Facebook management as a tool for client communication and research recruitment in a companion animal university veterinary teaching and research hospital setting.

BACKGROUND: There is little peer-reviewed information about the strategic use of Facebook in companion animal veterinary settings, including for research recruitment. This study evaluates the implementation and execution of a Facebook strategy in a University Teaching and Research Hospital setting and the use of Facebook as a veterinary communication and recruitment tool. METHODS: All posts published on the hospital's Facebook page, messages sent via Messenger, and Facebook insight data from April 2017 to November 2019 (31 months) were reviewed and categorized. Facebook as a recruitment tool was evaluated through a survey among the faculty. RESULTS: A total of 113 posts were published, the Facebook page had 3485 followers and altogether 590,877 users were reached. The use of a Facebook strategy supported consistent management of the Facebook page. The content was well aligned with the strategy. The survey showed that the faculty experienced a facilitation effect by their recruitment posts, although the actual recruitment varied and ranged from none to the vast majority of all recruited subjects. CONCLUSIONS: This case-based, descriptive study gives insight and generates awareness about the possibilities and limitations of communication and research recruitment via Facebook. Further research is needed to evaluate if the findings can be generalized.

Neurotrophin-3 attenuates human peripheral blood T cell and monocyte activation status and cytokine production post stroke.

BACKGROUND AND PURPOSE: Stroke therapy still lacks successful measures to improve post stroke recovery. Neurotrophin-3 (NT-3) is one promising candidate which has proven therapeutic benefit in motor recovery in acute experimental stroke. Post stroke, the immune system has opposing pathophysiological roles: pro-inflammatory cascades and immune cell infiltration into the brain exacerbate cell death while the peripheral immune response has only limited capabilities to fight infections during the acute and subacute phase. With time, anti-inflammatory mechanisms are supposed to support recovery of the ischemic damage within the brain parenchyma. However, interestingly, NT-3 can improve recovery in chronic neurological injury when combined with the pro-inflammatory stimulus lipopolysaccharide (LPS). AIM: We elucidated the impact of NT-3 on human monocyte and T cell activation as well as cytokine production ex vivo after stroke. In addition, we investigated the age-dependent availability of the high affinity NT-3 receptor TrkC upon LPS stimulation. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from acute stroke patients and controls and incubated with different dosages of NT-3 (10 and 100 ng/mL) and with or without LPS or anti-CD3/CD28 for 48 h. Total TrkC expression and cell activation (CD25, CD69 and HLA-DR) were assessed by FACS staining. IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21 and IL-22 were quantified by cytometric bead array. RESULTS: Most monocytes and only a small proportion of T cells expressed TrkC in blood from humans without stroke. Activation of cells from young humans (without strokes) using anti-CD3/CD28 or LPS partially reduced the proportion of monocytes expressing TrkC whilst they increased the proportion of T cells expressing TrkC. In contrast, activation of cells from elderly humans (without strokes) did not affect the proportion of monocytes expressing TrkC and only anti-CD3/CD28 led to an increase in the proportion of CD4+ T cells expressing TrkC. In blood from stroke patients or controls, NT-3 treatment reduced the percentage of monocytes and CD4+ and CD8+ T cells that were activated and reduced all cytokines investigated besides IL-21. CONCLUSIONS: NT-3 attenuated immune responses in cells from stroke patients and controls. The mechanism whereby human immune cells respond to NT-3 may be via TrkC receptors whose levels are regulated by stimulation. Further work is required to determine whether the induction of sensorimotor recovery in rodents by NT-3 after CNS injury is caused by this attenuation of the immune response.

Characterising thermal water circulation in fractured bedrock using a multidisciplinary approach: a case study of St. Gorman's Well, Ireland.

A hydrogeological conceptual model of the source, circulation pathways and temporal variation of a low-enthalpy thermal spring in a fractured limestone setting is derived from a multidisciplinary approach. St. Gorman's Well is a thermal spring in east-central Ireland with a complex and variable temperature profile (maximum of 21.8 °C). Geophysical data from a three-dimensional(3D)audio-magnetotelluric(AMT) survey are combined with time-lapse hydrogeological data and information from a previously published hydrochemical analysis to investigate the operation of this intriguing hydrothermal system. Hydrochemical analysis and time-lapse measurements suggest that the thermal waters flow within the fractured limestones of the Carboniferous Dublin Basin at all times but display variability in discharge and temperature. The 3D electrical resistivity model of the subsurface revealed two prominent structures: (1) a NW-aligned faulted contact between two limestone lithologies; and (2) a dissolutionally enhanced, N-aligned, fault of probable Cenozoic age. The intersection of these two structures, which has allowed for karstification of the limestone bedrock, has created conduits facilitating the operation of relatively deep hydrothermal circulation (likely estimated depths between 240 and 1,000 m) within the limestone succession of the Dublin Basin. The results of this study support a hypothesis that the maximum temperature and simultaneous increased discharge observed at St. Gorman's Well each winter is the result of rapid infiltration, heating and recirculation of meteoric waters within a structurally controlled hydrothermal circulation system. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10040-021-02393-1.

Un modèle conceptuel hydrogéologique de la source, des voies de circulation et de la variation temporelle d'une source thermale à faible enthalpie dans un contexte de calcaire fracturé est dérivé d'une approche multidisciplinaire. St. Gorman's Well est une source thermale du centre-est de l'Irlande avec un profil de température complexe et variable (maximum de 21.8 °C). Les données géophysiques d'un levé audio-magnétotellurique (AMT) en trois dimensions (3D) sont combinées avec des données hydrogéologiques à intervalles de temps et des informations provenant d'une analyse hydrochimique publiée précédemment pour étudier le fonctionnement de cet intrigant système hydrothermal. L'analyse hydrochimique et les mesures à différentes périodes suggèrent que les eaux thermales s'écoulent à tout moment dans les calcaires fracturés du bassin carbonifère de Dublin, mais présentent une variabilité de débit et de température. Le modèle de résistivité électrique 3D du sous-sol a révélé deux structures importantes: (1) un contact faillé orienté NW entre deux lithologies calcaires; et (2) une faille alignée au Nord, améliorée par dissolution, d'âge cénozoïque probable. L'intersection de ces deux structures, qui a permis la karstification du socle calcaire, a créé des conduits facilitant le fonctionnement d'une circulation hydrothermale relativement profonde (profondeurs estimées vraisemblablement entre 240 et 1,000 m) au sein de la succession calcaire du bassin de Dublin. Les résultats de cette étude appuient l'hypothèse selon laquelle la température maximale et l'augmentation simultanée du débit observés à St. Gorman's Well chaque hiver sont le résultat d'une infiltration, d'un réchauffement et d'une recirculation rapides des eaux météoriques dans un système de circulation hydrothermale structurellement contrôlé.

Se deriva un modelo conceptual hidrogeológico de la fuente, las vías de circulación y la variación temporal de un manantial termal de baja entalpía en un entorno de caliza fracturada a partir de un enfoque multidisciplinar. Gorman's Well es un manantial termal en el centro-este de Irlanda con un perfil de temperatura complejo y variable (máximo de 21.8 °C). Los datos geofísicos de un estudio audio-magnetotelúrico (AMT) tridimensional (3D) se combinan con los datos hidrogeológicos de un lapso de tiempo y la información de un análisis hidroquímico previamente publicado para investigar el funcionamiento de este intrigante sistema hidrotermal. El análisis hidroquímico y las mediciones a intervalos de tiempo sugieren que las aguas termales fluyen dentro de las calizas fracturadas de la cuenca carbonífera de Dublín en todo momento, pero muestran variabilidad en la descarga y la temperatura. El modelo de resistividad eléctrica tridimensional del subsuelo reveló dos estructuras prominentes: (1) un contacto de falla alineado al NW entre dos litologías calcáreas; y (2) una falla de disolución incrementada, alineada al N, de probable edad cenozoica. La intersección de estas dos estructuras, que ha permitido la karstificación del lecho rocoso calcáreo, ha creado conductos que facilitan el funcionamiento de una circulación hidrotermal relativamente profunda (probablemente a profundidades estimadas entre 240 y 1,000 m) dentro de la sucesión calcárea de la cuenca de Dublín. Los resultados de este estudio apoyan la hipótesis de que la temperatura máxima y el aumento simultáneo de la descarga observados en St. Gorman's Well cada invierno son el resultado de una rápida infiltración, calentamiento y recirculación de aguas meteóricas dentro de un sistema de circulación hidrotermal estructuralmente controlado.

Um modelo hidrogeológico conceitual da fonte, vias de circulação e variação temporal de uma fonte termal de baixa entalpia em um ambiente de calcário fraturado é derivado de uma abordagem multidisciplinar. O poço de St. Gorman é uma fonte termal no centro-leste da Irlanda com um perfil de temperatura complexo e variável (máximo de 21.8°C). Os dados geofísicos de uma pesquisa áudio-magnetotelúrica (AMT) tridimensional (3D) são combinados com dados hidrogeológicos em intervalos de tempo e informações de uma análise hidroquimica publicada anteriormente, para investigar a operação deste intrigante sistema hidrotérmico. A análise hidroquimica e as medições em intervalos de tempo sugerem que as águas termais fluem de dentro dos calcários fraturados da Bacia Carbonifera de Dublin o tempo todo, mas exibem variabilidade na descarga e na temperatura. O modelo de resistividade elétrica 3D da subsuperfície revelou duas estruturas proeminentes: (1) um contato defeituoso alinhado a NO entre duas litologias de calcário; e (2) uma falha dissolucionalmente aumentada, alinhada a N, de provável idade Cenozóica. A intersecção dessas duas estruturas, que permitiu a carstificação da rocha calcária, criou condutos que facilitam a operação de circulação hidrotérmica relativamente profunda (profundidade estimada entre 240 e 1,000 m) dentro da sucessão de calcário da Bacia Dublin. Os resultados desse estudo suportam a hipótese de que a temperatura máxima e o aumento simultâneo da descarga observada no poço de St. Gorman a cada inverno é o resultado da rápida infiltração, aquecimento e recirculação de águas meteóricas dentro de um sistema de circulação hidrotérmica estruturalmente controlado.

Nanoscopic X-ray tomography for correlative microscopy of a small meiofaunal sea-cucumber.

In the field of correlative microscopy, light and electron microscopy form a powerful combination for morphological analyses in zoology. Due to sample thickness limitations, these imaging techniques often require sectioning to investigate small animals and thereby suffer from various artefacts. A recently introduced nanoscopic X-ray computed tomography (NanoCT) setup has been used to image several biological objects, none that were, however, embedded into resin, which is prerequisite for a multitude of correlative applications. In this study, we assess the value of this NanoCT for correlative microscopy. For this purpose, we imaged a resin-embedded, meiofaunal sea cucumber with an approximate length of 1 mm, where microCT would yield only little information about the internal anatomy. The resulting NanoCT data exhibits isotropic 3D resolution, offers deeper insights into the 3D microstructure, and thereby allows for a complete morphological characterization. For comparative purposes, the specimen was sectioned subsequently to evaluate the NanoCT data versus serial sectioning light microscopy (ss-LM). To correct for mechanical instabilities and drift artefacts, we applied an alternative alignment procedure for CT reconstruction. We thereby achieve a level of detail on the subcellular scale comparable to ss-LM images in the sectioning plane.

Functional morphology of a lobopod: case study of an onychophoran leg.

Segmental, paired locomotory appendages are a characteristic feature of Panarthropoda-a diversified clade of moulting animals that includes onychophorans (velvet worms), tardigrades (water bears) and arthropods. While arthropods acquired a sclerotized exoskeleton and articulated limbs, onychophorans and tardigrades possess a soft body and unjointed limbs called lobopods, which they inherited from Cambrian lobopodians. To date, the origin and ancestral structure of the lobopods and their transformation into the jointed appendages are all poorly understood. We therefore combined high-resolution computed tomography with high-speed camera recordings to characterize the functional anatomy of a trunk lobopod from the onychophoran Euperipatoides rowelli. Three-dimensional reconstruction of the complete set of muscles and muscle fibres as well as non-muscular structures revealed the spatial relationship and relative volumes of the muscular, excretory, circulatory and nervous systems within the leg. Locomotory movements of individual lobopods of E. rowelli proved far more diverse than previously thought and might be governed by a complex interplay of 15 muscles, including one promotor, one remotor, one levator, one retractor, two depressors, two rotators, one flexor and two constrictors as well as muscles for stabilization and haemolymph control. We discuss the implications of our findings for understanding the evolution of locomotion in panarthropods.

3D Imaging of Soft-Tissue Samples using an X-ray Specific Staining Method and Nanoscopic Computed Tomography.

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described protocol is easy to apply, fast and suitable for larger soft-tissue samples. The presented methodology enables the characterization of crucial tissue structures in three dimensions and is demonstrated on a whole mouse kidney. The multiscale approach allows to image the entire mouse kidney and supports the selection of further volumes of interest, which are acquired with higher resolutions ranging into the nanometer range. Thereby, soft-tissue morphology with a similar detail level as the corresponding histological light microscopy images is reproduced. Deeper insights into the 3D configuration of tissue structures are achieved without impeding further investigations through histological methods.

Kinetic Study of DNA Topoisomerases by Supercoiling-Dependent Fluorescence Quenching.

DNA topoisomerases are essential enzymes for all living organisms and important targets for anticancer drugs and antibiotics. Although DNA topoisomerases have been studied extensively, steady-state kinetics has not been systematically investigated because of the lack of an appropriate assay. Previously, we demonstrated that newly synthesized, fluorescently labeled plasmids pAB1_FL905 and pAB1_FL924 can be used to study DNA topoisomerase-catalyzed reactions by fluorescence resonance energy transfer (FRET) or supercoiling-dependent fluorescence quenching (SDFQ). With the FRET or SDFQ method, we performed steady-state kinetic studies for six different DNA topoisomerases including two type IA enzymes (Escherichia coli and Mycobacterium smegmatis DNA topoisomerase I), two type IB enzymes (human and variola DNA topoisomerase I), and two type IIA enzymes (E. coli DNA gyrase and human DNA topoisomerase IIα). Our results show that all DNA topoisomerases follow the classical Michaelis-Menten kinetics and have unique steady-state kinetic parameters, K M, V max, and k cat. We found that k cat for all topoisomerases are rather low and that such low values may stem from the tight binding of topoisomerases to DNA. Additionally, we confirmed that novobiocin is a competitive inhibitor for adenosine 5'-triphosphate binding to E. coli DNA gyrase, demonstrating the utility of our assay for studying topoisomerase inhibitors.

X-ray imaging of a water bear offers a new look at tardigrade internal anatomy.

BACKGROUND: Tardigrades (water bears) are microscopic invertebrates of which the anatomy has been well studied using traditional techniques, but a comprehensive three-dimensional reconstruction has never been performed. In order to close this gap, we employed X-ray computed tomography (CT), a technique that is becoming increasingly popular in zoology for producing high-resolution, three-dimensional (3D) scans of whole specimens. While CT has long been used to scan larger samples, its use in some microscopic animals can be problematic, as they are often too small for conventional CT yet too large for high-resolution, optics-based soft X-ray microscopy. This size gap continues to be narrowed with advancements in technology, with high-resolution imaging now being possible using both large synchrotron devices and, more recently, laboratory-based instruments. RESULTS: Here we use a recently developed prototype lab-based nano-computed tomography device to image a 152 µm-long tardigrade at high resolution (200-270 nm pixel size). The resulting dataset allowed us to visualize the anatomy of the tardigrade in 3D and analyze the spatial relationships of the internal structures. Segmentation of the major structures of the body enabled the direct measurement of their respective volumes. Furthermore, we segmented every storage cell individually and quantified their volume distribution. We compare our measurements to those from published studies in which other techniques were used. CONCLUSIONS: The data presented herein demonstrate the utility of CT imaging as a powerful supplementary tool for studies of tardigrade anatomy, especially for quantitative volume measurements. This nanoCT study represents the smallest complete animal ever imaged using CT, and offers new 3D insights into the spatial relationships of the internal organs of water bears.

Author Correction: Nucleus-specific X-ray stain for 3D virtual histology.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Nucleus-specific X-ray stain for 3D virtual histology.

Histological investigations are indispensable with regards to the identification of structural tissue details but are limited to two-dimensional images, which are often visualized in one and the same plane for comparison reasons. Nondestructive three-dimensional technologies such as X-ray micro- and nanoCT have proven to provide valuable benefits for the understanding of anatomical structures as they allow visualization of structural details in 3D and from arbitrary viewing angles. Nevertheless, low attenuation of soft tissue has hampered their application in the field of 3D virtual histology. We present a hematein-based X-ray staining method that specifically targets the cell nuclei of cells, as demonstrated for a whole liver lobule of a mouse. Combining the novel staining protocol with the high resolving power of a recently developed nanoCT system enables the 3D visualization of tissue architecture in the nanometer range, thereby revealing the real 3D morphology and spatial distribution of the cell nuclei. Furthermore, our technique is compatible with conventional histology, as microscopic slides can be derived from the very same stained soft-tissue sample and further counter staining is possible. Thus, our methodology demonstrates future applicability for modern histopathology using laboratory X-ray CT devices.

Blood Serum From Head and Neck Squamous Cell Carcinoma Patients Induces Altered MicroRNA and Target Gene Expression Profile in Treated Cells.

The head and neck squamous cell carcinoma (HNSCC) represents one of the most common cancers in humans. Close to 600,000 new diagnoses are made every year worldwide and over half of diagnosed patients will not survive. In view of this low survival rate, the development of novel cell-based assays for HNSCC will allow more mechanistic approaches for specific diagnostics for each individual patient. The cell-based assays will provide more informative data predicting cellular processes in treated patient, which in effect would improve patient follow up. More importantly, it will increase the specificity and effectiveness of therapeutic approaches. In this study, we investigated the role of serum from HNSCC patients on the regulation of microRNA (miRNA) expression in exposed cells in vitro. Next-generation sequencing of miRNA revealed that serum from HNSCC patients induced a different miRNA expression profile than the serum from healthy individuals. Out of 377 miRNA detected, we found that 16 miRNAs were differentially expressed when comparing cells exposed to serum from HNSCC or healthy individuals. The analysis of gene ontologies and pathway analysis revealed that these miRNA target genes were involved in biological cancer-related processes, including cell cycle and apoptosis. The real-time PCR analysis revealed that serum from HNSCC patients downregulate the expression level of five genes involved in carcinogenesis and two of these genes-P53 and SLC2A1-are direct targets of detected miRNAs. These novel findings provide new insight into how cancer-associated factors in circulation regulate the expression of genes and regulatory elements in distal cells in favor of tumorigenesis. This has the potential for new therapeutic approaches and more specific diagnostics with tumor-specific cell lines or single-cell in vitro assays for personalized treatment and early detection of primary tumors or metastasis.

Three-dimensional virtual histology enabled through cytoplasm-specific X-ray stain for microscopic and nanoscopic computed tomography.

Many histological methods require staining of the cytoplasm, which provides instrumental details for diagnosis. One major limitation is the production of 2D images obtained by destructive preparation of 3D tissue samples. X-ray absorption micro- and nanocomputed tomography (microCT and nanoCT) allows for a nondestructive investigation of a 3D tissue sample, and thus aids to determine regions of interest for further histological examinations. However, application of microCT and nanoCT to biological samples (e.g., biopsies) is limited by the missing contrast within soft tissue, which is important to visualize morphological details. We describe an eosin-based preparation overcoming the challenges of contrast enhancement and selectivity for certain tissues. The eosin-based staining protocol is suitable for whole-organ staining, which then enables high-resolution microCT imaging of whole organs and nanoCT imaging of smaller tissue pieces retrieved from the original sample. Our results demonstrate suitability of the eosin-based staining method for diagnostic screening of 3D tissue samples without impeding further diagnostics through histological methods.

Myoanatomy of the velvet worm leg revealed by laboratory-based nanofocus X-ray source tomography.

X-ray computed tomography (CT) is a powerful noninvasive technique for investigating the inner structure of objects and organisms. However, the resolution of laboratory CT systems is typically limited to the micrometer range. In this paper, we present a table-top nanoCT system in conjunction with standard processing tools that is able to routinely reach resolutions down to 100 nm without using X-ray optics. We demonstrate its potential for biological investigations by imaging a walking appendage of Euperipatoides rowelli, a representative of Onychophora-an invertebrate group pivotal for understanding animal evolution. Comparative analyses proved that the nanoCT can depict the external morphology of the limb with an image quality similar to scanning electron microscopy, while simultaneously visualizing internal muscular structures at higher resolutions than confocal laser scanning microscopy. The obtained nanoCT data revealed hitherto unknown aspects of the onychophoran limb musculature, enabling the 3D reconstruction of individual muscle fibers, which was previously impossible using any laboratory-based imaging technique.

Data sets for author name disambiguation: an empirical analysis and a new resource.

Data sets of publication meta data with manually disambiguated author names play an important role in current author name disambiguation (AND) research. We review the most important data sets used so far, and compare their respective advantages and shortcomings. From the results of this review, we derive a set of general requirements to future AND data sets. These include both trivial requirements, like absence of errors and preservation of author order, and more substantial ones, like full disambiguation and adequate representation of publications with a small number of authors and highly variable author names. On the basis of these requirements, we create and make publicly available a new AND data set, SCAD-zbMATH. Both the quantitative analysis of this data set and the results of our initial AND experiments with a naive baseline algorithm show the SCAD-zbMATH data set to be considerably different from existing ones. We consider it a useful new resource that will challenge the state of the art in AND and benefit the AND research community.

High-coverage methylation data of a gene model before and after DNA damage and homologous repair.

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.

Non-homologous end joining induced alterations in DNA methylation: A source of permanent epigenetic change.

In addition to genetic mutations, epigenetic revision plays a major role in the development and progression of cancer; specifically, inappropriate DNA methylation or demethylation of CpG residues may alter the expression of genes that promote tumorigenesis. We hypothesize that DNA repair, specifically the repair of DNA double strand breaks (DSB) by Non-Homologous End Joining (NHEJ) may play a role in this process. Using a GFP reporter system inserted into the genome of HeLa cells, we are able to induce targeted DNA damage that enables the cells, after successfully undergoing NHEJ repair, to express WT GFP. These GFP+ cells were segregated into two expression classes, one with robust expression (Bright) and the other with reduced expression (Dim). Using a DNA hypomethylating drug (AzadC) we demonstrated that the different GFP expression levels was due to differential methylation statuses of CpGs in regions on either side of the break site. Deep sequencing analysis of this area in sorted Bright and Dim populations revealed a collection of different epi-alleles that display patterns of DNA methylation following repair by NHEJ. These patterns differ between Bright and Dim cells which are hypo- and hypermethylated, respectively, and between the post-repair populations and the original, uncut cells. These data suggest that NHEJ repair facilitates a rewrite of the methylation landscape in repaired genes, elucidating a potential source for the altered methylation patterns seen in cancer cells, and understanding the mechanism by which this occurs could provide new therapeutic targets for preventing this process from contributing to tumorigenesis.

Correction: DNA Damage, Homology-Directed Repair, and DNA Methylation.

[This corrects the article DOI: 10.1371/journal.pgen.0030110.].

An "Unlikely" Pair: The Antimicrobial Synergy of Polymyxin B in Combination with the Cystic Fibrosis Transmembrane Conductance Regulator Drugs KALYDECO and ORKAMBI.

Novel combination therapies are desperately needed for combating lung infections caused by bacterial "superbugs". This study aimed to investigate the synergistic antibacterial activity of polymyxin B in combination with the cystic fibrosis (CF) drugs KALYDECO (ivacaftor) and ORKAMBI (ivacaftor + lumacaftor) against Gram-negative pathogens that commonly colonize the CF lung, in particular, the problematic Pseudomonas aeruginosa. The in vitro synergistic activity of polymyxin B combined with ivacaftor or lumacaftor was assessed using checkerboard and static time-kill assays against a panel of polymyxin-susceptible and polymyxin-resistant P. aeruginosa isolates from the lungs of CF patients. Polymyxin B, ivacaftor, and lumacaftor were ineffective when used individually against polymyxin-resistant (MIC ≥ 4 mg/L) isolates. However, when used together, the combination of clinically relevant concentrations of polymyxin B (2 mg/L) combined with ivacaftor (8 mg/L) or ivacaftor (8 mg/L) + lumacaftor (8 mg/L) displayed synergistic killing activity against polymyxin-resistant P. aeruginosa isolates as demonstrated by a 100-fold decrease in the bacterial count (CFU/mL) even after 24 h. The combinations also displayed excellent antibacterial activity against P. aeruginosa under CF relevant conditions in a sputum medium assay. The combination of lumacaftor (alone) with polymyxin B showed additivity against P. aeruginosa. The potential antimicrobial mode of action of the combinations against P. aeruginosa was investigated using different methods. Treatment with the combinations induced cytosolic GFP release from P. aeruginosa cells and showed permeabilizing activity in the nitrocefin assay, indicating damage to both the outer and inner Gram-negative cell membranes. Moreover, scanning and transmission electron micrographs revealed that the combinations produce outer membrane damage to P. aeruginosa cells that is distinct from the effect of each compound per se. Ivacaftor was also shown to be a weak inhibitor of the bacterial DNA gyrase and topoisomerase IV with no effect on either human type I or type IIα topoisomerases. Lumacaftor displayed the ability to increase the cellular production of damaging reactive oxygen species. In summary, the combination of polymyxin B with KALYDECO or ORKAMBI exhibited synergistic activity against highly polymyxin-resistant P. aeruginosa CF isolates and can be potentially useful for otherwise untreatable CF lung infections.

DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism.

We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells.

Text messaging between clinicians and patients - Hve we got thngs unda cntrl?

INTRODUCTION Patients are interested in receiving text messages (texts) related to their health care. However, anecdotes are emerging of associated problems and it is possible that many of the potential pitfalls are not recognised. AIM To assess clinicians' attitudes and behaviours towards text messaging (texting) with patients. METHODS A voluntary, anonymous, online survey was created and distributed to general practitioners and physiotherapists in New Zealand and to Sports Medicine Fellows and Sports Medicine Registrars in New Zealand and Australia. RESULTS In total, 322 clinicians completed the survey. Texting behaviours relating to accuracy, privacy and security were identified. A range of sensitive and important medical information was frequently conveyed and at times forwarded to third parties. The clinicians generally felt uneasy communicating this way and some felt pressured into it. Most thought that guidelines are insufficient and that they had not received sufficient education on the issues. Most were interested in further education. CONCLUSION This study has demonstrated frequent texting between clinicians and patients. It has highlighted potential risks to the privacy, accuracy and security of medical information. Current guidelines and education may be insufficient. Clinicians were interested in receiving readily available best practice guidelines and education regarding texting.