Preclinical characterisation of fingolimod as a potential therapeutic agent for stroke

Stroke is the leading cause for death and disability worldwide, and the search for novel drug treatments has been affected by repeated clinical trial failures. One novel drug that has garnered promising results in preclinical and clinical stroke studies is fingolimod, an FDA approved drug for the treatment of multiple sclerosis. Even though there are several studies supporting the effectiveness of fingolimod for the treatment of stroke, the recommended characterisation based on the Stroke Treatment Academic Industry Roundtable (STAIR) guidelines is incomplete. Furthermore, the quality of the preclinical studies supporting fingolimod has been poor, thus rigorous studies are required to validate the effectiveness of fingolimod prior to evaluation in clinical trials. This thesis aimed to inform whether fingolimod is effective for the treatment of stroke in intracerebral haemorrhage and ischaemic stroke, and to inform whether fingolimod is a good candidate for evaluation in large randomised clinical trials. This goal was achieved by first using a model of intracerebral haemorrhage to evaluate the effect of administering fingolimod at 30 min, 24 and 48 h after stroke on lesion size and behaviour in a 14-day study on male and female mice. This was followed up by a series of studies using middle cerebral artery occlusion to cause a focal ischaemia. First an optimal dose of fingolimod was determined in a dose response study; then we evaluated the optimal drug dose in two animal model of common comorbidities associated with stroke, age and hyperlipidaemia; and the last study evaluated the effect of an extended treatment duration on stroke. For the ischaemic stroke studies we focused on lesion and behavioural measurements as primary outcomes, and secondary data was collected from daily scores, plus additional measures where necessary. The intracerebral haemorrhage study fingolimod treatment had no measurable effect on either lesion size or behavioural outcomes irrespective of sex, the only finding was that fingolimod treatment reduced mortality in female mice. The dose response study showed no difference in lesion size or behavioural outcomes between the two fingolimod doses (0.5 and 1.0 mg/kg) and control mice, the study did show that saline treated mice had a significantly larger atrophy compared to the lower dose of fingolimod. The lower dose was selected as optimal for further studies. The study evaluating the effect of 0.5 mg/kg fingolimod on stroke in aged mice showed that fingolimod-treated mice had a significantly larger atrophy and a significant improvement in the grid score 7 d after stroke compared to saline-treated mice. The study evaluating the effect of fingolimod on stroke in hyperlipidaemic mice showed that fingolimod-treated mice had a significantly reduced lesion size, without an effect on any other outcome measures. The final study evaluated two fingolimod treatment durations compared to saline controls, the study showed no difference between any of the outcome measures, with a trend towards improved behaviour outcomes in mice receiving 10 d of fingolimod treatment. Lastly, considering the fact that the results of these studies were inconclusive, we decided to pool the data of the ischaemic studies and evaluate whether fingolimod had an effect on the primary outcome measures in a heterogenous animal population. The pooled data showed that fingolimod treatment improved behaviour 7 d after stroke without an effect on lesion size or atrophy. The results of this thesis cast a doubt on the effectiveness of fingolimod and its suitability for translation into larger clinical trials. Furthermore, they highlight the need for thorough preclinical studies for promising drugs as well as the need for studies to meet the proposed STAIR guidelines prior to translation. Confirmatory studies, like those presented here, performed with measures intended to control for internal and external biases are all good measures to be implemented for future studies of novel and highly promising drugs for stroke treatment

The effect of fingolimod on regulatory T cells in a mouse model of brain ischaemia

Background: The role of the immune system in stroke is well-recognised. Fingolimod, an immunomodulatory agent licensed for the management of relapsing-remitting multiple sclerosis, has been shown to provide benefit in rodent models of stroke. Its mechanism of action, however, remains unclear. We hypothesised fingolimod increases the number and/or function of regulatory T cells (Treg), a lymphocyte population which promotes stroke recovery. The primary aim of this study was to rigorously investigate the effect of fingolimod on Tregs in a mouse model of brain ischaemia. The effect of fingolimod in mice with common stroke-related comorbidities (ageing and hypercholesteremia) was also investigated. Methods: Young (15–17 weeks), aged C57BL/6 mice (72–73 weeks), and ApoE?/? mice fed a high-fat diet (20–21 weeks) underwent permanent electrocoagulation of the left middle cerebral artery. Mice received either saline or fingolimod (0.5 mg/kg or 1 mg/kg) at 2, 24, and 48 h post-ischaemia via intraperitoneal injection. Another cohort of young mice (8–9, 17–19 weeks) received short-term (5 days) or long-term (10 days) fingolimod (0.5 mg/kg) treatment. Flow cytometry was used to quantify Tregs in blood, spleen, and lymph nodes. Immunohistochemistry was used to quantify FoxP3+ cell infiltration into the ischaemic brain. Results: Fingolimod significantly increased the frequency of Tregs within the CD4+ T cell population in blood and spleen post-ischaemia in all three mouse cohorts compared to untreated ischemic mice. The highest splenic Treg frequency in fingolimod-treated mice was observed in ApoE?/? mice (9.32 ± 1.73% vs. 7.8 ± 3.01% in young, 6.09 ± 1.64% in aged mice). The highest circulating Treg frequency was also noted in ApoE?/? mice (8.39 ± 3.26% vs. 5.43 ± 2.74% in young, 4.56 ± 1.60% in aged mice). Fingolimod significantly increased the number of FoxP3+ cells in the infarct core of all mice. The most pronounced effects were seen when mice were treated for 10 days post-ischaemia. Conclusions: Fingolimod increases Treg frequency in spleen and blood post-ischaemia and enhances the number of FoxP3+ cells in the ischaemic brain. The effect of fingolimod on this regulatory cell population may underlie its neuroprotective activity and could be exploited as part of future stroke therapy

Commercial hybrid yield of green asparagus (Asparagus officinalis L. var. altilis) processed in two spear-lengths, during the stable productivity stage, in the Province of Buenos Aires, Argentina

The perennial crop asparagus reaches maximum yield in its fifth season, exhibits marked genotype-environment interaction and requires productivity evaluation of its hybrids. To determine the behavior of green asparagus genotypes, a randomized complete block design trial (begun 16/11/11, density 23810 pl ha-1, area 1690 m2) was carried out in Azul (36°48’ S, lat. 59°51’ W, long.) within the framework of the ISHS’s Fourth International Asparagus Cultivar Trial, in which the following hybrids were harvested between 5/8/17 and 26/10/17 and evaluated for agronomic performance: ‘Italo’, ‘Vittorio’, ‘Eros’, ‘Ercole’, ‘Giove’, ‘Franco’, ‘Chino’, ‘Early-California’, ‘UC-157’, ‘Patrón’, ‘NJ-1189’, ‘NJ-1123’ and ‘NJ-1192’. Their response to pre-harvest diammonium phosphate (DAP) fertilisation was also evaluated (100 kg ha-1 vs. control). Harvested spears were cut, conditioned to two lengths (long 22 cm, short 17 cm), weighed, counted, washed and calibrated. The following characters were evaluated: total fresh commercial productivity (TFP) and that of long and short commercial spears (CFP-L and CFP-S); total commercial spear number (TSN) and that of long and short commercial spears (CSN-L and CSN-S); mean spear weight (MSW); calibre distribution (CD): (J: Jumbo; XL: Extra-Large; L: Large; M: Medium; S: Small and A: Asparagine). ANOVA-LSD test P≥0.05 was employed. DAP fertilisation raised yield by 3-10%, though not significantly. The following mean values were obtained: TFP: 8678.46 kg ha-1; TSN: 440298 spears ha-1; MSW: 20 g; CFP-L: 3109; CFP-S: 1945 kg ha-1; CSN-L: 165857 and CSN-S: 130467 spears ha-1. The following hybrids performed well: for CFP-L: ‘Vittorio’: 4379(a); ‘Franco’: 4033(ab); ‘NJ-1123’: 3849(abc); for CFP-C: ‘NJ-1123’: 2726(a); ‘Ercole’: 2483(ab); ‘Vittorio’: 2325(abc); for CSN-L: ‘NJ-1123’: 225260(a); ‘Franco’: 214161(a); ‘Vittorio’: 204358(ab); ‘Giove’: 200229(abc); for CSN-C: ‘NJ-1123’: 2725(a); ‘Ercole’: 2482(ab); ‘Vittorio’: 2325(abc); for CD: J: ‘UC-157’: 12767(a); ‘Eros’: 9882(ab); ‘NJ-1123’: 9005(abc); for XL: ‘NJ-1123’: 41124(a); ‘Franco’: 38958(ab); ‘NJ-1192’: 37644(ab); for L: ‘Ercole’: 85083(a); ‘NJ-1123’: 84700(a); ‘Franco’: 83872(a); ‘Italo’: 83095(a); for M: ‘NJ-1123’: 118198(a); ‘Franco’: 116369(a); ‘Vittorio’: 109645(ab); ‘Ercole’: 105984(ab); for S: ‘Giove’: 102748(a); ‘NJ-1123’: 80530(ab). In summary, ‘NJ-1123’ would be chosen for spear number productivity and ‘Vittorio’ and ‘Franco’ for yield.Fil: Castagnino, Ana Maria. Cresca-faa-unicen; Argentina. Asaho; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Diaz, K. E.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Rosini, M. B.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Guisolis, Andrea Paola. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Dussi, M. C.. Universidad Nacional del Comahue; ArgentinaFil: Rogers, William John. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; Argentin

Lack of aprataxin impairs mitochondrial functions via downregulation of the APE1/NRF1/NRF2 pathway.

https://v2.sherpa.ac.uk/id/publication/335Ataxia oculomotor apraxia type 1 (AOA1) is an autosomal recessive disease caused by mutations in APTX, which encodes the DNA strand-break repair protein aprataxin (APTX). CoQ10 deficiency has been identified in fibroblasts and muscle of AOA1 patients carrying the common W279X mutation, and aprataxin has been localized to mitochondria in neuroblastoma cells, where it enhances preservation of mitochondrial function. In this study, we show that aprataxin deficiency impairs mitochondrial function, independent of its role in mitochondrial DNA repair. The bioenergetics defect in AOA1-mutant fibroblasts and APTX-depleted Hela cells is caused by decreased expression of SDHA and genes encoding CoQ biosynthetic enzymes, in association with reductions of APE1, NRF1 and NRF2. The biochemical and molecular abnormalities in APTX-depleted cells are recapitulated by knockdown of APE1 in Hela cells and are rescued by overexpression of NRF1/2. Importantly, pharmacological upregulation of NRF1 alone by 5-aminoimidazone-4-carboxamide ribonucleotide does not rescue the phenotype, which, in contrast, is reversed by the upregulation of NRF2 by rosiglitazone. Accordingly, we propose that the lack of aprataxin causes reduction of the pathway APE1/NRF1/NRF2 and their target genes. Our findings demonstrate a critical role of APTX in transcription regulation of mitochondrial function and the pathogenesis of AOA1 via a novel pathomechanistic pathway, which may be relevant to other neurodegenerative diseases

Rv2577 of mycobacterium tuberculosis Is a virulence factor with dual phosphatase and phosphodiesterase functions

Tuberculosis, a lung disease caused by Mycobacterium tuberculosis (Mtb), is one of the ten leading causes of death worldwide affecting mainly developing countries. Mtb can persist and survive inside infected cells through modulation of host antibacterial attack, i.e., by avoiding the maturation of phagosome containing mycobacteria to more acidic endosomal compartment. In addition, bacterial phosphatases play a central role in the interplay between host cells and Mtb. In this study, we characterized the Rv2577 of Mtb as a potential alkaline phosphatase/phosphodiesterase enzyme. By an in vitro kinetic assay, we demonstrated that purified Rv2577 expressed in Mycobacterium smegmatis displays both enzyme activities, as evidenced by using the artificial substrates p-NPP and bis-(p-NPP). In addition, a three-dimensional model of Rv2577 allowed us to define the catalytic amino acid residues of the active site, which were confirmed by site-directed mutagenesis and enzyme activity analysis, being characteristic of a member of the metallophosphatase superfamily. Finally, a mutation introduced in Rv2577 reduced the replication of Mtb in mouse organs and impaired the arrest of phagosomes containing mycobacteria in early endosomes; which indicates Rv2577 plays a role in Mtb virulence

Rv2577 of Mycobacterium tuberculosis is a virulence factor with dual phosphatase and phosphodiesterase functions

Tuberculosis, a lung disease caused by Mycobacterium tuberculosis (Mtb), is one of the ten leading causes of death worldwide affecting mainly developing countries. Mtb can persist and survive inside infected cells through modulation of host antibacterial attack, i.e., by avoiding the maturation of phagosome containing mycobacteria to more acidic endosomal compartment. In addition, bacterial phosphatases play a central role in the interplay between host cells and Mtb. In this study, we characterized the Rv2577 of Mtb as a potential alkaline phosphatase/phosphodiesterase enzyme. By an in vitro kinetic assay, we demonstrated that purified Rv2577 expressed in Mycobacterium smegmatis displays both enzyme activities, as evidenced by using the artificial substrates p-NPP and bis-(p-NPP). In addition, a three-dimensional model of Rv2577 allowed us to define the catalytic amino acid residues of the active site, which were confirmed by site-directed mutagenesis and enzyme activity analysis, being characteristic of a member of the metallophosphatase superfamily. Finally, a mutation introduced in Rv2577 reduced the replication of Mtb in mouse organs and impaired the arrest of phagosomes containing mycobacteria in early endosomes; which indicates Rv2577 plays a role in Mtb virulence.Instituto de BiotecnologíaFil: Forrellad, Marina Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marrero Diaz de Villegas, Rubén. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vazquez, Cristina Lourdes. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Yaneff, Agustín. Universidad de Buenos Aires. Instituto de Investigaciones Farmacológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garcia, Elizabeth Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gutierrez, Maximiliano Gabriel. The Francis Crick Institute, Host-Pathogen Interactions in Tuberculosis Laboratory; Reino UnidoFil: Durán, Rosario. Institut Pasteur de Montevideo; Uruguay. Instituto de Investigaciones Biológicas Clemente Estable; UruguayFil: Villarino, Andrea. Universidad de la República (UdelaR). Facultad de Ciencias. Sección Bioquímica; UruguayFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de investigaciones Científicas y Tecnológicas; Argentin

Quantum simulation of conductivity plateaux and fractional quantum Hall effect using ultracold atoms

We analyze the role of impurities in the fractional quantum Hall effect using a highly controllable system of ultracold atoms. We investigate the mechanism responsible for the formation of plateaux in the resistivity/conductivity as a function of the applied magnetic field in the lowest Landau level regime. To this aim, we consider an impurity immersed in a small cloud of an ultracold quantum Bose gas subjected to an artificial magnetic field. We consider scenarios corresponding to experimentally realistic systems with gauge fields induced by rotation of the trapping parabolic potential. Systems of this kind are adequate to simulate quantum Hall effects in ultracold atom setups. We use exact diagonalization for few atoms and to emulate transport equations, we analyze the time evolution of the system under a periodic perturbation. We provide a theoretical proposal to detect the up-to-now elusive presence of strongly correlated states related to fractional filling factors in the context of ultracold atoms. We analyze the conditions under which these strongly correlated states are associated with the presence of the resistivity/conductivity plateaux. Our main result is the presence of a plateau in a region, where the transfer between localized and non-localized particles takes place, as a necessary condition to maintain a constant value of the resistivity/conductivity as the magnetic field increases

Assessment of peritoneal microbial features and tumor marker levels as potential diagnostic tools for ovarian cancer

Epithelial ovarian cancer (OC) is the most deadly cancer of the female reproductive system. To date, there is no effective screening method for early detection of OC and current diagnostic armamentarium may include sonographic grading of the tumor and analyzing serum levels of tumor markers, Cancer Antigen 125 (CA-125) and Human epididymis protein 4 (HE4). Microorganisms (bacterial, archaeal, and fungal cells) residing in mucosal tissues including the gastrointestinal and urogenital tracts can be altered by different disease states, and these shifts in microbial dynamics may help to diagnose disease states. We hypothesized that the peritoneal microbial environment was altered in patients with OC and that inclusion of selected peritoneal microbial features with current clinical features into prediction analyses will improve detection accuracy of patients with OC. Blood and peritoneal fluid were collected from consented patients that had sonography confirmed adnexal masses and were being seen at SIU School of Medicine Simmons Cancer Institute. Blood was processed and serum HE4 and CA-125 were measured. Peritoneal fluid was collected at the time of surgery and processed for Next Generation Sequencing (NGS) using 16S V4 exon bacterial primers and bioinformatics analyses. We found that patients with OC had a unique peritoneal microbial profile compared to patients with a benign mass. Using ensemble modeling and machine learning pathways, we identified 18 microbial features that were highly specific to OC pathology. Prediction analyses confirmed that inclusion of microbial features with serum tumor marker levels and control features (patient age and BMI) improved diagnostic accuracy compared to currently used models. We conclude that OC pathogenesis alters the peritoneal microbial environment and that these unique microbial features are important for accurate diagnosis of OC. Our study warrants further analyses of the importance of microbial features in regards to oncological diagnostics and possible prognostic and interventional medicine.Ope

TOROS optical follow-up of the advanced LIGO–VIRGO O2 second observational campaign

We present themethods and results of the optical follow-up, conducted by the Transient Optical Robotic Observatory of the South Collaboration, of gravitational wave events detected during the Advanced LIGO–Virgo second observing run (2016 November–2017 August). Given the limited field of view (∼100 arcmin) of our observational instrumentation, we targeted galaxies within the area of high localization probability that were observable from our sites. We analysed the observations using difference imaging, followed by a random forest algorithm to discriminate between real and spurious transients. Our observations were conducted using telescopes at Estacion Astrofısica de Bosque Alegre, Cerro Tololo Inter-American Observatory, the Dr. Cristina V. Torres Memorial Astronomical Observatory, and an observing station in Salta, Argentina

Structural insight into the substrate specificity of DNA polymerase μ.

DNA polymerase l (Pol l) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol l's unusual substrate specificity, we describe the 2.4 Å crystal structure of the polymerase domain of murine Pol l bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol l that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol l from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during NHEJ of intermediates whose 3¢ ends lack complementary template strand nucleotides. These results provide insight into the substrate specificity and differing functions of four closely related mammalian family X DNA polymerases. To accomplish the many DNA transactions involved in stably maintaining and replicating large genomes, mammals encode 16 DNA polymerases classified in families A, B, X and Y 1 . Clues to their functions include their substrate specificity, which can differ substantially, even for members of the same family. For example, two family X enzymes with intrinsic 5¢-deoxyribose 5¢-phosphate (dRP) lyase activities, DNA polymerase b (Pol b) and Pol l, are template dependent and efficiently fill short gaps, consistent with synthesis during short patch base excision repair 2,3 . Unlike Pol b, Pol l has a BRCT domain and probably participates in NHEJ of double-strand breaks (DSBs) in DNA 4-6 . Specifically, Pol l has a role in V(D)J recombination at immunoglobulin heavy-chain loci Pol m has an unusual primer-template specificity. Like Pol b and Pol l, Pol m can fill short gaps in a template-dependent manner 12 , yet it also shares with TdT the ability to catalyze template-independent synthesis 13 . Pol m has an unusually high capacity to extend misaligned primer termini 14 ; it can perform translesion synthesis (TLS) in vitro To test the relationship between substrate specificity and physiological function, one can compare structures of these polymerases bound to primer-templates. Structures already exist for Pol b 19-22 , Pol l 23-26 and TdT 27 , but not yet for Pol m. Here we fill this knowledge gap by describing a 2.4-Å crystal structure of a ternary complex of the polymerase domain of murine Pol m. This structure reveals substrate interactions unique to Pol m, compared with Pol b, Pol l and TdT. To test whether such differences are important for unusual substrate use by Pol m, we examined the properties of the wild-type enzyme and an H329A mutant that perturbs interactions with the DNA that are not found in Pol b and Pol l. A similar substitution was performed on the homologous histidine (H342A) in human TdT. These mutations were made to test whether the histidine is important for templateindependent synthesis and for template-dependent synthesis with substrates lacking a template nucleotide at the 3¢ primer terminus. RESULTS Overall structure of a ternary Pol l-DNA-ddTTP complex The polymerization domain (Pro132-Ala496) of murine Pol m was crystallized in a ternary complex with a gapped template-primer and a correctly paired nucleoside triphosphate bound in the nascent base pair-binding pocket. The DNA contained an 11-nucleotide (nt