Cross-correlation image analysis for real-time single particle tracking.

Accurately measuring the translations of objects between images is essential in many fields, including biology, medicine, chemistry, and physics. One important application is tracking one or more particles by measuring their apparent displacements in a series of images. Popular methods, such as the center of mass, often require idealized scenarios to reach the shot noise limit of particle tracking and, therefore, are not generally applicable to multiple image types. More general methods, such as maximum likelihood estimation, reliably approach the shot noise limit, but are too computationally intense for use in real-time applications. These limitations are significant, as real-time, shot-noise-limited particle tracking is of paramount importance for feedback control systems. To fill this gap, we introduce a new cross-correlation-based algorithm that approaches shot-noise-limited displacement detection and a graphics processing unit-based implementation for real-time image analysis of a single particle.

A chip-based supersonic microfluidic nebulizer for efficient sample introduction into inductively coupled plasma - Mass spectrometry.

As the use of microfluidic chips for handling biological samples is increasing, so is the need for combining them with powerful analytical techniques for metal determination such as inductively-coupled plasma mass spectrometry (ICP-MS). So far, coupling a microfluidic chip to an ICP-MS has been demonstrated mainly through the use of conventional pneumatic micro-flow nebulizers. However, disadvantages associated with the use of such nebulizers entail dead volume issues and liquid suction exerted on the outlet channel of the chip. Herein, we propose that a microfluidic chip, bearing a pneumatic nozzle for liquid nebulization, has the potential to advance metal determination in chip-based ICP-MS. More specifically, we demonstrate for the first time that the coupling of a chip-based supersonic microfluidic nebulizer (chip-µf-Neb) to an ICP-MS can be conveniently achieved through the use of a spray chamber with a laminar flow makeup gas. Operation of the combined system was evaluated at low liquid flow rates across 0.5-20 µL min-1, while nebulization and makeup argon (Ar) gas flow rates were optimized with respect to maximizing indium (In) sensitivity and minimizing oxide formation; a maximum sensitivity of 40000 cps (µg L-1)-1 was achieved at 10 µL min-1. The system was further evaluated for its performance in single-particle analysis, featuring a transport efficiency of 46% for Ag nanoparticles. Finally, the capabilities for conducting single-cell analysis were demonstrated with the detection of 80Se and 75As in individual Chlamydomas reinhardtii cells, which were previously incubated in 20 µM of selenate and 300 µM of arsenate, respectively. Efficient operation at low liquid flow rates along with the absence of self-aspiration render this nebulizer a promising tool for combining the powerful field of microfluidics with metal quantitation by means of ICP-MS.

Structure-specific amyloid precipitation in biofluids.

The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. Here we report a method to capture amyloid-containing aggregates in human biofluids in an unbiased way, a process we name amyloid precipitation. We use a structure-specific chemical dimer, a Y-shaped, bio-inspired small molecule with two capture groups, for amyloid precipitation to increase affinity. Our capture molecule for amyloid precipitation (CAP-1) consists of a derivative of Pittsburgh Compound B (dimer) to target the cross ß-sheets of amyloids and a biotin moiety for surface immobilization. By coupling CAP-1 to magnetic beads, we demonstrate that we can target the amyloid structure of all protein aggregates present in human cerebrospinal fluid, isolate them for analysis and then characterize them using single-molecule fluorescence imaging and mass spectrometry. Amyloid precipitation enables unbiased determination of the molecular composition and structural features of the in vivo aggregates formed in neurodegenerative diseases.

Multigenerational laboratory culture of pelagic ctenophores and CRISPR-Cas9 genome editing in the lobate Mnemiopsis leidyi.

Despite long-standing experimental interest in ctenophores due to their unique biology, ecological influence and evolutionary status, previous work has largely been constrained by the periodic seasonal availability of wild-caught animals and difficulty in reliably closing the life cycle. To address this problem, we have developed straightforward protocols that can be easily implemented to establish long-term multigenerational cultures for biological experimentation in the laboratory. In this protocol, we describe the continuous culture of the Atlantic lobate ctenophore Mnemiopsis leidyi. A rapid 3-week egg-to-egg generation time makes Mnemiopsis suitable for a wide range of experimental genetic, cellular, embryological, physiological, developmental, ecological and evolutionary studies. We provide recommendations for general husbandry to close the life cycle of Mnemiopsis in the laboratory, including feeding requirements, light-induced spawning, collection of embryos and rearing of juveniles to adults. These protocols have been successfully applied to maintain long-term multigenerational cultures of several species of pelagic ctenophores, and can be utilized by laboratories lacking easy access to the ocean. We also provide protocols for targeted genome editing via microinjection with CRISPR-Cas9 that can be completed within ~2 weeks, including single-guide RNA synthesis, early embryo microinjection, phenotype assessment and sequence validation of genome edits. These protocols provide a foundation for using Mnemiopsis as a model organism for functional genomic analyses in ctenophores.

Biomembranes in bioelectronic sensing.

Cell membranes are integral to the functioning of the cell and are therefore key to drive fundamental understanding of biological processes for downstream applications. Here, we review the current state-of-the-art with respect to biomembrane systems and electronic substrates, with a view of how the field has evolved towards creating biomimetic conditions and improving detection sensitivity. Of particular interest are conducting polymers, a class of electroactive polymers, which have the potential to create the next step-change for bioelectronics devices. Lastly, we discuss the impact these types of devices could have for biomedical applications.

Outcomes, complications and risk factors following fluoroscopically guided transcondylar screw placement for humeral intracondylar fissure.

OBJECTIVES: To describe the surgical technique and complications for fluoroscopically guided transcondylar screw placement for humeral intracondylar fissure in dogs. MATERIALS AND METHODS: A retrospective review was undertaken of cases from two hospitals where identical surgical technique was employed. Factors were analysed for any association with postoperative complications. RESULTS: Sixty-two dogs (82 elbows) were reviewed for which the postoperative complication rate was 45%; a total of 15% of cases required revision surgery. Complications were more likely in cases operated on earlier in the case series and with increasing dog bodyweight. Both increasing surgical time and being a neutered female were protective against postoperative complications. CLINICAL SIGNIFICANCE: Fluoroscopically guided transcondylar screw placement for humeral intracondylar fissure is associated with a high postoperative complication rate (45%) with 15% of cases requiring revision surgery.

The role of clearance mechanisms in the kinetics of pathological protein aggregation involved in neurodegenerative diseases.

The deposition of pathological protein aggregates in the brain plays a central role in cognitive decline and structural damage associated with neurodegenerative diseases. In Alzheimer's disease, the formation of amyloid-ß plaques and neurofibrillary tangles of the tau protein is associated with the appearance of symptoms and pathology. Detailed models for the specific mechanisms of aggregate formation, such as nucleation and elongation, exist for aggregation in vitro where the total protein mass is conserved. However, in vivo, an additional class of mechanisms that clear pathological species is present and is believed to play an essential role in limiting the formation of aggregates and preventing or delaying the emergence of disease. A key unanswered question in the field of neuro-degeneration is how these clearance mechanisms can be modeled and how alterations in the processes of clearance or aggregation affect the stability of the system toward aggregation. Here, we generalize classical models of protein aggregation to take into account both production of monomers and the clearance of protein aggregates. We show that, depending on the specifics of the clearance process, a critical clearance value emerges above which accumulation of aggregates does not take place. Our results show that a sudden switch from a healthy to a disease state can be caused by small variations in the efficiency of the clearance process and provide a mathematical framework to explore the detailed effects of different mechanisms of clearance on the accumulation of aggregates.

Analysis of risk factors for dermatitis and cellulitis in turkeys using abattoir post mortem data.

1. Cellulitis and dermatitis are often seen in turkeys at slaughter and are, in many cases, reasons for rejection at meat inspection. Using the results of meat inspections and information about the housing conditions of the animals may provide information on causative factors in the development of cellulitis and dermatitis. 2. A census of two years of turkey production in a company including both fattening farms and an abattoir in the north of England was used to study the relationship between the prevalence of cellulitis/dermatitis and mechanical versus natural ventilation in the poultry house, litter type (long straw, short straw and wood shavings) and stocking density. 3. The overall prevalence of cellulitis and dermatitis in this population was 0.12 %, which was extremely low compared to reports in the literature. In mechanically ventilated houses, the odds of developing cellulitis and/or dermatitis was significantly higher than in naturally ventilated houses (Odds Ratio 3.18). Long straw had a protective effect compared to wood shavings (Odds Ratio 0.18). 4. The study showed that using slaughter data can be helpful in studying on-farm risk factors for disease development.

Survey of husbandry and health on UK commercial dairy goat farms.

Published research relevant to the UK dairy goat industry is scarce. Current practices and concerns within the UK dairy goat industry must be better understood if research is to have optimal value. A postal survey was conducted of the farmer membership of the Milking Goat Association as a first step in addressing gaps in knowledge. Questions were asked about husbandry practices, farmer observations of their goats and their priorities for further research. Seventy-three per cent of Milking Goat Association members responded, representing 38 per cent of commercial dairy goat farms and 53 per cent of the commercial dairy goat population in England and Wales. Findings were comprehensive and showed extensive variation in farm practices. Farmers reported pneumonia and scours (diarrhoea) as the most prevalent illnesses of their kids. Pneumonia, diarrhoea, failure to conceive and poor growth were the most prevalent observations of youngstock. Overly fat body condition, assisted kidding, failure to conceive and difficulty drying off were the most prevalent observations of adult milking goats. Farmers' top priorities for further research were kid health (79.5 per cent of farmers), Johne's disease (69.5 per cent of farmers), tuberculosis (59 per cent of farmers) and nutrition (47.7 per cent of farmers).

Internal roosting location is associated with differential use of the outdoor range by free-range laying hens.

1. In commercial free-range systems for laying hens, popholes to the outdoor range are often installed on one side of the house only. In multi-tier systems, it is possible that some individuals fail to access the range due to internal barriers to movement. 2. Five commercial multi-tier flocks from different units were studied. For each flock, two different colour markers were used to distinguish 200 birds roosting near the popholes (NP-Roost) and 200 birds roosting far from the popholes (FP-Roost) at night. The following day, counts of marked birds on the range and inside the house were performed. 3. Significantly more NP-Roost birds were observed in all areas of the outdoor range than FP-Roost birds the next day. Distance of FP area from the popholes was very strongly positively correlated with effect size in the adjacent range area. 4. Additionally, in the indoor area far from the popholes (FP) more FP-Roost birds were observed the next day than NP-Roost birds. In the indoor area near to the popholes (NP) more NP-Roost birds were observed the next day than FP-Roost birds. 5. These results suggest that roosting location is associated with differential range use when popholes are only available on one side of the shed as birds that roosted far from the popholes used the range less.

Modelling-based identification of factors influencing campylobacters in chicken broiler houses and on carcasses sampled after processing and chilling.

AIMS: To identify production and processing practices that might reduce Campylobacter numbers contaminating chicken broiler carcasses. METHODS AND RESULTS: The numbers of campylobacters were determined on carcass neck skins after processing or in broiler house litter samples. Supplementary information that described farm layouts, farming conditions for individual flocks, the slaughterhouse layouts and operating conditions inside plants was collected, matched with each Campylobacter test result. Statistical models predicting the numbers of campylobacters on neck skins and in litter were constructed. Carcass microbial contamination was more strongly influenced by on-farm production practices compared with slaughterhouse activities. We observed correlations between the chilling, washing and defeathering stages of processing and the numbers of campylobacters on carcasses. There were factors on farm that also correlated with numbers of campylobacters in litter. These included bird gender, the exclusion of dogs from houses, beetle presence in the house litter and the materials used to construct the house frame. CONCLUSIONS: Changes in farming practices have greater potential for reducing chicken carcass microbial contamination compared with processing interventions. SIGNIFICANCE AND IMPACT OF THE STUDY: Routine commercial practices were identified that were correlated with lowered numbers of campylobacters. Consequently, these practices are likely to be both cost-effective and suitable for adoption into established farms and commercial processing.

Lameness assessment with automatic monitoring of activity in commercial broiler flocks.

The possibility of using automatic recordings of broiler chicken activity in commercial flocks to assess the birds΄ walking ability (lameness) was investigated. Data were collected from 5 commercial broiler farms in 4 European countries, using 16 flocks and 33 assessment occasions. Lameness was assessed using established gait scoring methods (Kestin et al., 1992; Welfare Quality®, 2009) and took place at 3, 4, and 5 wk of age. Gait score (GS) was used to assess the birds' walking ability, and automatic recordings of bird activity were collected using the eYeNamic™ camera system before, during, and after an assessor walked through the house. The variables used to predict the level of GS extracted from the camera system were: baseline activity, time from assessor leaving the house to resumption of baseline activity, average activity over that period, and Δ Amplitude (difference between highest activity peak after assessor left the house and baseline level). Age (<0.001) and Δ Amplitude (P = 0.0002) were significantly related to GS, with the gait getting poorer with increased age and Δ Amplitude decreasing with declining walking ability. Both measures are thus included in a predictive equation. The results demonstrate a potential method using image analysis techniques to realize an automated assessment of the level of lameness in commercial broiler flocks. This could be of use in future animal welfare assessment schemes.

Kinetics of fragmentation and dissociation of two-strand protein filaments: Coarse-grained simulations and experiments.

While a significant body of investigations have been focused on the process of protein self-assembly, much less is understood about the reverse process of a filament breaking due to thermal motion into smaller fragments, or depolymerization of subunits from the filament ends. Indirect evidence for actin and amyloid filament fragmentation has been reported, although the phenomenon has never been directly observed either experimentally or in simulations. Here we report the direct observation of filament depolymerization and breakup in a minimal, calibrated model of coarse-grained molecular simulation. We quantify the orders of magnitude by which the depolymerization rate from the filament ends koff is larger than fragmentation rate k- and establish the law koff/k- = exp[(ε‖ - ε⊥)/kBT] = exp[0.5ε/kBT], which accounts for the topology and energy of bonds holding the filament together. This mechanism and the order-of-magnitude predictions are well supported by direct experimental measurements of depolymerization of insulin amyloid filaments.

Secondary structure and (1)H, (13)C and (15)N resonance assignments of Skint-1: a selecting ligand for a murine γδ T cell subset implicated in tumour suppression.

A study describing the (1)H, (13)C and (15)N backbone and side chain chemical shift assignments and secondary structure of Skint-1 a prototypic member of a family of mouse genes, of which Skint-1 is involved in the development of the dendritic epidermal T cell (DETC) subset of γδ T cells.

A preliminary study on the use and effect of hand antiseptics in veterinary practice.

OBJECTIVES: To assess use and effect of hand antiseptics in veterinary clinical practice. MATERIALS AND METHODS: Veterinary practice nurses were questioned concerning their use of hand antiseptics, in particular waterless hand rubs. Subsequent clinical trials assessed the effect of single applications of alcohol-based and quaternary ammonium compound-based hand rubs at reducing bacterial counts on the hands of theatre nurses in a neutering clinic. RESULTS: The majority of responding practices used waterless hand rubs (alcohol-based, 67.5% and quaternary ammonium compound-based, 9.5%) as their primary hand hygiene agent and believed them to be effective. 23% of practices favoured an antiseptic hand-wash. In clinical trials, alcohol-based rubs were potentially more effective at reducing bacterial counts than quaternary ammonium compound-based rubs especially in the period immediately after application. However, over 3 hours there was no significant change between these and a control group. There were more adverse skin effects in the group using alcohol-based than in the quaternary ammonium compound-based and control groups. The bacterial counts after application were unaffected by variables such as the number of animals contacted. CLINICAL SIGNIFICANCE: The studies provide useful baseline data for evaluation of efficacy of more frequent applications of the most common antiseptic hand rubs used in veterinary practice.

Prescription of perioperative analgesics by UK small animal veterinary surgeons in 2013.

Data from a survey conducted in 1996-1997 suggested a low level of perioperative analgesic administration to cats and dogs in the UK. In order to evaluate current practice and attitudes with regards to perioperative analgesic prescription, a cross-sectional survey of UK practising small animal veterinary surgeons was undertaken in spring 2013. Four thousand one hundred paper questionnaires were distributed and the survey was made available online. Seven hundred and twenty valid responses were received and analysed. All respondents had access to at least one non-steroidal anti-inflammatory drug (NSAID) and one opioid within their practice. Respondents considered analgesic efficacy, and degree of intraoperative pain, the most important factors governing their selection of NSAID and opioid analgesics. Perioperative NSAIDs were administered by approximately 98 per cent of respondents to dogs and cats undergoing neutering. Multimodal (opioid+NSAID) analgesia was prescribed by the majority of respondents. Neutering was considered more painful in dogs than in cats, and lower rates of opioid and postdischarge NSAID prescription were reported for cats. Orthopaedic, abdominal and dental surgeries were considered equally painful in dogs and cats. Local analgesic techniques were not commonly used. Analgesic prescription has increased since previous surveys, which should translate to improved animal welfare.

Infrared thermometry for lesion monitoring in cattle lameness.

Infrared thermometry is a non-invasive tool shown to be useful in detecting claw abnormalities in cattle at an individual and herd level. This study used the technology to monitor foot temperature and investigate the association with lesion presence over time. A 990 cow dairy herd was enrolled and followed for six months, with data collection fortnightly, lesions were identified by examination of any cow with a mobility score >2, using the 0-3 scale. Two level, multilevel analysis of the association between ambient temperature and foot temperature found that the former was a significant predictor of the latter (coefficient estimate (se)=0.277 (0.02)). Actual foot temperatures were calculated by adjusting for this covariate to allow monitoring over time. Presence of a lesion was also found to be a significant predictor of foot temperature (coefficient estimate (se)=0.623 (0.19)), when added to the model, furthermore some lesion types, claw horn and multiple lesions, were found to be associated with differential foot temperatures. When monitoring lesions over time, the mean adjusted foot temperature was highest at the point of lesion identification. A marked drop in temperature then followed after the lesion was trimmed, with the lowest mean temperature recorded six weeks after treatment, significantly different from the point of lesion identification (P=0.003). This temperature was also lower than the six weeks prior to diagnosis of the lesion, suggesting inflammation was present for at least six weeks prior to the behavioural sign of lameness was seen.

Measuring the response to therapeutic foot trimming in dairy cows with fortnightly lameness scoring.

Lameness scoring (0-3) was carried out on four UK dairy farms during the housing period over three consecutive years (2010-2012). At the start of the study cows were matched by parity and stage of lactation and randomly allocated into a treatment (TX) and a control (CX) group. Cows were enrolled when two sound scores (0 or 1) were followed by a lame score (2). Farmers were immediately notified of score 3 cows, which were then excluded from the study, irrespective of whether they were in treatment or control groups. The animals in the TX group received treatment 3-48 h after being scored lame. Farmers remained blind to the treatment group. Throughout the study the participating farmers continued to identify and treat lame cows according to their usual approaches, this included treating animals in the CX or TX group if they so chose. The fortnightly lameness scoring and treatment of the TX group resulted in higher cure rates at each scoring session following treatment when compared with the CX group (P < 0.001). Two weeks after inclusion, 78% (SE ± 3.2) of TX cows were sound, compared with 66% (SE ± 3.1) of CX cows. At 18 weeks following initial recruitment this had fallen to 41% (SE ± 6.3) (TX) and 13% (SE ± 4.7) (CX). The percentage of total scores which were sound scores in the TX and CX groups following inclusion in the trial was 81% and 66.1%, respectively (P < 0.001). The main lesions found on treatment in the TX group were sole haemorrhage (41% of cases) and digital dermatitis (33%). Severe lesions (sole ulcers and toe necrosis) were only found in 6.6% of cases. In the treated CX animals the percentage of severe lesions was 14%.