How much information can be obtained from tracking the position of the leading edge in a scratch assay?

Moving cell fronts are an essential feature of wound healing, development and disease. The rate at which a cell front moves is driven, in part, by the cell motility, quantified in terms of the cell diffusivity $D$, and the cell proliferation rate �$\lambda$. Scratch assays are a commonly-reported procedure used to investigate the motion of cell fronts where an initial cell monolayer is scratched and the motion of the front is monitored over a short period of time, often less than 24 hours. The simplest way of quantifying a scratch assay is to monitor the progression of the leading edge. Leading edge data is very convenient since, unlike other methods, it is nondestructive and does not require labeling, tracking or counting individual cells amongst the population. In this work we study short time leading edge data in a scratch assay using a discrete mathematical model and automated image analysis with the aim of investigating whether such data allows us to reliably identify $D$ and $\lambda$�. Using a naıve calibration approach where we simply scan the relevant region of the ($D$;$\lambda$�) parameter space, we show that there are many choices of $D$ and $\lambda$� for which our model produces indistinguishable short time leading edge data. Therefore, without due care, it is impossible to estimate $D$ and $\lambda$� from this kind of data. To address this, we present a modified approach accounting for the fact that cell motility occurs over a much shorter time scale than proliferation. Using this information we divide the duration of the experiment into two periods, and we estimate $D$ using data from the first period, while we estimate �$\lambda$ using data from the second period. We confirm the accuracy of our approach using in silico data and a new set of in vitro data, which shows that our method recovers estimates of $D$ and $\lamdba$� that are consistent with previously-reported values except that that our approach is fast, inexpensive, nondestructive and avoids the need for cell labeling and cell counting.

Mini-scale method for nuclear run-on transcription assay in plants

We present a mini-scale method for nuclear run-on transcription assay. In our method, all the centrifuge steps can be carried out by using micro-tubes for short time (5 min each) throughout the process, including isolation of transcriptionally active nuclei and purification of labeled RNA after synthesis of RNA in isolated nuclei. The assay can be performed using a small amount of plant tissue, which enables analysis of developmental changes in transcriptional status of given genes in a single individual plant. Successful results were obtained using the tissues of flower and leaf of petunia and embryo of pea, suggesting that the method is potentially applicable to a variety of plant tissues.

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

Background We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. Results We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. Conclusion In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

A transient assay for recombination demonstrates that Arabidopsis SNM1 and XRCC3 enhance non-homologous recombination

Replacement of endogenous genes by homologous recombination is rare in plants; the majority of genetic modifications are the result of transforming DNA molecules undergoing random genomic insertion by way of non-homologous recombination. Factors that affect chromatin remodeling and DNA repair are thought to have the potential to enhance the frequency of homologous recombination in plants. Conventional tools to study the frequencies of genetic recombination often rely on stable transformation-based approaches, with these systems being rarely capable of high-throughput or combinatorial analysis. We developed a series of vectors that use chemiluminescent (LUC and REN) reporter genes to assay the relative frequency of homologous and non-homologous recombination in plants. These transient assay vectors were used to screen 14 candidategenes for their effects on recombination frequencies in Nicotiana benthamiana plants. Over-expression of Arabidopsis genes with sequence similarity to SNM1 from yeast and XRCC3 from humans enhanced the frequency of non-homologous recombination when assayed using two different donor vectors. Transient N. benthamiana leaf systems were also used in an alternative assay for preliminary measurements of homologous recombination frequencies, which were found to be enhanced by over-expression of RAD52, MIM and RAD51 from yeast, as well as CHR24 from Arabidopsis. The findings for the assays described here are in line with previous studies that analyzed recombination frequencies using stable transformation. The assays we report have revealed functions in non-homologous recombination for the Arabidopsis SNM1 and XRCC3 genes, so the suppression of these genes' expression offers a potential means to enhance the gene targeting frequency in plants. Furthermore, our findings also indicate that plant gene targeting frequencies could be enhanced by over-expression of RAD52, MIM, CHR24, and RAD51 genes.

Human breast cancer cell metastasis to long bone and soft organs of nude mice : a quantitative assay

Bone is a common metastatic site in human breast cancer (HBC). Since bone metastasis occurs very rarely from current spontaneous or experimental metastasis models of HBC cells in nude mice, an arterial seeding model involving the direct injection of the cells into the left ventricle has been developed to better understand the mechanisms involved in this process. We present here a sensitive polymerase chain reaction (PCR) method to detect and quantitate bone and soft organ metastasis in nude mice which have been intracardially inoculated with Lac Z transduced HBC cells. Amplification of genomically incorporated Lac Z sequences in MDA-MB-231-BAG HBC cells enables us to specifically detect these cells in mouse organs and bones. We have also created a competitive template to use as an internal standard in the PCR reactions, allowing us to better quantitate levels of HBC metastasis. The results of this PCR detection method correlate well with cell culture detection from alternate long bones from the same mice, and are more sensitive than gross Lac Z staining with X-gal or routine histology. Comparable qualitative results were obtained with PCR and culture in a titration experiment in which mice were inoculated with increasing numbers of cells, but PCR is more quantifiable, less time consuming, and less expensive. This assay can be employed to study the molecular and cellular aspects of bone metastasis, and could easily be used in conjunction with RT-PCR-based analyses of gene products which may be involved with HBC metastasis.

A study, by fire-assay and ICP-MS, of the distribution of rhodium, platinum, and gold in iron-age pottery

Forty-six archaeological specimens were treated by fire-assay and subsequently analysed by ICP-MS for selected precious metals: Ph, Pt and Au. The investigation was prompted by the possibility that archaeological samples could serve as "indicators" of the precious metal composition of the clays from the excavated sites. Therefore, the experimentally obtained concentrations were carefully studied to determine if there were anomalous levels of these precious metals in the deposits from which the specimens originated. Furthermore, the analytical data were used to establish if it was feasible to distinguish ancient potsherds based on precious metal concentrations, for employment as a basis in provenance studies.

One-step homogeneous C-reactive protein assay for saliva

Background: Cardiovascular disease is the leading cause of death in the world. Human C-reactive protein (CRP) has been used in the risk assessment of coronary events. Human saliva mirrors the body's health and well-being and is non-invasive, easy to collect and ideal for third world countries as well as for large patient screening. The aim was to establish a saliva CRP reference range and to demonstrate the clinical utility of salivary CRP levels in assessing the coronary events in a primary health care setting. Methods: We have used a homogeneous bead based assay to detect CRP levels in human saliva. We have developed a rapid 15 min (vs 90 min), sequential, one-step assay to detect CRP in saliva. Saliva was collected from healthy volunteers (n = 55, ages 20-70 years) as well as from cardiac patients (n = 28, ages 43-86 years). Results: The assay incubation time was optimised from 90 min to 15 mm and generated a positive correlation (n = 29, range 10-2189 pg/mL, r2 = 0.94; Passing Bablok slope 0.885. Intercept 0, p>0.10), meaning we could decrease the incubation time and produce equivalent results with confidence. The mean CRP level in the saliva of healthy human volunteers was 285 pg/mL and in cardiac patients was 1680 pg/mL (p<0.01). Analysis of CRP concentrations in paired serum and saliva samples from cardiac patients gave a positive correlation (r2 = 0.84, p<0.001) and the salivary CRP concentration capable of distinguishing healthy from diseased patients. Conclusions: The results suggest that this minimally invasive, rapid and sensitive assay will be useful in large patient screening studies for risk assessment of coronary events. (C) 2011 Elsevier B.V. All rights reserved.

Split leverage : attacking the confidentiality of linked databases by partitioning

This article considers the risk of disclosure in linked databases when statistical analysis of micro-data is permitted. The risk of disclosure needs to be balanced against the utility of the linked data. The current work specifically considers the disclosure risks in permitting regression analysis to be performed on linked data. A new attack based on partitioning of the database is presented.

Inducible resistance to maize streak virus

Maize streak virus (MSV), which causes maize streak disease (MSD), is the major viral pathogenic constraint on maize production in Africa. Type member of the Mastrevirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic maize lines constitutively expressing ‘‘dominant negative mutant’’ versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or ‘‘leaky’’ expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence of an infecting MSV’s replication associated protein. We used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastrevirus species. The application of this new technology to MSV resistance in maize could allow a better, more acceptable product.

Incidence and predictors of lower limb split-skin graft failure and primary closure dehiscence in day-case surgical patients

BACKGROUND After general surgery, the lower limb experiences some of the highest complication rates. However, little is known about contributing factors to surgical site failure in the lower limb dermatological surgery population. OBJECTIVE To determine the incidence of lower limb surgical site failure and to explore the predictors that contribute to surgical site failure. METHODS A prospective observational study design was used to collect data from 73 participants, from July 2010, to March 2012. Incidence was determined as a percentage of surgical site failure from the total population. Predictors were determined by the use of a binary logistic regression model. RESULTS The surgical site failure rate was 53.4%. Split-skin grafting had a higher failure rate than primary closures, 66% versus 26.1%. Predictors of lower limb surgical site failure were identified as increasing age (p = .04) and the presence of postoperative hematoma (p = .01), with all patients who developed surgical site infection experiencing surgical site failure (p = .01). CONCLUSION Findings from this study confirmed that the lower limb is at high risk of surgical site failure. Two predictors of surgical site failure from this cohort were determined. However, to understand this phenomenon and make recommendations to assist and reduce surgical site complications, further research in this field is required.

An immunodiagnostic assay for quantitation of specific ige to the major pollen allergen component, pas n 1, of the subtropical bahia grass

Background Pollens of the Panicoideae subfamily of grasses including Bahia (Paspalum notatum) are important allergen sources in subtropical regions of the world. An assay for specific IgE to the major molecular allergenic component, Pas n 1, of Bahia grass pollen (BaGP) would have immunodiagnostic utility for patients with pollen allergy in these regions. Methods Biotinylated Pas n 1 purified from BaGP was coated onto streptavidin ImmunoCAPs. Subjects were assessed by clinical history of allergic rhinitis and skin prick test (SPT) to aeroallergens. Serum total, BaGP-specific and Pas n 1-specific IgE were measured. Results: Pas n 1 IgE concentrations were highly correlated with BaGP SPT (r = 0.795, p < 0.0001) and BaGP IgE (r = 0.915, p < 0.0001). At 0.23 kU/l Pas n 1 IgE, the diagnostic sensitivity (92.4%) and specificity (93.1%) for the detection of BaGP allergy was high (area under receiver operator curve 0.960, p < 0.0001). The median concentrations of Pas n 1 IgE in non-Atopic subjects (0.01 kU/l, n = 67) and those with other allergies (0.02 kU/l, n = 59) showed no inter-group difference, whilst grass pollen-Allergic patients with allergic rhinitis showed elevated Pas n 1 IgE (6.71 kU/l, n = 182, p < 0.0001). The inter-Assay coefficient of variation for the BaGP-Allergic serum pool was 6.92%. Conclusions Pas n 1 IgE appears to account for most of the BaGP-specific IgE. This molecular component immunoassay for Pas n 1 IgE has potential utility to improve the sensitivity and accuracy of diagnosis of BaGP allergy for patients in subtropical regions.

Prospective external validation of an accelerated (2-h) acute coronary syndrome rule-out process using a contemporary troponin assay

Background: Recently there have been efforts to derive safe, efficient processes to rule out acute coronary syndrome (ACS) in emergency department (ED) chest pain patients. We aimed to prospectively validate an ACS assessment pathway (the 2-Hour Accelerated Diagnostic Protocol to Assess Patients with Chest Pain Symptoms Using Contemporary Troponins as the Only Biomarker (ADAPT) pathway) under pragmatic ED working conditions. Methods: This prospective cohort study included patients with atraumatic chest pain in whom ACS was suspected but who did not have clear evidence of ischaemia on ECG. Thrombolysis in myocardial infarction (TIMI) score and troponin (TnI Ultra) were measured at ED presentation, 2 h later and according to current national recommendations. The primary outcome of interest was the occurrence of major adverse cardiac events (MACE) including prevalent myocardial infarction (MI) at 30 days in the group who had a TIMI score of 0 and had presentation and 2-h TnI assays <99th percentile. Results: Eight hundred and forty patients were studied of whom 177 (21%) had a TIMI score of 0. There were no MI, MACE or revascularization in the per protocol and intention-to-treat 2-h troponin groups (0%, 95% confidence interval (CI) 0% to 4.5% and 0%, 95% CI 0% to 3.8%, respectively). The negative predictive value (NPV) was 100% (95% CI 95.5% to 100%) and 100% (95% CI 96.2% to 100%), respectively. Conclusions: A 2-h accelerated rule-out process for ED chest pain patients using electrocardiography, a TIMI score of 0 and a contemporary sensitive troponin assay accurately identifies a group at very low risk of 30-day MI or MACE.