A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis

BACKGROUND: The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR. RESULTS: Here, we report the first combined use of Multiplex Tandem PCR (MT-PCR) and High Resolution Melt (HRM) analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker. CONCLUSIONS: A pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes

Women's experiences of factors affecting treatment engagement and adherence in internet delivered behavioural activation for postnatal depression

ArticleCrown Copyright © 2014 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).Introduction Women with postnatal depression (PND) face significant barriers to treatment that may be overcome by internet based delivery of treatment. Demand for a self-help internet postnatal treatment offered via a parenting site was high, but attrition rates were also high. Aims To gain patient perspectives on engagement and barriers to the Netmums' “Helping with Depression” treatment. Method Semi-structured interviews were conducted with 17 participants selected from the Netmums trial. Results Thematic analysis revealed motivators and barriers to treatment. Women reported that the flexibility and anonymity of internet interventions fit with their postnatal circumstances. They identified that the relevance of the intervention to their personal circumstances, expectations of motherhood, stigma about depression and motherhood, hopelessness about their ability to improve, previous negative experiences with treatment and treatment seeking, and a lack of practical and emotional support contributed to feelings of being overwhelmed. Women who felt more overwhelmed were more likely to discontinue treatment. Women suggested that support would reduce the impact of barriers and improve adherence. Discussion Open access, self-help internet interventions are acceptable to women with postnatal depression, but it is critical to provide tailoring and support to help overcome barriers and improve treatment adherence

BAC-HAPPY mapping (BAP mapping): a new and efficient protocol for physical mapping

Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical “contig” maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning ~10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual “BAC-HAPPY-mapping” to convert BAC landing data into BAC linkage contigs is possible.Giang T. H. Vu, Paul H. Dear, Peter D. S. Caligari and Mike J. Wilkinso

Hexane-1,6-diammonium bis­(pyridine-2-carboxyl­ate)

The title compound, C6H18N2 2+·2C6H4NO2 −, consists of a doubly protonated hexa­methyl­enediammonium dication and two pyridine-2-carboxyl­ate anions. These ions inter­act by means of inter­molecular N—H⋯O and N—H⋯N hydrogen bonds to form a two-dimensional array. The carboxyl­ate groups of the anions appear to be delocalized on the basis of the C—O bond lengths

Bilateral pedicle stress fracture in the lumbar spine of a sedentary office worker

A case of bilateral pedicle fracture in the lumbar spine of a sedentary office worker is being presented. No such case has been reported in the literature previously. Bilateral pedicle fracture is a rare entity. Few cases have been reported in literature. All the reported cases had some underlying causative factors like degenerative spine disease, previous spinal surgery or stress-related activities, e.g. athletes. Our case is a 36-year-old sedentary office worker with none of the factors mentioned. We present a case of a 36-year-old sedentary worker with long-standing low backache. There were no root tension signs. Plain radiographs were inconclusive. The patient had a CT scan. The CT scan revealed long-standing defects in the pedicles of L2 vertebra with pseudoarthrosis. Infiltration with anaesthetic relieved the symptoms. Our patient was managed conservatively with spine rehabilitation physiotherapy program. Pedicle fracture can develop due to abnormal stresses in the pedicle either because of previous spinal surgery or spondylitic changes in the spine. Bilateral pedicle fracture in the absence of these conditions is extremely rare

Cardiac dysfunction in cancer survivors unmasked during exercise

Introduction: The cardiac dysfunction associated with anthracycline-based chemotherapy cancer treatment can exist sub-clinically for decades before overt presentation. Stress echocardiography, the measurement of left ventricular (LV) deformation and arterial haemodynamic evaluation have separately been used to identify sub-clinical cardiovascular (CV) dysfunction in several patient groups including those with hypertension and diabetes. The purpose of the present cross-sectional study was to determine whether the combination of these techniques could be used to improve the characterisation of sub-clinical CV dysfunction in long-term cancer survivors previously treated with anthracyclines. Materials and methods: Thirteen long-term cancer survivors (36±10 years) with prior anthracycline exposure (11±8 years post-treatment) and 13 age-matched controls were recruited. Left ventricular structure, function and deformation were assessed using echocardiography. Augmentation index was used to quantify arterial haemodynamic load and was measured using applanation tonometry. Measurements were taken at rest and during two stages of low-intensity incremental cycling.Results: At rest, both groups had comparable global LV systolic, diastolic and arterial function (all P>0.05), however longitudinal deformation was significantly lower in cancer survivors (-18±2 v -20±2, P<0.05). During exercise this difference between groups persisted and further differences were uncovered with significantly lower apical circumferential deformation in the cancer survivors (-24±5 v -29±5, -29±5 v 35±8 for first and second stage of exercise respectively, both P<0.05). Conclusion: In contrast to resting echocardiography the measurement of LV deformation at rest and during exercise provides a more comprehensive characterisation of sub-clinical LV dysfunction. Larger studies are required to determine the clinical relevance of these preliminary findings

Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis

Chemotaxis has been studied by classical methods that measure chemotactic and random motility responses in vitro, but these methods do not evaluate the total number and phenotype of migrating leukocytes simultaneously. Our objective was to develop and validate a novel assay, combined Boyden-flow cytometry chemotaxis assay (CBFCA), for simultaneous quantification and phenotypification of migrating leukocytes. CBFCA exhibited several important advantages in comparison to the classic Boyden chemotaxis assay (CBCA): 1) improved precision (intra-assay coefficients of variation (CVs): CBFCA-4.7 and 4.8% vs. CBCA-30.1 and 17.3%; inter-observer CVs: CBFCA-3.6% vs. CBCA 30.1%); 2) increased recovery of cells, which increased assay to provide increased sensitivity; 3) high specificity for determining the phenotype of migrating/attracted leukocytes; and 4) reduced performance time (CBFCA 120 min vs. CBCA 265 min). Other advantages of CBFCA are: 5) robustness, 6) linearity, 7) eliminated requirement for albumin and, importantly, 8) enabled recovery of migrating leukocytes for subsequent studies. This latter feature is of great benefit in the study of migrating leukocyte subsets. We conclude that the CBFCA is a novel and improved technique for experiments focused on understanding leukocyte trafficking during the inflammatory response