention(s): Tripronuclear embryos were cultured in vitro for 6 days. Those with ≥ 6 cells were biopsied for ploidy analysis. Blastocysts were studied for ploidy or parental inheritance. Heteroparental inheritance was determined after comparing polymorphic loci in the genomic DNA of a blastocyst and in the parents' DNA.

Main Outcome Measure(s): Tripronuclear origin, cell number at biopsy, chromosome analysis using fluorescent in situ hybridization, parental inheritance analysis using polymerase chain reaction amplification and sequencing, in vitro development to the blastocyst stage, and percentage of diploid and triploid cleavage embryos and blastocysts.

Result(s): Half of ICSI-TPN embryos became self-corrected blastocysts whereas only one IVF-TPN embryo did.

Conclusion(s): Both ICSI-TPN and IVF-TPN embryos are capable of self-correction, but the latter to a lesser extent. Neither parental inheritance nor ploidy determines the ability of a TPN embryo to progress to the blastocyst stage. However, the ability of a TPN embryo to become self-corrected is determined by the parental origin of the extra pronucleus.
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http://dx.doi.org/10.1016/j.fertnstert.2011.07.1087DOI Listing
October 2011

[Skin necrosis in a patient with temporal arteritis].

Reumatol Clin 2011 May-Jun;7(3):198-9. Epub 2011 Feb 22.

Servicio de Reumatología, Hospital 12 de Octubre, Madrid, España.

We present the case of a 91 years old patient diagnosed through biopsy with temporal arteritis who, in addition, had scalp necrosis. We briefly review the literature for published cases.
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http://dx.doi.org/10.1016/j.reuma.2010.06.003DOI Listing
April 2012