Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing.

BACKGROUND: Targeted amplicon deep sequencing (TADS) of the 16S rRNA gene is commonly used to explore and characterize bacterial microbiomes. Meanwhile, attempts to apply TADS to the detection and characterization of entire parasitic communities have been hampered since conserved regions of many conserved parasite genes, such as the 18S rRNA gene, are also conserved in their eukaryotic hosts. As a result, targeted amplification of 18S rRNA from clinical samples using universal primers frequently results in competitive priming and preferential amplification of host DNA. Here, we describe a novel method that employs a single pair of universal primers to capture all blood-borne parasites while reducing host 18S rRNA template and enhancing the amplification of parasite 18S rRNA for TADS. This was achieved using restriction enzymes to digest the 18S rRNA gene at cut sites present only in the host sequence prior to PCR amplification. RESULTS: This method was validated against 16 species of blood-borne helminths and protozoa. Enzyme digestion prior to PCR enrichment and Illumina amplicon deep sequencing led to a substantial reduction in human reads and a corresponding 5- to 10-fold increase in parasite reads relative to undigested samples. This method allowed for discrimination of all common parasitic agents found in human blood, even in cases of multi-parasite infection, and markedly reduced the limit of detection in digested versus undigested samples. CONCLUSIONS: The results herein provide a novel methodology for the reduction of host DNA prior to TADS and establish the validity of a next-generation sequencing-based platform for universal parasite detection

Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition.

Collagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF) in bone forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we have used a murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Furthermore, we also deleted Vegf in vitro in OBs extracted from male and female mice in an attempt to link sex-specific matrix signatures to deviations in gene expression. Label-free and non-destructive polarisation-resolved second harmonic generation microscopy (p-SHG) revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied only in males by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-stand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure including Adamts2, Spp1, Mmp9 and Lama1 The current results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders which clinically manifest in both pathological nano and macro-level disorganisation

The effectiveness of neuromuscular warm-up strategies, that require no additional equipment, for preventing lower limb injuries during sports participation: a systematic review

PMCID: PMC3408383The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1741-7015/10/75. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Observed Reductions in Schistosoma mansoni Transmission from Large-Scale Administration of Praziquantel in Uganda: A Mathematical Modelling Study

To date schistosomiasis control programmes based on chemotherapy have largely aimed at controlling morbidity in treated individuals rather than at suppressing transmission. In this study, a mathematical modelling approach was used to estimate reductions in the rate of Schistosoma mansoni reinfection following annual mass drug administration (MDA) with praziquantel in Uganda over four years (2003-2006). In doing this we aim to elucidate the benefits of MDA in reducing community transmission.Age-structured models were fitted to a longitudinal cohort followed up across successive rounds of annual treatment for four years (Baseline: 2003, TREATMENT: 2004-2006; n = 1,764). Instead of modelling contamination, infection and immunity processes separately, these functions were combined in order to estimate a composite force of infection (FOI), i.e., the rate of parasite acquisition by hosts.MDA achieved substantial and statistically significant reductions in the FOI following one round of treatment in areas of low baseline infection intensity, and following two rounds in areas with high and medium intensities. In all areas, the FOI remained suppressed following a third round of treatment.This study represents one of the first attempts to monitor reductions in the FOI within a large-scale MDA schistosomiasis morbidity control programme in sub-Saharan Africa. The results indicate that the Schistosomiasis Control Initiative, as a model for other MDA programmes, is likely exerting a significant ancillary impact on reducing transmission within the community, and may provide health benefits to those who do not receive treatment. The results obtained will have implications for evaluating the cost-effectiveness of schistosomiasis control programmes and the design of monitoring and evaluation approaches in general

Scandinavian Flagship Universities: an Appraisal of Leading National Universities in the European Context

In the immediate post-Sputnik era, Clark Kerr saw America’s breed of universities as “multiversities,” characterized by growing enrollment, an increasingly diversified academic community, and as the emerging driver of knowledge production and innovation (Kerr 2001: xi). This is now a widely shared vision throughout the world. Governments have provided additional funding, fueling further growth in academic programs and attracting talent. One result: the number of perceived stakeholders has increased, along with their expectations

Synchronous malignant vagal paraganglioma with contralateral carotid body paraganglioma treated by radiation therapy

Paragangliomas are rare tumors and very few cases of malignant vagal paraganglioma with synchronous carotid body paraganglioma have been reported. We report a case of a 20-year old male who presented with slow growing bilateral neck masses of eight years duration. He had symptoms of dysphagia to solids, occasional mouth breathing and hoarseness of voice. Fine needle aspiration cytology (FNAC) performed where he lived showed a sinus histiocytosis and he was administered anti-tubercular treatment for six months without any improvement in his symptoms. His physical examination revealed pulsatile, soft to firm, non-tender swellings over the anterolateral neck confined to the upper-mid jugulo-diagastric region on both sides. Direct laryngoscopy examination revealed a bulge on the posterior pharyngeal wall and another over the right lateral pharyngeal wall. Magnetic resonance imaging (MRI), 99mTc-labeled octreotide scan and angiography diagnosed the swellings as carotid body paraganglioma, stage III on the right side with left-sided vagal malignant paraganglioma. Surgery was ruled out as a high morbidity with additional risk to life was expected due to the highly vascular nature of the tumor. The patient was treated with radiation therapy by image guided radiation to a dose of 5040cGy in 28 fractions. At a follow-up at 16 months, the tumors have regressed bilaterally and the patient can take solids with ease

Effects of Antiepileptic Drugs on GABA Responses and on Reduction of GABA Responses by PTZ and DMCM on Mouse Neurons in Cell Culture

The mechanisms of action of antiepileptic drugs effective against generalized absence seizures (antiabsence AEDs) remain uncertain. Antiabsence AEDs are generally effective against seizures induced in experimental animals by pentylenÉtÉtrazol (PTZ) and methyl-6,7-dimethoxy-4-ethyl-Β-carboline-3-carboxylate (DMCM), drugs which reduce GABAergic inhibition. Thus, antiabsence AEDs have been suggested to enhance GABAergic inhibition. We studied the effects of several AEDs on GABA responses recorded from mouse spinal cord neurons grown in primary dissociated cell culture. Four antiabsence AEDs were included: ethosuximide (ESM), dimethadione (DMO), sodium valproate (VPA), and diazepam (DZP). Two experimental AEDs, CGS 9896 and ZK 91296, with anticonvulsant action against PTZ- or DMCM-induced seizures were also included. Possible effects of the antiabsence and experimental AEDS on PTZ- and DMCM-induced inhibition of GABA responses were also evaluated. PTZ and DMCM revers-ibly reduced GABA responses in a concentration-dependent manner. PTZ complÉtÉly inhibited GABA responses at 10 mM (IC 50 of 1.1 mM), whereas DMCM-induced inhibition of GABA responses reached a plateau level of 39% of control values at 1 p.M (IC 50 of 33 nM). ESM (1,200 ΜM), DMO (6 mM), VPA (200 u.M), CGS 9896 (2 ΜM), and ZK 98% (2 Μ M ) did not alter GABA responses. DZP enhanced GABA responses in a concentration-dependent manner. The inhibition of GABA responses produced by PTZ 1 mM was unaltered by ESM (600 Μ M ), DMO (6 mM), CGS 9896 (1 Μ M), or ZK 9896 (1 ΜM)- Coapplication of VPA (200 ΜM) and PTZ (1 mM) slightly enhanced the PTZ effect. DZP (> 10 nM), however, reversed the PTZ-induced reduction of GABA responses. The DMCM (250 nM) inhibition of GABA-responses was unaltered by ESM (600 Μ.M), DMO (2 mM), or VPA (200 ΜM). CGS 9896 (2 Μ M ) and ZK 91296 (2 ΜM), however, antagonized the DMCM effect. DZP (> 10 nM) significantly reversed the DMCM-induced inhibition of GABA responses. The lack of effect of VPA, ESM, and DMO on postsynaptic GABA responses suggests that direct enhancement of postsynaptic GABA action is not a common mechanism of action of antiabsence AEDs. The AEDs DZP, CGS 98%, and ZK 912% all reversed DMCM, but not PTZ, reduction of GABA responses, suggesting that these AEDs blocked DMCM seizures by acting at benzodiazepine receptors. However, since only DZP enhanced GABA responses, it is unclear how CGS 98% and ZK 912% blocked PTZ seizures. Key Words: Anticonvulsants–GABA–Neuron culture–Cell culture–Spinal cord neurons–Convulsants. RESUMEN Los mecanismos de accidn de las medicaciones antiepilÉpticas eficaces contra los ataques generalizados de ausencia (AEDs antiausencia) permanecen inciertos. Los AEDs antiausencia son, generalmente, eficaces contra ataques experimentales inducidos por el pentilentetrazol (PTZ) y el metil-6,7-dimetoxy-4-etil-Pcarbolina-3-carboxilato (DMCM) en animates, medicaciones que reducen la inhibiciÓn GABAÉrgica. Hemos estudiado los efectos de varios AEDs sobre respuestas-GABA registradas en las neuronas de la mÉdula espinal de ratones que habian crecido en cultivos de cÉlulas primarieas disociadas. Cuatro AEDs antiausencia fueron incluidos: etoxusimida (ESM), dimetadiona (DMO), valproato sÓdico (VPA) y diazepan (DZP). TambtÉn se incluyeron dos AEDs experimentales, CGS 9896 y ZK 912%, con acciÓn anticonvulsiva contra los ataques inducidos por PTZ o DMCM. TambiÓn se valoraron los posibles efectos de los AEDs antiausencia y experimentales sobre el PTZ y la inhibiciÓn de las respuestas-GABA inducidas por el DMCM. El PTZ y el DMCM redujeron las respuestas-GABA de modo reversible y dependiendo de sus concentraciones. El PTZ inhibiÓ cmpleta-mente las respuestas-GABA a 10 mM (IC 50 de 1.1 mM) mientras que la inhibitiÓn de las respuestas GABA inducida por el DMCM alcanzÓ un nivel estable del 39% de los valores control con 1 Μ. M (IC 50 de 33 mM). La ESM (1200 Μ.M), la DMO (6 mM), el VPA (200 Μ M ), el CGS 98% (2 Μ M) y el ZK 98% (2 Μ M) no alteraron las respuestas-GABA. El DZP aumentÓ las respuestas GABA de una manera concentraciÓn-dependiente. La inhibition de las respuestas-GABA producidas por el PTZ (1 mM), no se altero con las ESM (600 Μ M), la DMO (6 mM), el CGS 98% (1 Μ M) o el ZK 98% (1 Μ .M). La co-aplicacion de VPA (200 Μ M) y el PTZ (1 mM) aument6 ligeramente los efectos del PTZ. Sin embargo el DZP (10 nM) revirtiÓ significativamente la inhibition de las respuestas GABA inducidas por el DMCM. La falta de efectos de CPA, ESM y DMO sobre las respuestas GABA post-sinÁpticas sugiere que el incremento de la acciÓn GABA post-sinÁptica no es un mecanismo comÚn de actuatiÓn de las AEDs antiausencia. Todas las AEDs DZP, CGS 98% y ZK 912% revirtieron la reduction de las respuestas GABA producidas por el DMCM pero no las inducidas por el PTZ lo que sugiere que estos AEDs bloquean los ataques DMCM actuando sobre los receptores de la benzodiazepina. Sin embargo, puesto que el incremento de las respuestas GABA sÓlÓ se produce por el DZP, permanece todavia sin aclarar el por quÉ el CGS 98% y el ZK 912% bloquean los ataques producidos por el PTZ. ZUSAMMENFASSUNG Der Wirkmechansimus von Antiepileptika gegen generalisierte Absencen ist unklar. Antiabsencemittel sind generell wirkungs-voll gegen PTZ- und Methyl-6,7-Dimethoxy-4-Äthyl-P-Carbolin-Β-Carboxylat (DMCM) induzierte tierexperimentelle AnfÄlle, also von Medikamenten, die die GABA-erge Inhibition reduzieren. Es wurde vermutet, daß Antiabsencemittel die GABA-erge Inhibition verstÄrken. Wir untersuchten die Wirkung von verschiedenen Antiepileptika auf GABA-Antworten in spinalen MÄuseneuronen, die in Zellkulturen gew-achsen waren. Es wurden 4 Absencemittel untersucht: Ethosux-imid (ESM), Dimethadion (DMD), Sodium Valproat (VPA) und Diazepam (DZP). ZusÄtzlich wurden 2 experimentelle Antiepileptika, CGS 98% und ZK 912%, die gegen PTZ0 oder DMCM-induzierte AnfÄlle wirkungsvoll sind, eingeschlossen. Mogliche Wirkungen der Antiabsence- und experimentellen Antiepileptika auf PTZ- und DMCM-induzierte Hemmung der GABA-Antworten wurden ebenfalls ausgewertet. PTZ und DMCM zeigten eine konzentrationsabhÄngige reversible Reduktion der GABA-Antworten. PTZ zeigte eine komplette Hemmung der GABA-Antworten bei 10 mM (IC 50 1,1 mM), DMCM-Hemmung der GABA-Antworten zeigte ein Plateau von 39% der Kontroll-werte bei 1 uJtf (ICJO von 33 mAfl. ESM (1200 uJtf), DMD (6 mM), VPA (200 Μ M), CGS 98% (2 Μ M) und ZK 98% (2 Μ M) anderten nicht die GABA-Antworten. DZP verstarkte die GABA-Antworten konzentrationsabhangig. Die durch PTZ (1 mM) hervorgerufene Hemmung der GABA-Antworten war bei ESM (600 Μ M), DMD (6 mM), CGS 98% (1 mAO und ZK 3836 (1 mM) unverÄndert. ZusÄtliche Anwendung von VPA (200 mM) und PTZ (1 mM) verstÄrkten geringfÜgig den PTZ-Effekt. DZP (10 nM) kehrte die durch PTZ hervorgerufene Reduktion der GABA-Antworten um. Die durch DMCM (250 nM) hervorgerufene Hemmung der GABA-Antworten war durch ESM (600 Μ .M), DMD (2 mM) und VPA (200 Μ M ) unbeeinflusst. CGS 98% (2 Μ M) und ZK 912% (2 Μ M ) antagonisierten die DMCM-Wirkung. DZP (>10 nM) kehrte die durch DMCM-induzierte Hemmung der GABA-Antworten um. Das Fehlen einer Wirkung von VPA. ESM und DMD auf die postsynaptischen GABA-Antworten legen nahe, daß eine direkte VerstÄrkung der postsynaptischen GABA-Aktion kein gemeinsamer Mechanis-mus der Antiabsencemittel darstellt. Die Antiepileptika DZP, CGS 98% und ZK 912% kehrten die DMCM-Wirkung auf die GABA-Antworten um, jedoch nicht die von PTZ, was vermuten lapt, daß diese Antiepileptika die DMCM-AnfÄlle Über die Wirkung an den Benzodiazipin-Rezeptoren verhinderte. Da jedoch nur DZP GABA-Antworten verstarkte, ist unklar, in welcher Weise CGS 98% und ZK 912% die PTZ-AnfaUe ver-hinderten.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65188/1/j.1528-1157.1989.tb05275.x.pd

The Role of For-Profit Actors in Implementing Targeted Sanctions:The Case of the European Union

The evolution of sanctions from comprehensive to targeted has favored the inclusion of for-profit actors in the policy process. Sanctions are used to deal with security challenges and while the role of for-profit actors in the provision of public goods has been investigated, less has been said about their role in the provision of security. This chapter investigates the role of for-profit actors in the implementation of sanctions. More specifically, this chapter suggests a typology of regulatory environments that facilitates explaining and understanding the behavior of for-profit actors in implementing targeted sanctions. By looking at the quality of instructions provided by state authorities and their capacity to monitor the implementation of such decisions, the chapter argues that overcompliance, uneven and lack of compliance are more likely in certain regulatory environments rather than in others. The theoretical framework is tested on the case study of the restrictive measures of the EU. The data for this research was collected through semi-opened interviews and focus groups held in Brussels from 2013 to 2015