Development of a Novel, Automated High-Throughput Device for Performing the Comet Assay.

A comet assay is a trusted and widely used method for assessing DNA damage in individual eukaryotic cells. However, it is time-consuming and requires extensive monitoring and sample manipulation by the user. This limits the throughput of the assay, increases the risk of errors, and contributes to intra- and inter-laboratory variability. Here, we describe the development of a device which automates high throughput sample processing for a comet assay. This device is based upon our patented, high throughput, vertical comet assay electrophoresis tank, and incorporates our novel, patented combination of assay fluidics, temperature control, and a sliding electrophoresis tank to facilitate sample loading and removal. Additionally, we demonstrated that the automated device performs at least as well as our "manual" high throughput system, but with all the advantages of a fully "walkaway" device, such as a decreased need for human involvement and a decreased assay run time. Our automated device represents a valuable, high throughput approach for reliably assessing DNA damage with the minimal operator involvement, particularly if combined with the automated analysis of comets.

Changes in Torque-Angle Profiles of the Hamstrings and Hamstrings-to-Quadriceps Ratio After Two Hamstring Strengthening Exercise Interventions in Female Hockey Players.

Delextrat, A, Bateman, J, Ross, C, Harman, J, Davis, L, Vanrenterghem, J, and Cohen, DD. Changes in torque-angle profiles of the hamstrings and hamstrings-to-quadriceps ratio after two hamstring strengthening exercise interventions in female hockey players. J Strength Cond Res 34(2): 396-405, 2020-The aim of this study was to compare the effects of 2 hamstring strengthening interventions (Nordic hamstrings [NHE] vs. eccentric leg curl [ELC]) on the hamstring torque-angle profiles and functional hamstrings-to-quadriceps ratio (Hecc:Qcon) in female hockey players. Female university-level players were randomly allocated to an NHE group (n = 9, 19.7 ± 1.4 years; 168.4 ± 4.4 cm; 66.2 ± 7.2 kg, 26.0 ± 4.4%), an ELC group (n = 8, 19.5 ± 1.0 years; 168.1 ± 3.4 cm; 66.7 ± 4.5 kg, 24.8 ± 3.5%), or a control (C) group (n = 8, 19.6 ± 1.4 years; 169.9 ± 7.5 cm; 70.7 ± 13.0 kg, 25.9 ± 5.2%). They performed baseline isokinetic concentric strength tests of the quadriceps (Qcon) and eccentric strength of the hamstrings (Hecc) at 120°·s, followed by a 6-week intervention with exercises (NHE or ELC) performed 3 times weekly, before post-tests. Analyses of variance with repeated measures were used to assess the effects of knee position angle (from 90° of knee flexion to 10° close to extension), group, and time on Qcon, Hecc, and Hecc:Qcon. There were no interactions between independent variables. Significant increases in Hecc and Hecc:Qcon were shown after NHE (+29.9 and +27.8%) and ELC (+30.5 and +38.3%) in the nondominant leg only. Furthermore, significant shifts in the hamstring eccentric angle of peak torque toward a longer muscle length were shown in both legs (14.3-28.6%). These findings suggest that NHE and ELC both resulted in significant improvements in peak and muscle-length-specific neuromuscular risk factors in the nondominant (ND) limb, thereby reducing interlimb peak strength asymmetries. Strength and conditioning specialists could therefore use both the NHE and ELC exercises in female hockey players.

The Relation between Catheter Occlusion and Backflow during Intraparenchymal Cerebral Infusions.

Background/Aims: The distribution of infusate into the brain by convection-enhanced delivery can be affected by backflow along the catheter shaft. This work assesses the following: (1) whether tissue coring and occlusion of the catheter lumen occurs when an open end-port catheter is inserted, (2) whether there is a relationship between intracatheter pressure and backflow, and (3) whether catheter occlusion increases backflow. Methods: Freshly excised monkey brains were used to assess tissue coring and its correlation with the behavior of the line pressure. In vivo infusions of gadolinium solution into monkey putamen at 1 µl/min were conducted with and without a stylet during insertion. The effect of flow during insertion was evaluated in vivo in the pig thalamus. MRI and line pressure were continuously monitored during in vivo infusions. Results: Ex vivo testing showed that open end-port insertions always cored tissue (which temporarily plugs the catheter tip) and increased pressure followed by a rapid fall after its expulsion. Catheter insertion with a stylet in place prevented coring but not flow insertion; neither affected backflow. Conclusion: Open end-port catheters occlude during insertion, which can be prevented by temporarily closing the port with a stylet but not by infusing while inserting. Backflow was not completely prevented by any insertion method. © 2015 S. Karger AG, Basel.

Strategies for the delivery of multiple collinear infusion clouds in convection-enhanced delivery in the treatment of Parkinson's disease.

BACKGROUND: Delivery of multiple collinear payloads utilizing convection-enhanced delivery (CED) has historically been performed by retraction of a needle or catheter from the most distal delivery site. Few studies have addressed end-infusion morphology and associated payload reflux in stacked and collinear infusions, and studies comparing the advancement with the retraction mode are lacking. OBJECTIVE: To compare advancement versus retraction mode infusion results. METHODS: Infusion cloud pairs were created with the advancement and retraction technique in agarose gel using both open end-port SmartFlow (SF) and valve tip (VT) catheter infusion systems. Backflow, radius of infusion, and morphology were assessed. RESULTS: Infusions with the SF catheter, in contrast to the VT catheter, exhibited significantly more backflow in retraction mode at the shallow infusion site. Infusion morphology differed with the second infusion after retraction: the infusate at the proximal site first filling the channel left by the retraction and then being convected into gel in a pronouncedly non-spherical shape during the second infusion. CONCLUSIONS: Significant differences in cloud morphology were noted with respect to external catheter geometry with retraction versus penetration between infusions in an agarose gel model of the brain. Further study is warranted to determine optimal protocols for human clinical trials employing CED with multiple collinear payloads.

EuPathDB: a portal to eukaryotic pathogen databases.

EuPathDB (http://EuPathDB.org; formerly ApiDB) is an integrated database covering the eukaryotic pathogens of the genera Cryptosporidium, Giardia, Leishmania, Neospora, Plasmodium, Toxoplasma, Trichomonas and Trypanosoma. While each of these groups is supported by a taxon-specific database built upon the same infrastructure, the EuPathDB portal offers an entry point to all these resources, and the opportunity to leverage orthology for searches across genera. The most recent release of EuPathDB includes updates and changes affecting data content, infrastructure and the user interface, improving data access and enhancing the user experience. EuPathDB currently supports more than 80 searches and the recently-implemented 'search strategy' system enables users to construct complex multi-step searches via a graphical interface. Search results are dynamically displayed as the strategy is constructed or modified, and can be downloaded, saved, revised, or shared with other database users.

TriTrypDB: a functional genomic resource for the Trypanosomatidae.

TriTrypDB (http://tritrypdb.org) is an integrated database providing access to genome-scale datasets for kinetoplastid parasites, and supporting a variety of complex queries driven by research and development needs. TriTrypDB is a collaborative project, utilizing the GUS/WDK computational infrastructure developed by the Eukaryotic Pathogen Bioinformatics Resource Center (EuPathDB.org) to integrate genome annotation and analyses from GeneDB and elsewhere with a wide variety of functional genomics datasets made available by members of the global research community, often pre-publication. Currently, TriTrypDB integrates datasets from Leishmania braziliensis, L. infantum, L. major, L. tarentolae, Trypanosoma brucei and T. cruzi. Users may examine individual genes or chromosomal spans in their genomic context, including syntenic alignments with other kinetoplastid organisms. Data within TriTrypDB can be interrogated utilizing a sophisticated search strategy system that enables a user to construct complex queries combining multiple data types. All search strategies are stored, allowing future access and integrated searches. 'User Comments' may be added to any gene page, enhancing available annotation; such comments become immediately searchable via the text search, and are forwarded to curators for incorporation into the reference annotation when appropriate.

The role of oct-1 in the regulation of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) expression in bovine mammary epithelial cells.

Lingual antimicrobial peptide (LAP) and tracheal antimicrobial peptide (TAP) are two important ß-defensins of antimicrobial peptide family, which are evolutionarily conserved effector molecules of the innate immune response. Although known to be sensitive to pathogenic challenge, the control of their expression remains unclear. Both LAP and TAP genes showed constitutive and inducible expression in bovine mammary epithelial tissues, and the aim of this study was to investigate the mechanisms underlying their expression and regulation. Reporter plasmids fused with 5' regions of the two gene promoter regions were constructed and transiently transfected into a bovine mammary epithelial (BME) cell line. Initial serial deletion of the promoter regions from both genes identified two positive regulatory elements within the 1 kb regions upstream the transcription start sites, which co-operatively contribute to LAP and TAP gene expression. Further luciferase reporter assays revealed that an enhancer and a 61-bp region proximal to both genes are important for basal expression and regulation of transcription. Electrophoretic mobility shift assays (EMSA) indicated the involvement of the Oct-1 protein-DNA complex in regulating the promoter activity, which was confirmed by super shift EMSA with Oct-1 antibody and by knockdown of Oct-1 with small interfering RNA. The Oct-1 binding motif was also shown to be responsive to phorbol 12-myristate 13-acetate but not LPS stimulation. The results from this study clearly demonstrate the involvement of the Oct-1 transcription factor in the regulation of LAP and TAP expression.

PlasmoDB: a functional genomic database for malaria parasites.

PlasmoDB (http://PlasmoDB.org) is a functional genomic database for Plasmodium spp. that provides a resource for data analysis and visualization in a gene-by-gene or genome-wide scale. PlasmoDB belongs to a family of genomic resources that are housed under the EuPathDB (http://EuPathDB.org) Bioinformatics Resource Center (BRC) umbrella. The latest release, PlasmoDB 5.5, contains numerous new data types from several broad categories--annotated genomes, evidence of transcription, proteomics evidence, protein function evidence, population biology and evolution. Data in PlasmoDB can be queried by selecting the data of interest from a query grid or drop down menus. Various results can then be combined with each other on the query history page. Search results can be downloaded with associated functional data and registered users can store their query history for future retrieval or analysis.

GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia lamblia and Trichomonas vaginalis.

GiardiaDB (http://GiardiaDB.org) and TrichDB (http://TrichDB.org) house the genome databases for Giardia lamblia and Trichomonas vaginalis, respectively, and represent the latest additions to the EuPathDB (http://EuPathDB.org) family of functional genomic databases. GiardiaDB and TrichDB employ the same framework as other EuPathDB sites (CryptoDB, PlasmoDB and ToxoDB), supporting fully integrated and searchable databases. Genomic-scale data available via these resources may be queried based on BLAST searches, annotation keywords and gene ID searches, GO terms, sequence motifs and other protein characteristics. Functional queries may also be formulated, based on transcript and protein expression data from a variety of platforms. Phylogenetic relationships may also be interrogated. The ability to combine the results from independent queries, and to store queries and query results for future use facilitates complex, genome-wide mining of functional genomic data.

Children's perceptions of environment and health in two Scottish neighbourhoods.

This article explores children's understanding of the role that neighbourhood plays in their health and well-being. Whilst evidence exists on the relationship between the environment and children's health, we have little knowledge of this from the perspective of children themselves. Children's experiences are all too frequently researched through the eyes of adults. Following a Rights of the Child framework, respecting children's views and giving them due weight, this paper reports from a project that worked with children from two relatively deprived urban neighbourhoods in Scotland. Using this framework, the children themselves were the researchers who designed the themes, decided upon the methods, conducted the research and analysed the resulting data. Using focus groups, visual mapping and community walks the children explored their local neighbourhoods and the findings reveal features of the environment that the children perceive as important for their health and well-being. The children selected three themes to explore in the research: safety, littering, and family and friends, through which they elicit their experiences, feelings and attitudes towards the environment and their well-being. The paper reveals that not only do the children have a deep understanding of the link between environment and health, but that they also understand how aspects of disadvantage, including place-based stigma, can limit their social participation and inclusion in society. We conclude with recommendations made by the children themselves, ranging from access to affordable activities, improved open spaces, 'support not stigma' and the need to be heard in local decision making.

Regional atrophy of the corpus callosum in dementia.

The regional distribution of degeneration of the corpus callosum (CC) in dementia is not yet clear. This study compared regional CC size in participants (n = 179) from the Cache County Memory and Aging Study. Participants represented a range of cognitive function: Alzheimer's disease (AD), vascular dementia (VaD), mild ambiguous (MA-cognitive problems, but not severe enough for diagnosis of dementia), and healthy older adults. CC outlines obtained from midsagittal magnetic resonance images were divided into 99 equally spaced widths. Factor analysis of these callosal widths identified 10 callosal regions. Multivariate analysis of variance revealed significant group differences for anterior and posterior callosal regions. Post-hoc pairwise comparisons of CC regions in patient groups as compared to the control group (controlling for age) revealed trends toward smaller anterior and posterior regions, but not all were statistically significant. As compared to controls, significantly smaller anterior and posterior CC regions were found in the AD group; significantly smaller anterior CC regions in the VaD group; but no significant CC regional differences in the MA group. Findings suggest that dementia-related CC atrophy occurs primarily in the anterior and posterior portions.

Molecular identification and functional expression of porcine Toll-like receptor (TLR) 3 and TLR7.

To investigate porcine Toll-like receptors (TLR) responding to viral pathogen associated molecular patterns, the full-length cDNA of porcine TLR3 and TLR7 were identified and characterized. Porcine TLR3 and TLR7 cDNA encode 904- and 1050-amnio-acid polypeptides, respectively. Both porcine TLR3 and TLR7 contain typical functional TLR domains and share about 80% sequence identity to other mammalian orthologues. Tissue expression profiles showed that TLR3 was highly expressed in kidney, duodenum, spleen and liver, and moderately expressed in bone marrow, lung, and skin. Conversely, TLR7 was moderately and constitutively expressed in all tissues evaluated. Stimulation of mammalian cells transfected with porcine TLR3 and TLR7 constructs with TLR3 and TLR7 agonists [poly (I:C) and imiquimod (R837), respectively], and adenovirus elicited activation of interferon regulatory factors (IRFs). These data provide molecular and functional information for porcine TLR3 and TLR7, and implicate their role in mediating immune protection against porcine viral diseases.

Porcine liver-expressed antimicrobial peptides, hepcidin and LEAP-2: cloning and induction by bacterial infection.

Hepcidin is a liver-expressed iron-regulating hormone that also is an antimicrobial peptide. Here we report the full-length cDNA sequences of porcine hepcidin and liver-expressed antimicrobial peptide-2 (LEAP-2). Porcine hepcidin and LEAP-2 cDNA sequences contain 411 and 525 bp, and encode predicted peptides of 82 and 77 amino acid residues, respectively. Both porcine hepcidin and LEAP-2 are highly expressed in liver and LEAP-2 also is expressed in intestinal tissues and kidney. Pigs infected with Salmonella enterica serovar Typhimurium showed inducible but differential expression of hepcidin and LEAP-2 in bone marrow and intestinal tissues. Conversely, although highly expressed in liver, expression of hepcidin mRNA in liver was not influenced by Salmonella infection. These findings provide fundamental comparative data showing the relationship of porcine hepcidin and LEAP-2 to other mammalian orthologs and indicate that bacterial infections influence their expression.

Antibacterial peptide PR-39 affects local nitric oxide and preserves tissue oxygenation in the liver during septic shock.

The effects of the antibacterial peptide PR-39 on nitric oxide (NO) and liver oxygenation (pO(2)) in a mouse model of endotoxaemia have been explored. In vivo electron paramagnetic resonance (EPR) spectroscopy was used to make direct measurements of liver NO and pO(2). Measurements of pO(2) were made at two different anatomical locations within hepatic tissue to assess effects on blood supply (hence oxygen supply) and lobule oxygenation; selectively from the liver sinusoids or an average pO(2) across the liver lobule. PR-39 induced elevated levels of liver NO at 6 h following injection of lipopolysaccharide (LPS) as a result of increased iNOS expression in liver, but had no effect on eNOS or circulatory NO metabolites. Sinusoidal oxygenation was preserved, and pO(2) across the hepatic tissue bed improved with PR-39 treatment. We propose that the beneficial effects of PR-39 on liver in this septic model were mediated by increased levels of local NO and preservation of oxygen supply to the liver sinusoids.

PU.1-mediated transcriptional regulation of prophenin-2 in primary bone marrow cells.

Prophenin-2 (PF-2) is a cathelicidin, 97-amino-acid antimicrobial protein stored in neutrophil secondary granules. PF-2 is expressed specifically in porcine immature myeloid cells; however, little is known about its regulation. In this study, we characterized the 5' regulatory regions of the PF-2 gene to understand the molecular mechanisms regulating its expression. Using bioinformatic approaches, site-directed mutagenesis, and transactivation experiments, we found that the PF-2 gene was regulated by transcription factor PU.1. In addition, PF-2 expression also is regulated by the cytokines GM-CSF and IL-3. Taken together, these results identify cis- and trans-acting factors involved in the regulation of PF-2 and clarify mechanisms of cathelidicin gene regulation.

Cloning of porcine triggering receptor expressed on myeloid cells-1 (TREM-1) and its induction by lipopolysaccharide, peptidoglycan, and Salmonella enterica serovar Typhimurium infection.

Triggering receptors expressed on myeloid cells (TREM) are a family of activating receptors expressed on neutrophils and monocytes. These receptors are involved in regulation of immunity by inducing the expression of inflammatory cytokines and adhesion molecules, augmenting dendritic cell maturation, and are implicated in septic shock. Here we report the cloning of full-length TREM cDNA from porcine bone marrow cells, which predicts a 238 amino-acid peptide. Treating porcine bone marrow cells with lipopolysaccharide or peptidoglycan caused an increase in TREM-1 expression. Moreover, bone marrow cells derived from pigs that were orally challenged with Salmonella enterica serovar Typhimurium showed increases in TREM-1 at 8 and 24 h post-infection, respectively. Complete down-regulation of TREM-1 expression was observed at 48 h post-infection. These findings provide fundamental comparative data indicating that bacterial infection induces TREM-1 expression.

Late preconditioning by ethanol is initiated via an oxidant-dependent signaling pathway.

Ingestion of alcoholic beverages at low to moderate levels 24 h prior to ischemia and reperfusion (I/R) prevents postischemic leukocyte/endothelial cell adhesive interactions, a phenomenon referred to as late ethanol preconditioning (EtOH-PC). The aim of this study was to determine whether oxidants act as initiators of late EtOH-PC. Ethanol was instilled into the stomachs of C57BL/6 mice as a bolus by gavage at a dose that produced a peak plasma concentration of 45 mg/dl 30 min after administration and returned to control levels 60 min after ingestion. Twenty four hours later, the superior mesenteric artery was occluded for 45 min followed by 70 min of reperfusion. The numbers of rolling and firmly adherent leukocytes were quantified in postcapillary venules of the small intestine in sham animals (no EtOH-PC, no I/R), in mice subjected to I/R alone or EtOH-PC + I/R, and in animals treated with Mn-TBAP (a cell-permeant superoxide dismutase mimetic), oxypurinol (a XO inhibitor), the NAD(P)H oxidase inhibitors PR-39 or apocynin, or oxypurinol plus PR39 during the period of EtOH-PC on Day 1 followed by I/R on Day 2. In separate groups of mice, oxypurinol or apocynin were also administered 1 h after ethanol ingestion on Day 1, with induction of I/R 24 h later. I/R induced marked increases in leukocyte rolling and adherence, effects that were completely prevented by EtOH-PC. Coincident treatment with Mn-TBAP, oxypurinol, PR-39, apocynin, or oxypurinol plus PR-39 with ethanol attenuated these anti-inflammatory actions of EtOH-PC. However, administration of oxypurinol or apocynin 1 h after ethanol ingestion failed to prevent these protective effects of EtOH-PC. Our results indicate that reactive oxygen species formed during the period of ethanol exposure on Day 1 trigger the development of an anti-inflammatory phenotype that renders the small bowel resistant to the proadhesive effects of I/R 24 h later.

Tissue hypoxia during bacterial sepsis is attenuated by PR-39, an antibacterial peptide.

Endotoxin (a lipopolysaccharide (LPS) component of the Gram negative bacterial cell wall) induces sepsis in laboratory animals and is the cause of septic shock in patients. Tissues often develop necrotic regions, particularly in kidney and liver, thought to be directly the result of endotoxin-induced release of nitric oxide (NO). These studies investigated the potential of PR-39, an antibacterial peptide, as an alternative treatment for sepsis. Our rationale for these experiments was based on the knowledge that PR-39 inhibits the superoxide-producing NADH/NADPH-oxidase system, and also inhibits NOS. In a mouse model of sepsis, we carried out EPR measurements of liver pO2 and NO simultaneously in vivo. Physiological parameters were also measured in these animals (blood pressure, heart rate). NO levels in blood were measured by EPR analysis of red blood cell nitrosyl-hemoglobin. We found PR-39 alleviated endotoxin-induced liver hypoxia 6 hrs after treatment. Tissue NO was higher in the PR-39 + LPS group compared to LPS alone. Circulating levels of NO were the same in these groups. Taken together, these results suggest PR-39 is effective in improving survival following a septic episode. The exact mechanism is unclear, but increased NO as a result of decreased superoxide production seems to play an important role in alleviating tissue hypoxia.

The safety of Canadian early discharge guidelines. Effects of discharge timing on readmission in the first year post-discharge and exclusive breastfeeding to four months.

BACKGROUND: Few studies have examined earlier discharge in relation to Canadian guidelines for earlier discharge and infant feeding. We addressed differences in readmission (1 year post-discharge) and exclusive breastfeeding (4 months) for newborns and mothers discharged within 48 hours compared to those with a longer hospital stay. METHOD: A cohort of 1,357 vaginally delivered singleton normal newborns and their mothers (births between January 1, 1996 and March 31, 1997) were studied by linking five databases and a chart audit. RESULTS: Overall there were no differences in infant and maternal readmission or rates of exclusive breastfeeding. CONCLUSION: Canadian guidelines for earlier discharge appear appropriate for vaginally delivered singleton normal newborns and their mothers with timely home visitation.

Convection Enhanced Delivery: A Comparison of infusion characteristics in ex vivo and in vivo non-human primate brain tissue.

BACKGROUND: Convection enhanced delivery (CED) is emerging as a promising infusion toolto facilitate delivery of therapeutic agents into the brain via mechanically controlled pumps. Infusion protocols and catheter design have an important impact on delivery. CED is a valid alternative for systemic administration of agents in clinical trials for cell and gene therapies. Where gel and ex vivo models are not sufficient in modeling the disease, in vivo models allow researchers to better understand the underlying mechanisms of neuron degeneration, which is helpful in finding novel approaches to control the process or reverse the progression. Determining the risks, benefits, and efficacy of new gene therapies introduced via CED will pave a way to enter human clinical trial. PURPOSE: The objective of this study is to compare volume distribution (Vd)/ volume infused (Vi) ratios and backflow measurements following CED infusions in ex vivo versus in vivo non-human primate brain tissue, based on infusion protocols developed in vitro. METHODS: In ex vivo infusions, the first brain received 2 infusions using a balloon catheter at rates of 1 µL/min and 2 µL/min for 30 minutes. The second and third brains received infusions using a valve-tip (VT) catheter at 1 µL/min for 30 minutes. The fourth brain received a total of 45 µL infused at a rate of 1 µL/min for 15 minutes followed by 2 µL/min for 15 minutes. Imaging was performed (SPGR FA34) every 3 minutes. In the in vivo group, 4 subjects received a total of 8 infusions of 50 µL. Subjects 1 and 2 received infusions at 1.0 µL/min using a VT catheter in the left hemisphere and a smart-flow (SF) catheter in the right hemisphere. Subjects 3 and 4 each received 1 infusion in the left and right hemisphere at 1.0 µL/min. RESULTS: MRI calculations of Vd/Vi did not significantly differ from those obtained on post-mortem pathology. The mean measured Vd/Vi of in vivo (5.23 + /-1.67) compared to ex vivo (2.17 + /-1.39) demonstrated a significantly larger Vd/Vi for in vivo by 2.4 times (p = 0.0017). CONCLUSION: We detected higher ratios in the in vivo subjects than in ex vivo. This difference could be explained by the extra cellular space volume fraction. Studies evaluating backflow and morphology use in vivo tissue as a medium are recommended. Further investigation is warranted to evaluate the role blood pressure and heart rate may play in human CED clinical trials.