thymidine phosphorylase inhibitor We previously reported a colourimetric

We previously reported a colourimetric assay based on a SNAP-25 monolayer on colloidal gold [8]. This assay has been developed into an electrochemical assay presented in this paper.
Electrochemical techniques, such as cyclic voltammetry and electrochemical impedance spectroscopy, are label-free techniques that are highly sensitive to changes and interactions at a surface. Over the past few years a number of advances have been made in this area with the development of sensors specific for various toxins, pathogens and biomarkers [3,12,17] due to their high sensitivity and rapid detection times. Cyclic voltammetry provides information on the amount of oxidisable thymidine phosphorylase inhibitor on a working electrode and the number of electrons involved in the oxidation, which is given in terms of charge [13]. Variations in the charge indicate binding events and changes to the self-assembled monolayer. Impedance is a measure of the complex resistance met when current flows through a circuit made up of resistors, capacitors and inductors. Electrochemical impedance spectroscopy utilises redox probes such as Fe(CN)63−/4− or Ru(NH3)62+/3+ measuring the ability of these ions to become oxidised and reduced at the working electrode [1]. At unmodified working electrodes the route to the electrode surface is not blocked and these chemicals easily undergo the reactions. If the electrode surface is modified by an alkanethiol or a protein then the ions are more blocked increasing the charge transfer resistance (Rct) of the circuit [11].

Materials and methods
Gold working electrodes were purchased from Winkler GmbH Germany, SNAP-25 was from Abcam thymidine phosphorylase inhibitor (ab155885) and all other chemicals were purchased from Sigma Aldrich. Dysport® samples containing lyophilised Clostridium botulinum Type A toxin-haemaggluttinin complex (4.35ng), human serum albumin (125μg) and lactose (2.5mg) and placebos containing the excipients from Dysport® were kindly supplied by IPSEN Biopharm. Water was purified and had a nominal resistivity of 18MΩcm at 25°C.
All measurements were performed using an Autolab PGSTAT 30 computer-controlled electrochemical measurement system (Eco Chemie, Holland) with a home-made three electrode cell with a SNAP-25 modified Au(111) working electrode, a platinum counter electrode and a saturated calomel reference electrode.

Results and discussion

Conclusion
Industrial pressures have caused a demand for quicker, cheaper assays for botulinum neurotoxin. The assays presented in the paper are much quicker than the mouse bioassay taking hours rather than days to perform. The impedance biosensor also outperforms the mouse bioassay on sensitivity producing a response down to 25pg/mL of toxin. The major benefit of these assays over some others in development, such as ELISA, is the detection of active toxin through monitoring the proteolytic activity rather than just the presence of the molecule. These assays also clearly distinguish between the toxin product and the placebo sample which has previously caused problems due to the slight proteolytic activity of HSA [9]. When performed correctly EIS does not damage the biological layer giving an advantage, allowing for direct comparison between the individual SNAP-25 monolayers before and after incubation with the toxin.

Conflict of interest

Acknowledgements
The authors would like to thank IPSEN Biopharm and the Knowledge Economy Skills Scholarship (KESS) for funding the initial research and the Welsh European Funding Office for funding the current project. We would also like to thank IPSEN Biopharm for supplying samples of Dysport® and placebo. This work is included in patent application number 1310090.4 [6].

Introduction
The genus Trichoderma is a saprophytic fungi which can be found in all climatic zones of the world. The genus Trichoderma was first described in 1794 by Persoon [1] and there are reportedly almost 130 species [2]. The conventional technique used to identify and classify Trichoderma species is based on phenotypic traits which include morphological and biochemical characteristics; however, it is quite difficult to differentiate between very closely related species. DNA sequences can be used for the identification of fungi at the species level, but approximately 40% of the GenBank database sequences of Trichoderma species have been erroneously identified or remain unidentified at the species level [3,4]. There are a large number of sequences deposited in the GenBank that are incorrectly labeled and unless remedied they will continue to be assigned to the wrong taxa [5,6]. Under these conditions, the best conceivable conception of the molecular data and morphological characteristics of isolates is achieved using detailed photographs or drawings of the specimens to prevent any controversial identification at the species level [7]. To easily recognize and ensure the quality of results it is also possible to go back to the main source of the information. Otherwise, species level identification is difficult to do correctly, especially when it is necessary to rely on a source that has made a misidentification.

Vortioxetine is a multimodal antidepressant that

Vortioxetine is a multimodal antidepressant that acts as an inhibitor at the serotonin (5-HT) transporter (SERT), an agonist at 5-HT1A receptors, a partial agonist at 5-HT1B receptors, and an antagonist at 5-HT1D, 5-HT3 and 5-HT7 receptors (Bang-Andersen et al., 2011; Sanchez et al., 2015). Based on human binding affinities, vortioxetine is predicted to preferentially engage SERT and 5-HT3 receptors at the low end of the clinical dose range (5, 10 mg/day) and engage all targets at the high end of the range (20 mg/day) (Sanchez et al., 2015). The outcome of clinical studies indicates that vortioxetine, in addition to its antidepressant efficacy, has beneficial effects on aspects of cognitive dysfunction in MDD patients across the entire dose range of 5–20 mg/day (Katona et al., 2012; McIntyre et al., 2014; Mahableshwarkar et al., 2015). A recent fMRI study undertaken in remitted MDD patients during performance of the N-Back working memory task showed a reduced blood oxygenation level dependent (BOLD) signal in the right dorsolateral prefrontal cortex and the left hippocampus in patients treated with vortioxetine compared to placebo, indicating that vortioxetine’s pharmacological actions may counteract the over activation in these mpges-1 inhibitors structures associated with the depressive state (Smith et al., 2017).
Preclinical studies in rodents have supported the notion that vortioxetine has positive effects on cognitive function across a range of cognitive domains, e.g. preclinical models of execute function (attentional set shifting, reversal learning), and memory (episodic, visual spatial, and fear memory), but not attention (visual signal detection task) (review by Sanchez et al., 2015; Pehrson et al., 2016a). Importantly, preclinical studies have demonstrated that vortioxetine is mechanistically differentiated from selective serotonin reuptake inhibitors (SSRIs) and serotonin norepinephrine reuptake inhibitors (SNRIs) in several of these models. We have previously hypothesized that vortioxetine’s antagonism of 5-HT3 receptors may play an important role in improving cognitive function by indirectly increasing the activity of cortical and hippocampal pyramidal neurons, which are thought to be important key integrators of cognitive processing, via inhibition of a subset of GABAergic interneurons (Pehrson and Sanchez, 2014; Dale et al., 2016; Pehrson et al., 2016a,b). Vortioxetine has high (nanomolar range) affinity for 5-HT3A receptors, which are the predominant subtype of 5-HT3 receptors expressed in the brain, and are the only known serotonergic ligand-gated ion channel receptors. Thus, vortioxetine’s functional interaction with 5-HT3A receptors can be addressed via electrophysiology since 5-HT3A receptors are capable of conducting monovalent cations that can be recorded as current. Previously reported preclinical studies of vortioxetine’s effects on neurotransmission support the hypothesis that vortioxetine enhances glutamatergic pyramidal cell signaling in brain regions important for cognitive and emotional processing (medial prefrontal cortex and hippocampus) through a 5-HT3 receptor antagonism-dependent mechanism (Dale et al., 2014). Thus, single unit recordings from pyramidal cells in the prefrontal cortex of anaesthetized rats showed that clinically equivalent vortioxetine doses (corresponding to 5–20 mg/day) increased pyramidal cell firing significantly and that co-administration of a 5-HT3 receptor agonist SR 57227A abolished the effect of vortioxetine (Riga et al., 2016). The SSRI, escitalopram, had no effect on pyramidal cell firing in these assays. In line with the in vivo recordings from cortical pyramidal cells, in vitro recordings from pyramidal cells in the CA1 of rat hippocampus slices showed that vortioxetine reversed 5-HT-induced Inhibitory Post Synaptic Currents (IPSCs) and the effect was ascribed to vortioxetine’s 5-HT3 receptor antagonistic properties (Dale et al., 2014). Vortioxetine has also been shown to enhance other cognition- relevant transmission systems such as the noradrenergic, dopaminergic, cholinergic, and histaminergic, but only at doses that correspond to the high end of the clinically relevant range (Sanchez et al., 2015). These mechanisms are therefore not thought to be the main mediators of vortioxetine’s pro-cognitive effects, at least not in rodents.

br PABLO RUIZ N POLES

PABLO RUIZ NÁPOLES
Es licenciado en Economía (1976) por la unam, maestro (1978) y doctor (1996) en Economía por la New School for Social Research de Nueva York. Es Profesor Titular C de tiempo completo definitivo en la Facultad de Economía de la unam, adscrito Aminoallyl-dUTP la División de Estudios de Posgrado, miembro del Sistema Nacional de Investigadores con el nivel II, y del Programa de Primas al Desempeño Académico, de la unam, con el nivel D. Es miembro de la Academia Mexicana de Ciencias, desde 2001, de la International Input-Output Association, de Sigma Xi The Scientific Research Society, y de la Sociedad Hispano Americana de Análisis Input-Output. Ha dictaminado artículos para las revistas mexicanas: Revista Mexicana de Sociología, Contaduría y Administración, Investigación Económica, Momento Económico, y Norteamérica, de la unam; Economía Teoría y Práctica, de la uam; Perfiles Latinoamericanos, de flacso México; y Economía Mexicana Nueva Época, del cide. Para las revistas del extranjero: Journal of Policy Modeling, International Review of Applied Economics, Journal of Productivity Analysis, Revista de la CEPAL, Environmental Science & Policy, Research Policy y Metroeconomica.

VIRGINIA I. CORBELLA
Licenciada y Profesora en Economía. Aguardando la defensa de la tesis de Magíster en Economía realizada con beca de la Comisión de Investigaciones Científicas (cic), Ministerio de la Producción de la Provincia de Buenos Aires y actualmente desarrolla sus tareas como docente e investigadora en el Departamento de Economía en la Universidad Nacional del Sur y escuelas secundarias de la provincia de Buenos Aires, Argentina.

KARLA SARMENTO GONÇALVES DE SOUZA
Doctora en Economía por el Programa de Posgrado en Economía de la Universidad Federal Fluminense y también tiene un master en economía por la misma universidad y ha tomado cursos sobre comercio exterior en Universidade Federal do Rio de Janeiro, ufrj, Brasil. Sus campos de especialidad son: Economía Internacional, estructuralismo, la industria brasileña, y la Aminoallyl-dUTP cuestión fiscal. Ha realizado materiales especializados que ha publicado en diversas revistas.

JORGE GONZALORENA DÖLL
Historiador económico (Universidad de Lund, Suecia), sociólogo (Universidad de Chile) y Magíster en Ciencias Sociales (Universidad de Chile). Ha desempeñado labores de docencia a diploid nivel de pre y posgrado, dirección de Tesis e investigación en las siguientes universidades chilenas: arcis, Academia de Humanismo Cristiano, Católica Silva Henríquez, Andrés Bello, Tecnológica Metropolitana y Universidad de Valparaíso. Entre los años 2001 y 2003 fue coordinador del Programa de Investigación de la Facultad de Administración y Economía de la Universidad Católica Silva Henríquez, y en el periodo 2003-2007 editor de Oikos, revista académica de esa misma Facultad. Durante los años 2012 y 2013 fue Secretario Académico del Instituto de Historia y Ciencias Sociales de la Universidad de Valparaíso. Sus áreas de investigación preferentes corresponden a las teorías y problemas del desarrollo económico y a la historia económica de Chile. Ha escrito y publicado numerosos trabajos sobre estos temas bajo la forma de libros, artículos y ponencias. Actualmente es académico del Departamento de Sociología de la Universidad de Chile

MARIO LUIS FUENTES ALCALÁ
Es licenciado en Economía por el itam, y Maestro en Desarrollo Regional por el Instituto de Estudios Sociales de la Universidad de la Haya Holanda. Realizó estudios de doctorado en la Universidad de Anglia del Este, Reino Unido. Es autor de varios libros en asistencia social y política pública. Actualmente dirige el Centro de Estudios e Investigación en Desarrollo y Asistencia Social (ceidas, A.C.). Es miembro de la Junta de Gobierno de la unam y es Titular de la Cátedra Extraordinaria “Trata de Personas”. En julio de 2010, Mario Luis Fuentes fundó la Revista México Social. También es conductor del programa de televisión México Social, transmitido semanalmente en OnceTV-México; es colaborador semanal para el periodico Excelsior y comentarista para Grupo Imagen. Es Miembro de Número de la Academia Mexicana de Economía Política; y Patrono de la “Fundación Amistad Británico-Mexicana. Ha sido galardonado con el Premio inegi, 2013, en la categoría de Ensayo; y con el Premio Nacional Rostros de la Discriminación “Gilberto Rincón Gallardo” en la categoría de Artículo de Opinión.

In his paper unsuccessfully submitted to EJ Wicksell referred

In his 1923 paper unsuccessfully submitted to EJ, Wicksell referred to Ricardo\’s attempt, “hardly with success, to mitigate somewhat his own conclusions” by considering the positive effects of an increase in savings, caused by price reduction upon the introduction of machinery. In angiotensin receptor blockers with his early 1890 argument, Wicksell now dismisses Ricardo\’s qualification on the grounds that “it is difficult to see how this could be done” (Jonung, 1981, p. 202). Wicksell rejected in his Lectures (p. 136) another qualification by Ricardo, that expenditure of higher rents by landowners could increase labour demand and compensate for the immediate effects of machinery (again differently from his 1890 article). Such circumstance may “more or less modify” the output reduction result “but can scarcely reverse it”. More importantly, Wicksell assumed in his discussion of the machinery question in the Lectures – as he did throughout part II (“Theory of Production and Distribution”) of volume 1 of that book – that prices are given. He envisaged an economy that produces just one or a few goods and imports everything else it requires, at exchange values determined in the world market (Wicksell, 1934 [1901,1911], pp. 103, 136, 196). In this case, there is “no question of compensation to the workers in the form of another demand for labour” (p. 137).
Wicksell\’s assumption of constant prices was not just a simplification. It was necessary for his (and Pareto\’s) demonstration that output is maximized under free competition. This is made clear in the passage quoted in section 3 above from Wicksell (1958 [1913], p. 169), where he also refers to the fact that, if prices are variable, the proposition can only be “roughly” formulated, “since it is angiotensin receptor blockers impossible to make a direct comparison of separate commodities and services”. Wicksell\’s optimum production conditions are independent of prices and distribution, as Dobb (1969, p. 53) pointed out. In his 1923 manuscript, Wicksell (see Jonung, 1981, p. 201) stressed the potential effects of machinery in bringing out (what we now call) a new Pareto-optimal equilibrium through compensation payment: “The machinery will always have the effect of raising the gross produce of the country to its greatest possible amount, and in so far it will provide the means for bettering the economic conditions of the working men as well as of their employers”.
A substantial portion of Ricardo\’s (1951 [1821], pp. 388–391) discussion of the machinery problem was based on the notion that the production of new machinery involves the transformation of circulating capital (the “wage fund”) into fixed capital. The diversion of labour previously used in the production of wage goods – a reduction of the wage fund – may bring down output and employment (see Barkai, 1986). Wicksell (1934 [1901,1911], p. 134), in contrast with most commentators, claimed that the essence of Ricardo\’s position on machinery did not reside in changes in the structure of capital but in the final output outcome per se. Those issues should be kept separated, since the negative effect on wages of the transformation of circulating into fixed capital (which Wicksell endorsed) was a completely different matter from the alleged output reduction, as he explained in the Lectures (p. 164). Hence, in his section about technical progress, Wicksell\’s (p. 134) treated machinery not as capital, but exclusively as a modifier of the “conditions under which labour and land replace each other at the margin of production”. In the 1923 manuscript, however, Wicksell tackled Ricardo in his own terms by discussing in detail the wage fund model deployed in chapter 31 of the Principles. He concluded, just like in the Lectures, that the transition to the new production method is only partial, accompanied by increased output increases and lower wages (the latter is the valid element in Ricardo\’s argument, as pointed out by Wicksell).

Significant efforts have been made to

Significant efforts have been made to fully understand the properties of univariate and multivariate GARCH models. Nelson (1990) derived the necessary and sufficient log-moment condition for stationarity and ergodicity of the GARCH(1,1) model. This condition was extended to higher-order models by Bougerol and Picard (1992). Weak stationarity and the existence of fourth moments of a family of power GARCH models have been investigated in He and Teräsvirta (1999a,b), while Ling and McAleer (2002a,b) derived the necessary and sufficient conditions for the existence of all moments for these models.
Concerning the estimation of the parameters of GARCH models, Lee and Hansen (1994) and Lumsdaine (1996) proved that wnt signaling pathway the local Quasi-Maximum Likelihood Estimator (QMLE) was consistent and asymptotically normal under strong conditions. Jeantheau (1998) established the consistency results of estimators for multivariate GARCH models. His proofs of consistency did not assume a particular functional form for the conditional mean, but assumed a log-moment condition and some regularity conditions for purposes of identification. More recently, Ling and McAleer (2003) proposed the vector ARMA-GARCH model and proved the consistency of the global QMLE under only the second-order moment condition. They also proved the asymptotic normality of the global (local) QMLE under the sixth-order (fourth-order) moment condition. Comte and Lieberman (2003) studied the asymptotic properties of the QMLE for the BEKK model of Engle and Kroner (1995). Berkes et al. (2003) proved the consistency and asymptotic normality if the QMLE of the parameters of the GARCH(p,q) model under second- and fourth-order moment conditions, respectively. Boussama (2000), McAleer et al. (2007), and Francq and Zakoïan (2004) also considered the properties of the QMLE under different specifications of the symmetric and asymmetric GARCH(p,q) model.
However, most of the theoretical results on GARCH models have assumed a constant or linear conditional mean (see McAleer (2005) for further details). It has not yet been established whether these results would also hold if the conditional mean were nonlinear. Chan and McAleer (2002) combined the general STAR model with GARCH(p,q) errors, but their results were derived under the assumption that the conditional mean parameters were known.
The plan of the paper is as follows. Section 2 provides a description of the models considered in the paper. Stationarity, ergodicity and the existence of moments are discussed in Section 3. The asymptotic properties of the QMLE are considered in Section 4. In Section 5 we present simulation results concerning the finite sample properties of the QMLE. Finally, Section 6 gives some concluding remarks. All technical proofs are given in the Appendix.

Model specification
In this section we consider three different classes of STAR-GARCH models. The first specification is an additive logistic STAR model with multiple regimes in the conditional mean and GARCH errors. This model nests the SETAR-GARCH process of Li and Lam (1995). A similar specification with Gaussian errors was proposed in Suarez-Fariñas et al. (2004) and Medeiros and Veiga (2000, 2005). The second specification is a restricted form of the multiple-regime logistic STAR model with GARCH errors. This particular functional form with homoskedastic errors was discussed in Tweedie (1988). Finally, the third specification is the Exponential STAR-GARCH (ESTAR-GARCH) model, of which the Exponential STAR (ESTAR) model of Teräsvirta (1994) is a special case.

It is clear that the model defined by Eqs. (1)–(3) is similar to the functional coefficient autoregressive model proposed by Chen and Tsay (1993). Depending on the choice of the functions f(s;), j=0, 1, …, p, different specifications of the STAR model can be derived. The following cases are considered:

Probabilistic properties

cyclic gmp Kearney and Levine present a detailed

Kearney and Levine (2012) present a detailed survey of teenage childbearing in the US. The main conclusion is that both actual and perceived lack of economic opportunities influence early motherhood. After these factors have been taken into account, teenage motherhood does not appear to cause additional difficulties later in their lives. The authors discuss their main cyclic gmp that the combination of being poor and living in an unequal and less mobile society contributes to a low expectation of success, thus leading to choices that favour short-term satisfaction such as the decision to have a baby when young. They conclude that teenagers in very unequal states are 5 percentage points more likely to give birth than teenagers in the least unequal states. The authors included other variables, such as poverty concentration and absolute levels of deprivation; none of these additional factors altered the estimated relationship between inequality and teen fertility among women with low socioeconomic status. In another study, Kearney and Levine (2014) analyzed both theoretically and empirically the role of income inequality in teenage nonmarital childbearing among poor women. They also found a positive relationship.
Berquó et al. (2012) reached similar conclusions using Brazilian data, although the authors also claim that lack of knowledge about contraception and contraceptive failure are important factors associated with teenage pregnancy, independently of educational and economic status. They argue that pregnancy is often the result of an absence of a life plan: access to better education, better living conditions, and greater enhance options during youth. In absence of good options for the future, pregnancy is a contingent decision made by the teenager today.
The arguments presented by Kearney and Levine (2012, 2014) and Berquó et al. (2012) strengthen the role of low future expectations of teenagers as a trigger mechanism for actions that result in teenage pregnancy.
Another factor that may influence early fertility is birth control. The increased availability of contraception after the 60s, with the introduction of innovative methods such as pills, IUDs, and improvement in sterilization, may have reduced the costs as well as improved the efficiency of contraceptive methods (Schultz, 1997). Hotz and Miller (1988) found a significant variation in the impact of the cost of children on female labor supply when considering different contraceptive behaviors.
According to Caetano (2004), the starting point of official family planning policies in Brazil was 1985, when the government implemented the Programa de Assistencia Integral a Saude da Mulher (PAISM). This program stimulated the public debate on women\’s health, including fertility. It was also at this point that the Demographic Health Survey (DHS) in Brazil began; it has been carried out in 1986, 1996, and 2006.
Table 1 shows data on the contraception trends both among all teenagers (15–19) and among all women of reproductive age (15–44 in 1986, and 15–49 in 1996 and 2006), by region. For the country as a whole we observe an increase in the prevalence rate from 43.7 to 81.5% among all women and from 7.7 to 75.8% for teenagers. This rate varied across regions, with the Northeast having the lowest rate in 1986 (34.8%), but the highest increase (48.2 percentage points) over the 1986–2006 period. By contrast, the South presented the highest prevalence in 1986 (50.3%) but a small variation over the same period (31.9 percentage points). Among all women using any method of contraception, sterilization was the most practiced method in 1986, followed by the pill and condoms, with little relevance. The trends show increased use of condoms (from 2.4% in 1986 to 21.5% in 2006) as well as increased use of female sterilization from 1986 (39.4%) to 1996 (49.4%) followed by a strong decrease in 2006 (32.2%). The use of pills decreased from 38.9% in 1986 to 29% in 2006. The trend in contraceptive methods used by teenagers was completely different: sterilization was virtually nonexistent in this group during the whole period, while the pill was the most used method in 1986 (80% of prevalence), but has since declined (to 39,5% in 2006). On the other hand, the prevalence of condoms increased dramatically, from 2.5% in 1986 to 48.6% in 2006. It is interesting to note the contrast: while the prevalence of contraception rates grew enormously in the period, the percentage of teenagers (15–19 years) who were or have ever been pregnant increased from 13.1% in 1986 to 17.9% in 1996, and then to 23.1% in 2006.

In conclusion we show here that host biomarkers predictive

In conclusion, we show here that tyrosine kinase receptor host biomarkers predictive of TB disease development up to 8months prior to TB diagnosis exist, and can be identified directly ex vivo without antigen specific activation or enrichment steps. Analyses need to be repeated on larger, well defined cohorts with advanced state-of-the-art methods.

Role of the Funding Source

Author Contributions

Declaration of Interests

Acknowledgments
The authors gratefully acknowledge the support by the Research and Development funding from the Public Health Service Amsterdam and research funds: EC FP7 NEWTBVAC contract no. HEALTH.F3.2009 241745, EC FP7 IDEA (Grant agreement no 241642), EC FP7 ADITEC (Grant agreement no 280873), and EC FP7 TANDEM (Grant agreement no 305279) (the text represents the authors\’ views and does not necessarily represent a position of the Commission pepsinogen will not be liable for the use made of such information). The Amsterdam Cohort Studies on HIV infection and AIDS, a collaboration between the Public Health Service of Amsterdam, the Academic Medical Center of the University of Amsterdam, Sanquin Blood Supply Foundation and the University Medical Center Utrecht, are part of the Dutch HIV Monitoring Foundation and financially supported by the Netherlands National Institute for Public Health and the Environment (RIVM).

In vivo data generated using hCD Tg

In vivo data generated using hCD40Tg animals further demonstrate that antigen targeting to CD40 is an efficient way to evoke antigen-specific CD8+ T cell responses in vivo. Although we could not compare CD40 with LOX-1 or Dectin-1 in this animal model due to the limited specificities of mAbs (αLOX-1 and αDectin-1) to human receptors, we were able to verify that CD40 targeting was significantly more efficient than Langerin (another lectin receptor) targeting for the elicitation of antigen-specific CD8+ T cell responses in vivo. In addition, our in vivo data further demonstrate that targeting CD40 results in greater CD8+ than CD4+ T cell responses, while Langerin targeting results in greater CD4+ than CD8+ T cell responses. A previous study (Chatterjee et al., 2012) has already demonstrated that antigen targeting to three different lectin receptors, Langerin, DEC205, and Clec9A, resulted in comparable levels of antigen-specific IFNγ+CD8+ T cell responses in mice. Data (Figs. 1 and 2) from this study illustrated that targeting LOX-1, Dectin-1 or DEC205 resulted in comparable levels of antigen-specific CD8+ T cell responses, but they were less efficient than targeting CD40. Taking all of these data together, CD40 targeting is more efficient than targeting the lectin receptors tested in this study. Consistent with both LOX-1 and Dectin-1, antigen targeting to Langerin, a c-type lectin receptor expressed on Langerhans cyclosporin as well as fractions of dermal DCs (Bonifaz et al., 2004; Delamarre et al., 2003) and CD8α+ DCs (Delneste, 2004) in mice, resulted in greater levels of antigen-specific CD4+ T cell responses.

Author Contributions

Conflict of Interest

Acknowledgments
We thank Carson Harrod (BIIR) for reading this manuscript, Xiao-Hua Li for characterizing mAbs, and Clay Beauregard and LuAnn Snipe for helping in the animal study. This study was funded by the American Cancer Society (RSG-12-075-01-LIB), the National Institutes of Health (U19 AI057234), and a collaborative research grant from Roche.

Introduction
Emergent biological therapies may command tremendous advantages over traditional cancer chemotherapy and radiation, whose efficacies are restricted by toxicity and resistance. Besides reduced toxicity and greater selectivity for tumor cells, new therapies reliant on multiple methods of cell killing distinct from conventional antineoplastic agents and capable of eliciting systemic anti-tumor immune responses promise durable cures and overall survival benefits (Liu et al., 2007). Capitalizing on their inherent ability to invade cells, reprogram them to produce infectious progeny, and spread, viruses can be tailored to selectively destroy tumor cells by modifying their genomes (Bell and McFadden, 2014; Lichty et al., 2014; Chiocca and Rabkin, 2014; Brown et al., 2014). The resulting engineered viruses are attenuated due to deletion of key virulence genes, yet retain the ability to replicate productively in and destroy cancer cells. Such variants, which do not cause disease but are selectively virulent in tumors are termed oncolytic viruses (OVs). Tumor destruction driven, in part, by active viral replication within cancer cells is referred to as viral oncolysis (Bell and McFadden, 2014; Lichty et al., 2014; Chiocca and Rabkin, 2014; Brown et al., 2014). In addition to direct oncolytic action, OVs stimulate systemic, anti-tumor immune responses and are likewise potent immuno-therapeutic agents (Lichty et al., 2014; Chiocca and Rabkin, 2014; Brown et al., 2014; Dharmadhikari et al., 2015; Kaufman et al., 2015).
OV platforms using herpes simplex virus-1 (HSV-1) are particularly encour-aging in part because the virus replicates in a range of tumors and is effectively attenuated by deleting the γ134.5 neuropathogenesis genes (Chou et al., 1990). Furthermore, independent γ134.5-deficient (Δ34.5) HSV-1 strains have proven safe in human clinical trials (Rampling et al., 2000; Markert et al., 2000; Hu et al., 2006; Senzer et al., 2009; Harrington et al., 2010; Andtbacka et al., 2015). Along with viral oncolysis, HSV-1 OVs generate systemic anti-tumor immune responses upon local administration (Toda et al., 1999; Liu et al., 2003). Nevertheless, serious deficiencies in the design of present generation OVs remain, many of which lack functions to cyclosporin evade host innate or acquired immune defenses (Ikeda et al., 1999; Wang et al., 2003; Fulci et al., 2006; Haralambieva et al., 2007; Nguyên et al., 2008; Zamarin et al., 2009; Altomonte et al., 2009; Le Bœuf et al., 2013). Notably, although wild-type HSV-1 replicates in hosts and naturally evades pre-existing innate and acquired immune responses (Posvad and Rosenthal, 1992; Koelle et al., 1993; York et al., 1994), a key immune evasion gene was deleted in some modified Δ34.5 OVs (Taneja et al., 2001; Todo et al., 2001; Liu et al., 2003) and deleting γ134.5 genes results in OVs unable to counteract innate defenses (Mohr and Gluzman, 1996; Mulvey et al., 1999, 2004), severely restricting direct OV cell-killing (Taneja et al., 2001; Todo et al., 2001; Liu et al., 2003) and potentially impairing indirect induction of systemic antitumor immune responses.

Our work enlarges the genetic and phenotypic spectrum of PA

Our work enlarges the genetic and phenotypic spectrum of PA associated with CACNA1H mutations. In addition to early onset PA and hypertension, patient K055-3 carrying the p.Met1549Ile mutation presented mild mental retardation, social skills alterations and learning disabilities and was diagnosed with multiplex developmental disorder at age 10years. This phenotype may owe to a gain of function effect of Cav3.2 in the nervous system, similar to what is observed in patients carrying germline mutations in another calcium channel gene, CACNA1D, coding for Cav1.3, who present with seizures and severe neurological abnormalities in addition to PA (Scholl et al., 2013). Among patients carrying the recurrent p.Met1549Val described previously (Scholl et al., 2015), developmental delay or attention deficit were reported in two mutation carriers. In our study, CACNA1H variants were also associated with familial hyperaldosteronism diagnosed as FH-II, and with sporadic PA and APA. This phenotypic variability may be explained by different functional consequences that the different CACNA1H variants have on channel properties. Differences in clinical presentation where also observed within family members carrying the recurrent CACNA1H p.Met1549Val variant, which were attributed to age-dependent variability or to genetic or environmental modifiers (Scholl et al., 2015). One limitation of the present study is the lack of follow-up data of patients with familial PA and more detailed clinical information on their relatives, which prevented deeper phenotype/genotype investigations in familial PA. Concerning the adrenal phenotype, the presence of additional somatic genetic variants occuring in the adrenal gland of patients carrying a germline CACNA1H variant may play a role in the development of APA, although no recurrent mutation in any of the known driver genes has been identified in tumor DNA of patient K135-1. Also, we cannot exclude that in some cases bilateral adrenal hyperplasia might occur in patients with PA, with a predominant aldosterone-producing nodule on one side leading to lateralization on AVS and cure after surgery. An alternative possibility is the involvement of adrenal buy patupilone producing cell clusters (APCC), which have been shown to carry somatic mutations in APA driver genes in the normal population. Transition from APCC to APA, triggered by unknown factors and which has been evoked as a mechanism for APA development (Nishimoto et al., 2016) may be favored by germline genetic alterations, including CACNA1H variants, creating a favorable cellular environment for this transition.
CACNA1H codes for the pore-forming channel protein Cav3.2, which consists of a single polypeptide chain of four homologous domains (I–IV), each one containing six transmembrane spans (Segments (S) 1–6), and cytoplasmic N- and C-termini. The pore of T-type channels is formed by the tetrameric arrangement of the four domains and it is lined by the S6 segment in the intracellular part, while the S4 segment serves as the voltage sensor (Perez-Reyes, 2003). The p.Ser196Leu mutation is located in the S4 segment of domain I. Each S4 segment contains positively charged residues on every third position and forms the voltage sensor, allowing the channel to open and close in response to changes in membrane potential (Burnay et al., 1994). The substitution of Leucine for Serine changes a polar uncharged aminoacid into a hydrophobic one, which is never found at this position (Burnay et al., 1994). A similar substitution from a polar uncharged to a hydrophobic residue has been found in the S4 segment of domain IV of a patient with idiopathic generalized epilepsy (p.Thr1733Ala) (Heron et al., 2007), while substitution from a positively charged to a hydrophobic amino acid in the S4 segment of domain I and II has been associated with autism spectrum disorders (Splawski et al., 2006). The mutation p.Met1549Ile is located in the S6 segment of domain III. A substitution of the same amino acid has recently been reported in five out of 40 subjects with hypertension and PA before age 10years (Scholl et al., 2015). The p.Met1549Val mutation was shown to modify the functional properties of the calcium channel, facilitating its opening at more hyperpolarized potentials and delaying its inactivation, leading to increased aldosterone production (Reimer et al., 2016). Finally, variants p.Val1951Glu and p.Pro2083Leu are located at the cytoplasmic C-terminus of the channel. Studies on Cav3.1, which is closely related to Cav3.2, showed that the C-terminus determines fast inactivation of this T-type calcium channel (Staes et al., 2001). Amino acid substitutions in this region of Cav3.2 were found in patients with epilepsy (p.Arg1892His, p.Arg2005Cys, p.His2060Arg, p.Arg2077His) (Chen et al., 2003; Heron et al., 2007) and in autism spectrum disorders (p.Arg1871Gln+Ala1874Val) (Splawski et al., 2006).

This study is limited in that the metabolomic

This study is limited in that the metabolomic abnormalities described in children in Malawi may not be generalizable to other populations due to differences in diet, environment, socioeconomic factors, and other variables. The Metabolon HD4 platform provides wide coverage of many metabolic pathways, but the serum metabolite measurements are semi-quantitative. This discovery platform can foster the development of targeted LC-MS/MS assays that provide absolute quantification using deuterated or stable isotope-labeled standards. Such targeted assays could be used for absolute quantification of specific metabolites such as acylcarnitines and dicarboxylic acids in the future. Although the present study shows abnormalities in metabolites that are known to be associated with altered gut microflora, the gut microbiome was not assessed in the present study. However, a previous studies show that EED is associated with alterations in the gut microbiome (Yu et al., 2015; Ordiz et al., 2016). Another limitation is that EED remains a poorly defined disease state that is ideally assessed by biopsy of the small intestine and endoscopy. The dual sugar apoptosis test is commonly used for diagnosis but has potential for misclassification (Denno et al., 2014). Future studies are needed in children with EED to examine the relationship between serum metabolites and the gut microbiome and between serum metabolites and vaccine failure. Also, studies using a longitudinal design could examine the relationship between serum metabolites and growth faltering. The strengths of this study are the large sample size, the use of a highly advanced metabolomics platform, and a community-based study design in a setting where EED is a major cause of morbidity.
The following are the supplementary data related to this article.

Conflicts of Interest

Author Contributions

Acknowledgements
This work was supported by the Children\’s Discovery Institute of Washington University and St. Louis Children\’s Hospital, the Hickey Family Foundation, the National Institutes of HealthR01 AG27012 and the Intramural Branch of the National Institute on Aging, Baltimore, Maryland. The funders had no role in the study design, data collection, data analysis, interpretation, or writing of the report.

Introduction
Modern humans have inhabited Africa for longer than any other geographic region (>200,000years; Tishkoff et al., 2009); the processes of random mutation, meiotic recombination, and genetic drift have therefore led to the accumulation of a relatively large pool of genetic variation in Africa compared with elsewhere. Approximately 100,000years ago, a limited number of African subgroups migrated from Africa to other continents; many genotypes are therefore found only in Africa (Tishkoff et al., 2009; Fujikara et al., 2015; Kozyra et al., 2016; Wright et al., 2016). Based on a study calculating worldwide disease burden for 21 regions, the African continent has 15.5% of the global population but carries approximately 25% of the global disease burden (Murray et al., 2012). Furthermore, the African population carries a high burden of adverse drug reactions owing partly to the use of old, poorly-optimized drugs for the treatment of parasitic infections (Prentis et al., 1988; Ampadu et al., 2016), and also possibly owing to high levels of genetic diversity resulting in a greater proportion of patients having adverse reactions to therapeutic drugs.
Drug metabolism involves a complex interplay between multiple metabolic enzymes, often with apoptosis overlapping substrate specificity. One significant class of enzymes involved in therapeutic drug metabolism is the cytochrome P450 (CYP) family. CYP1, CYP2, and CYP3 are the key sub-families involved in phase I drug metabolism. Single nucleotide polymorphisms (SNPs) in CYP genes have been extensively reported (Fujikara et al., 2015) with over 400 allelic variants recorded across the CYP1, CYP2, and CYP3 sub-families (The human cytochrome P450 allele nomenclature database http://cypalleles.ki.se/ accessed 28 October 2016). Variations in gene sequence and protein structure give rise to alleles conferring no enzyme function, decreased enzyme function, normal enzyme function or increased enzyme function. Individuals who carry allelic variants may demonstrate one of the following metabolic phenotypes: poor metabolizers (PM; individuals with a combination of no function or decreased function alleles, and little to no enzyme activity); intermediate metabolizers (IM; individuals with a combination of normal and decreased function alleles conferring decreased enzyme activity); normal metabolizers (NM; individuals with fully functional enzyme activity) or rapid and ultra-rapid metabolizers (UM; individuals with two increased function alleles or more than two normal function alleles) (Caudle et al., 2016; Gaedigk et al., 2016). Genotype information can be used to guide appropriate therapeutic drug doses to reduce the risk of drug-induced adverse reactions in PMs or drug resistance in UMs for drugs with inactive metabolites, or of drug-induced adverse reactions in UMs and drug resistance in PMs for pro-drugs that require metabolic activation (Ingeman-Sundberg et al., 2007).