Targeted activation of toll-like receptors: conjugation of a toll-like receptor 7 agonist to a monoclonal antibody maintains antigen binding and specificity

Therapeutic activation of Toll-like receptors (TLR) has potential for cancer immunotherapy, for augmenting the activity of anti-tumor monoclonal antibodies (mAbs), and for improved vaccine adjuvants. A previous attempt to specifically target TLR agonists to dendritic cells (DC) using mAbs failed because conjugation led to non-specific binding and mAbs lost specificity. We demonstrate here for the first time the successful conjugation of a small molecule TLR7 agonist to an anti-tumour mAb (the anti-hCD 20 rituximab) without compromising antigen specificity. The TLR7 agonist UC-1V150 was conjugated to rituximab using two conjugation methods and yield, molecular substitution ratio, retention of TLR7 activity and specificity of antigen binding were compared. Both conjugation methods produced rituximab-UC-1V150 conjugates with UC-1V150 : rituximab ratio ranging from 1:1 to 3:1 with drug loading quantified by UV spectroscopy and drug substitution ratio verified by MALDI TOF mass spectroscopy. The yield of purified conjugates varied with conjugation method, and dropped as low as 31% using a method previously described for conjugating UC-1V150 to proteins, where a bifunctional crosslinker was firstly reacted with rituximab, and secondly to the TLR7 agonist. We therefore developed a direct conjugation method by producing an amine-reactive UV active version of UC-1V150, termed NHS:UC-1V150. Direct conjugation with NHS:UC-1V150 was quick and simple and gave improved conjugate yields of 65-78%. Rituximab-UC-1V150 conjugates had the expected pro-inflammatory activity in vitro (EC50 28-53 nM) with a significantly increased activity over unconjugated UC-1V150 (EC50 547 nM). Antigen binding and specificity of the rituxuimab-UC-1V150 conjugates was retained, and after incubation with human peripheral blood leukocytes, all conjugates bound strongly only to CD20-expressing B cells whilst no non-specific binding to CD20-negative cells was observed. Selective targeting of Toll-like receptor activation directly within tumors or to DC is now feasible.

Mapping power law distributions in digital health social networks: methods, interpretations, and practical implications

BACKGROUND: Social networks are common in digital health. A new stream of research is beginning to investigate the mechanisms of digital health social networks (DHSNs), how they are structured, how they function, and how their growth can be nurtured and managed. DHSNs increase in value when additional content is added, and the structure of networks may resemble the characteristics of power laws. Power laws are contrary to traditional Gaussian averages in that they demonstrate correlated phenomena. OBJECTIVES: The objective of this study is to investigate whether the distribution frequency in four DHSNs can be characterized as following a power law. A second objective is to describe the method used to determine the comparison. METHODS: Data from four DHSNs—Alcohol Help Center (AHC), Depression Center (DC), Panic Center (PC), and Stop Smoking Center (SSC)—were compared to power law distributions. To assist future researchers and managers, the 5-step methodology used to analyze and compare datasets is described. RESULTS: All four DHSNs were found to have right-skewed distributions, indicating the data were not normally distributed. When power trend lines were added to each frequency distribution, R(2) values indicated that, to a very high degree, the variance in post frequencies can be explained by actor rank (AHC .962, DC .975, PC .969, SSC .95). Spearman correlations provided further indication of the strength and statistical significance of the relationship (AHC .987. DC .967, PC .983, SSC .993, P<.001). CONCLUSIONS: This is the first study to investigate power distributions across multiple DHSNs, each addressing a unique condition. Results indicate that despite vast differences in theme, content, and length of existence, DHSNs follow properties of power laws. The structure of DHSNs is important as it gives insight to researchers and managers into the nature and mechanisms of network functionality. The 5-step process undertaken to compare actor contribution patterns can be replicated in networks that are managed by other organizations, and we conjecture that patterns observed in this study could be found in other DHSNs. Future research should analyze network growth over time and examine the characteristics and survival rates of superusers.

Microbial Recognition Via Toll-Like Receptor-Dependent and -Independent Pathways Determines the Cytokine Response of Murine Dendritic Cell Subsets to CD40 Triggering

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8alpha(+) and CD8alpha(-)CD4(-) DC to make IL-12 p70. In contrast, exposure of CD8alpha(+), CD4(+) and CD8alpha(-)CD4(-) DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.

Soil phosphorus dynamics and phytoavailability from sewage sludge at different stages in a treatment stream

The effect of different stages of sewage sludge treatment on phosphorus (P) dynamics in amended soils was determined using samples of undigested liquid (UL), anaerobically digested liquid (AD) and dewatered anaerobically digested (DC) sludge. Sludges were taken from three points in the same treatment stream and applied to a sandy loam soil in field-based mesocosms at 4, 8 and 16t ha−1 dry solids. Mesocosms were sown with perennial ryegrass (Lolium perenne cv. Melle), and the sward was harvested after 35 and 70 days to determine yield and foliar P concentration. Soils were also sampled during this period to measure P transformations and the activities of acid phosphomonoesterase and phosphodiesterase. Data show that the AD amended soils had the greatest plant-available and foliar P content up to the second harvest, but the UL amended soils had the greatest enzyme activity. Characterisation of control and 16t ha−1 soils and sludge using solution 31P nuclear magnetic resonance (NMR) spectroscopy after NaOH–EDTA extraction revealed that P was predominantly in the inorganic pool in all three sludge samples, with the highest proportion (of the total extracted P) as inorganic P in the anaerobically digested liquid sludge. After sludge incorporation, P was immobilised to organic species. The majority of organic P was in monoester-P forms, while the remainder of organic P (diester P and phosphonate P) was more susceptible to transformations through time and showed variation with sludge type. These results show that application of sewage sludge at rates as low as 4t ha−1 can have a significant nutritional benefit to ryegrass over an initial 35-day growth and subsequent 35-day re-growth periods. Differences in P transformation, and hence nutritional benefit, between sludge types were evident throughout the experiment. Thus, differences in sludge treatment process alter the edaphic mineralisation characteristics of biosolids derived from the same source material.

Post-conflict recovery and peacebuilding

Civil wars are the most common type of large scale violent conflict. They are long, brutal and continue to harm societies long after the shooting stops. Post-conflict countries face extraordinary challenges with respect to development and security. In this paper we examine how countries can recover economically from these devastating conflicts and how international interventions can help to build lasting peace. We revisit the aid and growth debate and confirm that aid does not increase growth in general. However, we find that countries experience increased growth after the end of the war and that aid helps to make the most of this peace dividend. However, aid is only growth enhancing when the violence has stopped, in violent post-war societies aid has no growth enhancing effect. We also find that good governance is robustly correlated with growth, however we cannot confirm that aid increases growth conditional on good policies. We examine various aspects of aid and governance by disaggregating the aid and governance variables. Our analysis does not provide a clear picture of which types of aid and policy should be prioritized. We find little evidence for a growth enhancing effect of UN missions and suggest that case studies may provide better insight into the relationship between security guarantees and economic stabilization.

Echoes of 1992: the NAFTA negotiations and North America now

Two decades ago, Canada, Mexico, and the United States created a continental economy. The road to integration from the signing of the North American Free Trade Agreement has not been a smooth one. Along the way, Mexico lived through a currency crisis, a democratic transition, and the rising challenge of Asian manufacturing. Canada stayed united despite surging Quebecois nationalism during the 1990s; since then, it has seen dramatic economic changes with the explosion of hydrocarbon production and a much stronger currency. The United States saw a stock-market bust, the shock of 9/11, and the near-collapse of its financial system. All of these events have transformed the relationships that emerged after NAFTA entered into force in 1994. Given the tremendous changes, one might be skeptical that the circumstances and details of the negotiation and ratification of NAFTA hold lessons for the future of North America. However, the road to NAFTA had its own difficulties, and many of the issues involved in the negotiations underpin today's challenges. NAFTA was conceived at a time of profound change in the international system. When Mexican leaders surveyed the world two decades ago, they saw emerging regional groupings in Europe, Asia, and South America. Faced with a lack of interest or compatibility, they instead doubled down on North America. How did Mexican leaders reconsider their national interests and redefine Mexico's role in the world in light of those transformations? Unpublished Mexican documents from SECOFI, the secretariate most involved in negotiating NAFTA, help illustrate Mexican thinking about its interests and role at that time. Combining those insights with analysis of newly available evidence from U.S. presidential archives, this paper sheds light on the negotiations that concluded two decades ago.

Second-order model for remote and close-up short-circuit faults currents on DC traction supply

A technique to calculate the current waveform for both close-up and remote short-circuit faults on DC supplied railways and subways is presented. Exact DC short-circuit current calculation is best performed by sophisticated computer transient simulations. However, an accurate simplified calculation method based on second-order approximation which can be easily executed with the help of a calculator or a spreadsheet program is proposed.

Development and Validation of a Mice liar Electrokinetic Chromatography Method for Quantitative Determination of Butenolides in Piper malacophyllum (C. Presl) C. DC.

Introduction - A large number of natural and synthetic compounds having butenolides as a core unit have been described and many of them display a wide range of biological activities. Butenolides from P. malacophyllum have presented potential antifungal activities but no specific, fast, and precise method has been developed for their determination. Objective - To develop a methodology based on micellar electrokinetic chromatography to determine butenolides in Piper species. Methodology - The extracts were analysed in an uncoated fused-silica capillaries and for the micellar system 20 mmol/L SDS, 20% (v/v) acetonitrile (ACN) and 10 mmol/L STB aqueous buffer at pH 9.2 were used. The method was validated for precision, linearity, limit of detection (LOD) and limit of quantitation (LOQ) and the standard deviations were determined from the standard errors estimated by the regression line. Results - A micellar electrokinetic chromatography (MEKC) method for determination of butenolides in extracts gave full resolution for 1 and 2. The analytical curve in the range 10.0-50.0 mu g/mL (r(2) = 0.999) provided LOD and LOQ for 1 and 2 of 2.1/6.3 and 1.1/3.5 mu g/mL, respectively. The RSD for migration times were 0.12 and 1.0% for peak area ratios with 100.0 +/- 1.4% of recovery. Conclusions - A novel high-performance MEKC method developed for the analysis of butenolides 1 and 2 in leaf extracts of P. malacophyllum allowed their quantitative determined within an analysis time shorter than 5 min and the results indicated CE to be a feasible analytical technique for the quantitative determination of butenolides in Piper extracts. Copyright (C) 2010 John Wiley & Sons, Ltd.

Prenylated benzoic acid derivatives from Piper aduncum L. and P. hostmannianum C. DC. (Piperaceae)

The bioactivity-guided fractionation of the crude extracts from leaves of Brazilian species Piper aduncum and Piper hostmannianum by means of bioautography using the fungi Cladosporium cladosporioides and C. sphaerospermum afforded prenylated methyl benzoate, chromenes, and dihydrobenzopyran derivatives as antifungal compounds. The isolation and structural elucidation of a new compound methyl 4-hydroxy-3-(2`-hydroperoxy-3`-methyl-3`-butenyl) benzoate were performed by application of chromatographic techniques and spectroscopic analyses. (C) 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)). Piper solmsianum is a shrub from Southeast Brazil in which many biologically active compounds were identified. The aim of this work was to establish a cell suspension culture system for this species. With this in mind, petiole and leaf explants obtained from in vitro plantlets were cultured in the presence of different plant growth regulator combinations (IAA, NAA, 2,4-D and BA). Root and indirect shoot adventitious formation, detected by histological analysis, was observed. Besides the different combinations of plant growth regulators, light regime and the supplement of activated charcoal (1.5 mg.l(-1)) were tested for callus induction and growth. Cultures maintained in light, on a 0.2 mg.l(-1) 2,4-D and 2 mg.l(-1) BA supplemented medium, and in the absence of activated charcoal, showed the highest calli fresh matter increment. From a callus culture, cell suspension cultures were established and their growth and metabolite accumulation studied. The achieved results may be useful for further characterization of the activated secondary metabolites pathways in in vitro systems of P. solmsianum.

Energy Analysis within Industrial Hydraulics and Correspondent Solar PV System Design

Energy efficiency and renewable energy use are two main priorities leading to industrial sustainability nowadays according to European Steel Technology Platform (ESTP). Modernization efforts can be done by industries to improve energy consumptions of the production lines. These days, steel making industrial applications are energy and emission intensive. It was estimated that over the past years, energy consumption and corresponding CO2 generation has increased steadily reaching approximately 338.15 parts per million in august 2010 [1]. These kinds of facts and statistics have introduced a lot of room for improvement in energy efficiency for industrial applications through modernization and use of renewable energy sources such as solar Photovoltaic Systems (PV).The purpose of this thesis work is to make a preliminary design and simulation of the solar photovoltaic system which would attempt to cover the energy demand of the initial part of the pickling line hydraulic system at the SSAB steel plant. For this purpose, the energy consumptions of this hydraulic system would be studied and evaluated and a general analysis of the hydraulic and control components performance would be done which would yield a proper set of guidelines contributing towards future energy savings. The results of the energy efficiency analysis showed that the initial part of the pickling line hydraulic system worked with a low efficiency of 3.3%. Results of general analysis showed that hydraulic accumulators of 650 liter size should be used by the initial part pickling line system in combination with a one pump delivery of 100 l/min. Based on this, one PV system can deliver energy to an AC motor-pump set covering 17.6% of total energy and another PV system can supply a DC hydraulic pump substituting 26.7% of the demand. The first system used 290 m2 area of the roof and was sized as 40 kWp, the second used 109 m2 and was sized as 15.2 kWp. It was concluded that the reason for the low efficiency was the oversized design of the system. Incremental modernization efforts could help to improve the hydraulic system energy efficiency and make the design of the solar photovoltaic system realistically possible. Two types of PV systems where analyzed in the thesis work. A method was found calculating the load simulation sequence based on the energy efficiency studies to help in the PV system simulations. Hydraulic accumulators integrated into the pickling line worked as energy storage when being charged by the PV system as well.

Inhibition of the myotoxic activity of Bothrops jararacussu venom and its two major myotoxins, BthTX-I and BthTX-II, by the aqueous extract of Tabernaemontana catharinensis A. DC. (Apocynaceae)

Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA(2) activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 mug/ml) plus AE (400 mug/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 mug/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 mug/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 mug/ml, I h) did not reveal any change in muscle fibers, but severe necrosis induced by -BV or toxins (20 mug/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA(2), activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 mug of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 mug), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with I h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors. (C) 2004 Elsevier GmbH. All rights reserved.

Molecular architecture and electrical properties in evaporated films of cobalt phthalocyanine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Magnetic response and critical current properties of mesoscopic-size YBCO superconducting samples

In this contribution superconducting specimens of YBa(2)Cu(3)O(7-delta) were synthesized by a modified polymeric precursor method, yielding a ceramic powder with particles of mesoscopic-size. Samples of this powder were then pressed into pellets and sintered under different conditions. The critical current density was analyzed by isothermal AC-susceptibility measurements as a function of the excitation field, as well as with isothermal DC-magnetization runs at different values of the applied field. Relevant features of the magnetic response could be associated to the microstructure of the specimens and, in particular, to the superconducting intra- and intergranular critical current properties.

Biased Atmospheric, Sub-Atmospheric, and Low-Pressure Air Plasmas for Material Surface Improvements

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)