Prediction of m6A and m5C at single-molecule resolution reveals a transcriptome-wide co-occurrence of RNA modifications.

The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytosine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI's capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome.

Rhizosphere metabolism and its effect on phosphorus pools in the root zone of a submerged macrophyte, Isoëtes kirkii.

The presence of oxygen in lake sediments reduces sediment oxygen demand, and potentially improves sediment phosphorus retention and coupled nitrification/denitrification. However, the release of oxygen from the roots of macrophytes has not previously been measured in highly reducing sediments. Here, in the highly reducing environments of a commercial garden soil and sediment from a hyper-eutrophic lake, we used nine oxygen optodes, placed onto scintillation vials to detect oxygen in the rhizosphere of Isoetes kirkii referred to as quillworts. We calculated rhizosphere metabolism using "night-time regression" a method designed to estimate stream metabolism at the reach scale. After the incubations, sediment was collected from each vial (with and without macrophytes) and was subjected to sequential phosphorus extractions. A lag period between light availability and increasing oxygen concentration, that varied between individual optodes, was used to improve the accuracy of metabolism estimates as it was postulated to represent the distance between the root and the optode. Higher sediment oxygen demand in the lake sediment caused I. kirkii to have higher root oxygen release than those plants grown in the garden soil and may have pushed plants in lake sediment close to their ability to survive. This was evident as a significant, negative relationship between root oxygen release and increasing sediment oxygen demand, indicating that if photosynthesis decreased or sediment oxygen demand increased, the plants would no longer being able to oxygenate the sediment surrounding their roots, which would likely lead to death. Finally, the presence of quillworts in lake sediments significantly increased stores of metal oxide and recalcitrant phosphorus in the lake sediment but not the garden soil.

Sensory-motor network functional connectivity in children with unilateral cerebral palsy secondary to perinatal stroke.

BACKGROUND: Perinatal stroke is the most common cause of unilateral cerebral palsy. Mechanisms of post-stroke developmental plasticity in children are poorly understood. To better understand the relationship between functional connectivity and disability, we used resting-state fMRI to compare sensorimotor connectivity with clinical dysfunction. METHODS: School-aged children with periventricular venous infarction (PVI) and unilateral cerebral palsy were compared to controls. Resting-state BOLD signal was acquired on 3 T MRI and analyzed using CONN in SPM12. Functional connectivity was computed between S1, M1, supplementary motor area (SMA), and thalamus of the left/non-lesioned and right/lesioned hemisphere. Primary outcome was connectivity expressed as a Fisher-transformed correlation coefficient. Motor function was measured using the Assisting Hand Assessment (AHA), and Melbourne Assessment (MA). Proprioceptive function was measured using a robotic position matching task (VarXY). RESULTS: Participants included 15 PVI and 21 controls. AHA and MA in stroke patients were negatively correlated with connectivity (increased connectivity = poorer performance). Position sense was inversely correlated with connectivity (increased connectivity = improved performance) between the non-lesioned S1 and thalamus/SMA. In controls, VarXY was positively correlated with connectivity between the thalamus and bilateral sensorimotor regions. CONCLUSIONS: Resting state fMRI measures of sensorimotor connectivity are associated with clinical sensorimotor function in children with unilateral cerebral palsy secondary to PVI. Greater insight into understanding reorganization of brain networks following perinatal stroke may facilitate personalized rehabilitation.

First detection of bovine noroviruses and detection of bovine coronavirus in Australian dairy cattle.

BACKGROUND AND OBJECTIVE: Noroviruses have been recognised as a significant cause of neonatal enteritis in calves in many countries, but there has been no investigation of their occurrence in Australian cattle. This study aimed to establish whether bovine noroviruses could be detected in faecal samples from Australian dairy cattle. It also sought to determine whether bovine coronaviruses, also associated with neonatal enteritis in calves, could be detected in the same faecal samples. METHODS: A selection of faecal samples that were negative for rotaviruses from dairy farms located in three geographically distinct regions of Victoria were pooled and tested by reverse transcription-PCR for the presence of noroviruses (genogroup III), neboviruses and bovine coronaviruses. RESULTS AND CONCLUSION: Genetically distinct genogroup III noroviruses were detected in two sample pools from different geographic regions and bovine coronavirus was detected in a third pool of samples. This is the first report of bovine norovirus infection in Australian cattle and suggests that future work is required to determine the significance of these agents as a cause of bovine enteric disease in Australia.

Do plastic surgery division heads and program directors have the necessary tools to provide effective leadership?

BACKGROUND: Effective leadership is imperative in a changing health care landscape driven by increasing expectations in a setting of rising fiscal pressures. Because evidence suggests that leadership abilities are not simply innate but, rather, effective leadership can be learned, it is prudent for plastic surgeons to evaluate the training and challenges of their leaders because there may be opportunities for further growth and support. OBJECTIVE: To investigate the practice profiles, education/training, responsibilities and challenges of leaders within academic plastic surgery. METHODS: Following research ethics board approval, an anonymous online survey was sent to division heads and program directors from all university-affiliated plastic surgery divisions in Canada. Survey themes included demographics, education/training, job responsibilities and challenges. RESULTS: A response rate of 74% was achieved. The majority of respondents were male (94%), promoted to their current position at a mean age of 48 years, did not have a leadership-focused degree (88%), directly manage 30 people (14 staff, 16 faculty) and were not provided with a job description (65%). Respondents worked an average of 65 h per week, of which 18% was devoted to their leadership role, 59% clinically and the remainder on teaching and research. A discrepancy existed between time spent on their leadership role (18%) and related compensation (10%). Time management (47%) and managing conflict (24%) were described as the greatest leadership challenges by respondents. CONCLUSIONS: Several gaps were identified among leaders in plastic surgery including predominance of male sex, limitations in formal leadership training and requisite skill set, as well as compensation and human resources management (emotional intelligence). Leadership and managerial skills are key core competencies, not only for trainees, but certainly for those in a position of leadership. The present study provides evidence that academic departments, universities and medical centres may benefit by re-evaluating how they train, promote and support their leaders in plastic surgery.

HISTORIQUE: Il est essentiel d'assumer un leadership efficace dans un paysage de la santé en mutation animé par des attentes toujours plus nombreuses, dans un contexte de pressions financières grandissantes. Selon les données probantes, les capacités de leadership ne sont pas innées, mais un leadership efficace peut être acquis. Ainsi, il est prudent pour les plasticiens d'évaluer la formation et les difficultés de leurs leaders, car ils profiteraient peut-être d'occasions de perfectionnement et de soutien. OBJECTIF: Examiner les profils de pratique, la formation, les responsabilités et les difficultés des leaders dans le milieu de la chirurgie plastique universitaire. MÉTHODOLOGIE: Après avoir été approuvé par un comité d'éthique de la recherche, un sondage virtuel anonyme a été expédié aux chefs et directeurs de programme de tous les services de chirurgie plastique associés à une université au Canada. Ce sondage portait sur la démographie, la formation, de même que les responsabilités et les difficultés liées à l'emploi. RÉSULTATS: Le sondage a suscité un taux de réponse de 74 %. La majorité des répondants étaient des hommes (94 %) promus dans leur fonction à un âge moyen de 48 ans, qui n'avaient pas de diplôme lié au leadership (88 %), géraient directement 30 personnes (14 membres du personnel, 16 professeurs) et n'avaient pas de description de tâches (65 %). Les répondants travaillaient en moyenne 65 heures par semaine, dont 18 % étaient consacrées à leur rôle de leadership, 59 % aux tâches cliniques et le reste à l'enseignement et à la recherche. Ils remarquaient un écart entre le temps consacré à leur rôle de leadership (18 %) et la rémunération s'y rapportant (10 %). Ils décrivaient la gestion du temps (47 %) et des conflits (24 %) comme leurs plus grosses difficultés de leadership. CONCLUSIONS: Les chercheurs ont constaté plusieurs lacunes chez les leaders en chirurgie plastique, y compris la prédominance du sexe masculin, les limites de la formation officielle et des compétences en leadership, la rémunération et la gestion des ressources humaines (intelligence émotionnelle). Le leadership et les connaissances en gestion sont des compétences de base essentielles, non seulement pour les stagiaires, mais aussi pour les personnes en position d'autorité. La présente étude démontre qu'il serait bénéfique aux services universitaires, aux universités et aux centres médicaux de réévaluer leur mode de formation, de promotion et de soutien des leaders en chirurgie plastique.

Posttransplantation systemic immunomodulation with SA-FasL-engineered donor splenocytes has robust efficacy in preventing cardiac allograft rejection in mice.

Apoptosis induced by the engagement of FasL with Fas receptor on the surface of lymphocytes is an important immune homeostatic mechanism that ensures tolerance to self-antigens under normal physiologic conditions. As such, FasL has been extensively tested as a tolerogenic molecule with the use of gene therapy in settings of autoimmunity and transplantation with conflicting outcomes. Although the mechanistic basis of these contradictory observations is largely unknown, the use of wild-type FasL and the means by which the gene was expressed may provide an explanation. To overcome these complications, we generated a chimeric FasL protein with streptavidin (SA-FasL) having potent apoptotic activity and displayed this molecule effectively and rapidly on biotinylated biologic membranes for immunomodulation. In the present study, we displayed SA-FasL on the surface of BALB/c splenocytes and injected 5 × 10(6) cells intraperitoneally into C57BL/6 recipients of BALB/c heart grafts on days 1, 3, and 5 after-transplantation. To control initial graft-reactive immune responses and facilitate FasL-mediated apoptosis, rapamycin was used as an immunosuppressant at 0.2 mg/kg daily for a total of 15 doses immediately after heart transplantation. All mice injected with SA-FasL-engineered donor splenocytes accepted their grafts during the 100-day observation period. In marked contrast, immunomodulation with control streptavidin protein-engineered BALB/c splenocytes had minimal effect on graft survival (mean survival, 21.4 ± 1.5 d). Taken together, these results establish posttransplantation systemic immunomodulation with SA-FasL-engineered donor splenocytes under transient cover of rapamycin as an effective regimen in preventing cardiac allograft rejection in rodents with important clinical implications.

Interobserver agreement after pipeline embolization device implantation.

BACKGROUND AND PURPOSE: Although flow diversion devices are popular in treatment of aneurysms, angiographic assessment with these devices has rarely been verified by interobserver variability study. The purpose of this study was to determine the interobserver agreement of a 3-point grading system for assessing the angiographic outcome after flow diversion therapy of intracranial, saccular aneurysms and to determine factors affecting such agreement. MATERIALS AND METHODS: After approval by the institutional review board, 5 independent readers assessed pretreatment and follow-up digital subtraction angiograms from 96 patients treated with the Pipeline embolization device by using a 3-point grading system (complete, near-complete, and incomplete occlusion). "Minor discrepancy" was defined as a difference between any 2 readers of 1 grade, that is, complete vs near-complete or near-complete vs incomplete. "Major discrepancy" was defined as a difference between any 2 readers in which 1 reader noted complete occlusion and the other reader noted incomplete occlusion. We performed statistical analysis for the interobserver agreement by using the intraclass correlation coefficient. Subgroup analyses for discrepancy rate and ICC were performed for previously coiled aneurysms. RESULTS: The interobserver agreement was excellent (ICC, 0.76; 95% CI, 0.69-0.92). Among 96 cases, there was absolute agreement in 74 (77%), of which 67 had unanimous consensus of "complete" occlusion, 2 "near-complete" occlusion, and 5 "incomplete" occlusion. Discordance between any 2 readers was noted in 22 cases (23%), of which 7 (7.3%) revealed a major discrepancy. Subgroup analysis showed that minor discrepancies were more common among patients previously treated with coils vs those not previously treated with coils (37.5% vs 11.2%; P < .05). CONCLUSIONS: The observer agreement regarding occlusion after PED therapy is excellent. Only a minority of cases demonstrated discrepancy considered as major in this study.

Toxicity in animals: target species.

The macrocyclic lactone endectocides typified by ivermectin are safe and effective drugs when used according to label directions. However, off-label use, misuse and overdosing can result in toxicity in animal patients as revealed by pharmacovigilance activities. Preclinical toxicity studies demonstrates that the major clinical signs of toxicity are those associated with neurotoxic effects and these are the most common adverse drug reactions noted in overdosed treated animals. Subpopulations of some strains or breeds of some species appear to be uniquely sensitive to the neurotoxic effects of the macrocyclic lactones due to enhanced brain penetration by these drugs as a result of a deficiency in P-glycoprotein arising as a result of a mutation in the MDR1 gene.

Assessment of user safety, exposure and risk to veterinary medicinal products in the European Union.

Safety is an important part of veterinary drug assessment while user safety is a critical part of the overall safety assessment. In the European Union (EU), user safety is addressed through preclinical studies and by relationships with exposure but a key part of the process is the user safety assessment. EU user safety guidelines are available and these make certain recommendations but in places they lack detail and clarity. This paper seeks to examine the relevant factors that lie behind user risk assessments for veterinary medicinal products in general while focusing on EU requirements, the determination of risk management and risk communication strategies and how this relates to user safety assessment and pharmacovigilance responsibilities.

The potential impact of the use of homeopathic and herbal remedies on monitoring the safety of prescription products.

The purpose of this paper is to explore the possibility that adverse reactions and drug interactions arising from the use of homeopathic and herbal medicines could lead to confusion when adverse reactions to conventional medicines are reported. An extensive literature review was conducted on the occurrence of adverse reactions and drug interactions following the use of homeopathic or herbal remedies, and the potential for these to confound adverse event reporting to conventional medicines considered. The survey demonstrates the potential for herbal remedies and homeopathic products, to produce adverse drug reactions or drug interactions, and shows the scope for potential for confusion with those arising from conventional medicines. There is a need for greater awareness that adverse reactions apparently due to a conventional medicine, might in reality be due to a herbal medicine or a drug interaction between a herbal medicine and a conventional drug, particularly when a health professional is unaware of the extent of a patient's self-medication with alternative therapies.

Veterinary pharmacovigilance. Part 1. The legal basis in the European Union.

Pharmacovigilance is a growing discipline and nowhere is this more true than in the context of the legislative requirements for veterinary pharmacovigilance within the European Union (EU), or more specifically, within the European Economic Area. Since 1995, the legislation governing the authorization of veterinary medicinal products in the EU has resulted in the older national procedures being replaced by the mutual recognition procedure and the centralized procedure. Also since 1995, the regulatory requirements for pharmacovigilance have developed and grown, as have the associated guidelines. The recent review of European veterinary medicines legislation, which concluded with the publication of an amending directive and a new regulation in March 2004, has introduced refinements to the pharmacovigilance system. This paper examines the EU legislation governing the authorization of veterinary medicinal products, including the elaboration of maximum residue limits, and the way in which this relates to the requirements of pharmacovigilance.

Veterinary pharmacovigilance. Part 2. Veterinary pharmacovigilance in practice -- the operation of a spontaneous reporting scheme in a European Union country -- the UK, and schemes in other countries.

Veterinary pharmacovigilance, as it operates in the European Union (EU), covers a very broad remit, including adverse effects in treated animals, exposed humans and the environment, and in addition, it extends to cover the violation of maximum residue limits. The mainstay of veterinary pharmacovigilance is the spontaneous reporting scheme working along side other systems such as those reporting on residues surveillance. One of the most well established schemes in the EU is that operating in the UK and this paper examines the evolution of that scheme and some of its findings, data from other countries, and information available from the literature. It also tentatively examines the ways that pharmacovigilance can be used for regulatory purposes, and the contribution from pharmacoepidemiology.

Veterinary pharmacovigilance. Part 3. Adverse effects of veterinary medicinal products in animals and on the environment.

Like humans, animals may experience adverse effects when treated with medicinal products. These effects may be related to the pharmacological or toxicological properties of the substances used or they may arise because of hypersensitivity. Veterinary medicinal products may also possess the ability to harm the environment. This paper reviews the potential of veterinary medicinal products to cause adverse effects in animals and on the environment.

Veterinary pharmacovigilance. Part 4. Adverse reactions in humans to veterinary medicinal products.

Although seemingly rare, adverse reactions to veterinary products do occur. These may arise from inadvertent exposure during use or as a result of occupational accidents. They are often mild in nature and include adverse effects such as minor skin reactions. However, more serious reactions may occur, and they are not restricted to the effects of the veterinary medicines themselves. For example, high-pressure injection injuries may occur as a result of accidents occurring during animal vaccination operations. This paper reviews some of these events, mentions where appropriate the regulatory actions taken, and describes some of the measures used to minimise such effects in the future, and serves to bring the issues discussed here to the attention of pharmacologists, pharmacoepidemiologists and others who train those who use veterinary medicinal products.

Veterinary pharmacovigilance. Part 5. Causality and expectedness.

In the European Union and elsewhere there is a requirement to ascribe causality to adverse drug reactions which occur in treated animals. In the EU, the ABON system of causality assessment is used but the assignment of causality assigned is not always self evident, and it may be complicated for a variety of reasons. In this paper, the approaches to causality assessment, based on a number of key criteria which examine the administration of the drug in relation to the sequence of ensuing events and the presence of biological plausibility are examined, along with the utility of using algorithms to facilitate this process. Unexpected adverse drug reactions usually require expedited reporting, depending on national or regional regulatory requirements. Again, deciding on what might constitute an expected (or unexpected) adverse reaction, particularly when a product may be intended for use in a number of species, and when within any one species a number of breeds may be treated, is not necessarily a straightforward issue. However, an approach to facilitate the decision- making process, based on a similar approach used in the pharmacovigilance of human medicinal products is discussed.

Veterinary pharmacovigilance. Part 6. Predictability of adverse reactions in animals from laboratory toxicology studies.

Toxicological studies are conducted on constituents of veterinary medicinal products for a number of reasons. Aside from being a requirement of legislation, they are carried out for predictive purposes in the assessment of user safety or for the determination of consumer safety, for example, in the elaboration of maximum residue limits or tolerances. Alternatively, the results of toxicology studies may be available as they have been generated for registration of the drug for human medicinal purposes. This paper examines if the results of such studies have any predictive value for adverse reactions, which might occur during clinical use in animals. A number of adverse reactions, notably the Type A (toxicology or pharmacology dependent) should be predictable from these laboratory studies. However, as with human pharmaceutical products, they have less utility in predicting Type-B reactions (idiosyncratic in nature).

DNA microarrays with stem-loop DNA probes: preparation and applications.

We have developed DNA microarrays containing stem-loop DNA probes with short single-stranded overhangs immobilized on a Packard HydroGel chip, a 3-dimensional porous gel substrate. Microarrays were fabricated by immobilizing self-complementary single-stranded oligonucleotides, which adopt a partially duplex structure upon denaturing and re-annealing. Hybridization of single-stranded DNA targets to such arrays is enhanced by contiguous stacking interactions with stem-loop probes and is highly sequence specific. Subsequent enzymatic ligation of the targets to the probes followed by stringent washing further enhances the mismatched base discrimination. We demonstrate here that these microarrays provide excellent specificity with signal-to-background ratios of from 10- to 300-fold. In a comparative study, we demonstrated that HydroGel arrays display 10-30 times higher hybridization signals than some solid surface DNA microarrays. Using Sanger sequencing reactions, we have also developed a method for preparing nested 3'-deletion sets from a target and evaluated the use of stem-loop DNA arrays for detecting p53 mutations in the deletion set. The stem-loop DNA array format is simple, robust and flexible in design, thus it is potentially useful in various DNA diagnostic tests.

CNS myelination and PLP gene dosage.

The phenomenon of gene dosage effects demonstrates that the mechanisms of some genetic diseases are best recognised at the genomic level. Classical gene mutation screening approaches utilising PCR are unsuccessful in unravelling the basis of disease because the gene sequence is unaltered and only the copy number is different. Techniques for detecting DNA dosage are required. Examples of haploinsufficiency and gene deletions are well documented, but increased gene dosage is also an important genetic mechanism in disorders involving myelin proteins in the central (CNS) and peripheral nervous system (PNS). Here we review the dosage effects and mutations of the proteolipid protein (PLP) gene that causes Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia Type 2 (SPG2) disorders of CNS myelination. Similarities are drawn with the peripheral neuropathies Charcot-Marie-Tooth disease Type 1 (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) that are also caused by dosage effects and mutations in a single myelin protein gene (peripheral myelin protein 22, PMP-22). We compare the different mutational mechanisms in man and analogous mouse models that suggest a function for PLP beyond its structural role in myelin. We focus on the increased dosage of the PLP gene that is the major cause of PMD and results from a submicroscopic duplication of Xq22. Other clinical phenotypes may arise from gene dosage imbalance with the potential effect of submicroscopic duplications and deletions of the genome being underestimated. Genome sequencing may identify intrinsic structural properties of the DNA with greater susceptibility to these rearrangements and thereby reflect structural changes in the genome.