Portable smartphone quantitation of prostate specific antigen (PSA) in a fluoropolymer microfluidic device

We present a new, power-free and flexible detection system named MCFphone for portable colorimetric and fluorescence quantitative sandwich immunoassay detection of prostate specific antigen (PSA). The MCFphone is composed by a smartphone integrated with a magnifying lens, a simple light source and a miniaturised immunoassay platform, the Microcapillary Film (MCF). The excellent transparency and flat geometry of fluoropolymer MCF allowed quantitation of PSA in the range 0.9 to 60 ng/ml with < 7 % precision in 13 minutes using enzymatic amplification and a chromogenic substrate. The lower limit of detection was further improved from 0.4 to 0.08 ng/ml in whole blood samples with the use of a fluorescence substrate. The MCFphone has shown capable of performing rapid (13 to 22 minutes total assay time) colorimetric quantitative and highly sensitive fluorescence tests with good %Recovery, which represents a major step in the integration of a new generation of inexpensive and portable microfluidic devices with commercial immunoassay reagents and off-the-shelf smartphone technology.

Climate variability and human impact in South America during the last 2000 years: synthesis and perspectives from pollen records

An improved understanding of present-day climate variability and change relies on high-quality data sets from the past 2 millennia. Global efforts to model regional climate modes are in the process of being validated against, and integrated with, records of past vegetation change. For South America, however, the full potential of vegetation records for evaluating and improving climate models has hitherto not been sufficiently acknowledged due to an absence of information on the spatial and temporal coverage of study sites. This paper therefore serves as a guide to high-quality pollen records that capture environmental variability during the last 2 millennia. We identify 60 vegetation (pollen) records from across South America which satisfy geochronological requirements set out for climate modelling, and we discuss their sensitivity to the spatial signature of climate modes throughout the continent. Diverse patterns of vegetation response to climate change are observed, with more similar patterns of change in the lowlands and varying intensity and direction of responses in the highlands. Pollen records display local-scale responses to climate modes; thus, it is necessary to understand how vegetation–climate interactions might diverge under variable settings. We provide a qualitative translation from pollen metrics to climate variables. Additionally, pollen is an excellent indicator of human impact through time. We discuss evidence for human land use in pollen records and provide an overview considered useful for archaeological hypothesis testing and important in distinguishing natural from anthropogenically driven vegetation change. We stress the need for the palynological community to be more familiar with climate variability patterns to correctly attribute the potential causes of observed vegetation dynamics. This manuscript forms part of the wider LOng-Term multi-proxy climate REconstructions and Dynamics in South America – 2k initiative that provides the ideal framework for the integration of the various palaeoclimatic subdisciplines and palaeo-science, thereby jump-starting and fostering multidisciplinary research into environmental change on centennial and millennial timescales.

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.

Prediction of metabolisable energy concentrations of fresh cut grass using digestibility data measured with non-pregnant non-lactating cows

Pasture-based ruminant production systems are common in certain areas of the world, but energy evaluation in grazing cattle is performed with equations developed, in their majority, with sheep or cattle fed total mixed rations. The aim of the current study was to develop predictions of metabolisable energy (ME) concentrations in fresh-cut grass offered to non-pregnant non-lactating cows at maintenance energy level, which may be more suitable for grazing cattle. Data were collected from three digestibility trials performed over consecutive grazing seasons. In order to cover a range of commercial conditions and data availability in pasture-based systems, thirty-eight equations for the prediction of energy concentrations and ratios were developed. An internal validation was performed for all equations and also for existing predictions of grass ME. Prediction error for ME using nutrient digestibility was lowest when gross energy (GE) or organic matter digestibilities were used as sole predictors, while the addition of grass nutrient contents reduced the difference between predicted and actual values, and explained more variation. Addition of N, GE and diethyl ether extract (EE) contents improved accuracy when digestible organic matter in DM was the primary predictor. When digestible energy was the primary explanatory variable, prediction error was relatively low, but addition of water-soluble carbohydrates, EE and acid-detergent fibre contents of grass decreased prediction error. Equations developed in the current study showed lower prediction errors when compared with those of existing equations, and may thus allow for an improved prediction of ME in practice, which is critical for the sustainability of pasture-based systems.

Prediction of nutrient digestibility and energy concentrations in fresh grass using nutrient composition

Improved nutrient utilization efficiency is strongly related to enhanced economic performance and reduced environmental footprint of dairy farms. Pasture-based systems are widely used for dairy production in certain areas of the world, but prediction equations of fresh grass nutritive value (nutrient digestibility and energy concentrations) are limited. Equations to predict digestible energy (DE) and metabolizable energy (ME) used for grazing cattle have been either developed with cattle fed conserved forage and concentrate diets or sheep fed previously frozen grass, and the majority of them require measurements less commonly available to producers, such as nutrient digestibility. The aim of the present study was therefore to develop prediction equations more suitable to grazing cattle for nutrient digestibility and energy concentrations, which are routinely available at farm level by using grass nutrient contents as predictors. A study with 33 nonpregnant, nonlactating cows fed solely fresh-cut grass at maintenance energy level for 50 wk was carried out over 3 consecutive grazing seasons. Freshly harvested grass of 3 cuts (primary growth and first and second regrowth), 9 fertilizer input levels, and contrasting stage of maturity (3 to 9 wk after harvest) was used, thus ensuring a wide representation of nutritional quality. As a result, a large variation existed in digestibility of dry matter (0.642-0.900) and digestible organic matter in dry matter (0.636-0.851) and in concentrations of DE (11.8-16.7 MJ/kg of dry matter) and ME (9.0-14.1 MJ/kg of dry matter). Nutrient digestibilities and DE and ME concentrations were negatively related to grass neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents but positively related to nitrogen (N), gross energy, and ether extract (EE) contents. For each predicted variable (nutrient digestibilities or energy concentrations), different combinations of predictors (grass chemical composition) were found to be significant and increase the explained variation. For example, relatively higher R(2) values were found for prediction of N digestibility using N and EE as predictors; gross-energy digestibility using EE, NDF, ADF, and ash; NDF, ADF, and organic matter digestibilities using N, water-soluble carbohydrates, EE, and NDF; digestible organic matter in dry matter using water-soluble carbohydrates, EE, NDF, and ADF; DE concentration using gross energy, EE, NDF, ADF, and ash; and ME concentration using N, EE, ADF, and ash. Equations presented may allow a relatively quick and easy prediction of grass quality and, hence, better grazing utilization on commercial and research farms, where nutrient composition falls within the range assessed in the current study.

Evaluating nitrogen utilization efficiency of nonpregnant dry cows offered solely fresh cut grass at maintenance levels

The present study aimed to identify key parameters influencing N utilization and develop prediction equations for manure N output (MN), feces N output (FN), and urine N output (UN). Data were obtained under a series of digestibility trials with nonpregnant dry cows fed fresh grass at maintenance level. Grass was cut from 8 different ryegrass swards measured from early to late maturity in 2007 and 2008 (2 primary growth, 3 first regrowth, and 3 second regrowth) and from 2 primary growth early maturity swards in 2009. Each grass was offered to a group of 4 cows and 2 groups were used in each of the 8 swards in 2007 and 2008 for daily measurements over 6 wk; the first group (first 3 wk) and the second group (last 3 wk) assessed early and late maturity grass, respectively. Average values of continuous 3-d data of N intake (NI) and output for individual cows ( = 464) and grass nutrient contents ( = 116) were used in the statistical analysis. Grass N content was positively related to GE and ME contents but negatively related to grass water-soluble carbohydrates (WSC), NDF, and ADF contents ( < 0.01), indicating that accounting for nutrient interrelations is a crucial aspect of N mitigation. Significantly greater ratios of UN:FN, UN:MN, and UN:NI were found with increased grass WSC contents and ratios of N:WSC, N:digestible OM in total DM (DOMD), and N:ME ( < 0.01). Greater NI, animal BW, and grass N contents and lower grass WSC, NDF, ADF, DOMD, and ME concentrations were significantly associated with greater MN, FN, and UN ( < 0.05). The present study highlighted that using grass lower in N and greater in fermentable energy in animals fed solely fresh grass at maintenance level can improve N utilization, reduce N outputs, and shift part of N excretion toward feces rather than urine. These outcomes are highly desirable in mitigation strategies to reduce nitrous oxide emissions from livestock. Equations predicting N output from BW and grass N content explained a similar amount of variability as using NI and grass chemical composition (excluding DOMD and ME), implying that parameters easily measurable in practice could be used for estimating N outputs. In a research environment, where grass DOMD and ME are likely to be available, their use to predict N outputs is highly recommended because they strongly improved of the equations in the current study.

The grass-free lawn: floral performance and management implications

Grass lawns are a ubiquitous feature of urban green-space throughout much of the temperate world. Species poor and intensively managed, lawns are ecologically impoverished, however environmentally aware lawn owners are reluctant to implement alternatives due to aesthetic concerns. Developing an alternative lawn format which is both biodiversity friendly and aesthetically pleasing is an imperative for urban greening. We suggest that such an alternative can be provided by replacing the grass lawn by a forb-based mix. To advance this, we tested the floral performance of three groups of clonal perennial forbs (native, non-native and mixed), each maintained using standard lawn management mowing regimes. Our findings show that both the frequency of mowing and the height at which mowing is applied influence floral performance and lawn aesthetics. Species origin was found to influence floral productivity, floral visibility and floral variety within grass-free lawns, with native species providing the greatest floral performance. The behaviour and management of grass lawns was not found to be a suitable analogue for the management of grass-free lawns and grass-free lawns are sufficiently different from grass lawns to require an entirely original management approach. We suggest that the grass-free lawn can provide an aesthetically and environmentally relevant replacement for the ubiquitous and ecologically-poor grass lawn.