The influence of molecular design on structure-property relationships of a supramolecular polymer prodrug.

Supramolecular self-assemblies of hydrophilic macromolecules functionalized with hydrophobic, structure-directing components have long been used for drug delivery. In these systems, loading of poorly soluble compounds is typically achieved through physical encapsulation during or after formation of the supramolecular assembly, resulting in low encapsulation efficiencies and limited control over release kinetics, which are predominately governed by diffusion and carrier degradation. To overcome these limitations, amphiphilic prodrugs that leverage a hydrophobic drug as both the therapeutic and structure-directing component can be used to create supramolecular materials with higher loading and controlled-release kinetics using biodegradable or enzymatically cleavable linkers. Here, we report the design, synthesis, and characterization of a library of supramolecular polymer prodrugs based on poly(ethylene glycol) (PEG) and the proregenerative drug 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (DPCA). Structure-property relationships were elucidated through experimental characterization of prodrug behavior in both the wet and dry states using scattering techniques and electron microscopy and corroborated by coarse-grained modeling. Molecular architecture and the hydrophobic-to-hydrophilic ratio of PEG-DPCA conjugates strongly influenced their physical state in water, ranging from fully soluble to supramolecular spherical assemblies and nanofibers. Molecular design and supramolecular structure, in turn, were shown to dramatically alter hydrolytic and enzymatic release and cellular transport of DPCA. In addition to potentially expanding therapeutic options for DPCA through control of supramolecular assemblies, the design principles elaborated here may inform the development of other supramolecular prodrugs based on hydrophobic small-molecule compounds.

Changes in infant porcine pulmonary tissue oxylipins induced by cardiopulmonary bypass.

BACKGROUND: Oxylipins are metabolites derived from fatty acids such as arachidonic acid (AA) and are key mediators in inflammation, host defense, and tissue injury. Serum oxylipins increase in adults after cardiopulmonary bypass (CPB) but tissue-level changes are poorly defined. The objective of this study was to characterize pulmonary tissue oxylipins in an infant porcine model of CPB with deep hypothermic circulatory arrest (DHCA). METHODS: Infant pigs underwent CPB with DHCA. Controls received anesthesia only. Right upper and lower lobes of the lung underwent oxylipin analysis via liquid chromatography-tandem mass spectrometry. One-way ANOVA was utilized to assess differences in oxylipin concentrations across groups, followed by pairwise comparisons. RESULTS: AA and multiple AA metabolites via cytochrome P450 (CYP450), lipoxygenase (LOX), and cyclooxygenase (COX) pathways were significantly increased in the upper and lower lobe of pigs exposed to CPB/DHCA as compared to controls. Multiple prostaglandin metabolites produced via COX were also significantly elevated in the lower lobes of control animals. CONCLUSIONS: CPB/DHCA induces a significant increase in pulmonary tissue AA, with subsequent metabolism via COX, LOX, and CYP450 pathways. Interestingly, prostaglandins were also elevated in the lower lobes of the controls, suggesting a mechanism separate from CPB/DHCA. Future oxylipin studies are needed to better understand CPB-induced acute lung injury. IMPACT: CPB/DHCA and, to a lesser extent, lung region influence pulmonary tissue-level AA metabolite production. Inflammatory mediator AA metabolites have been noted in previous studies to increase following CPB; however, this is the first study to look at pulmonary tissue-level differences following CPB/DHCA. Increases in many AA metabolites, including LOX- and CYP450-derived products, were seen in both upper and lower lobe of piglets following CPB/DHCA. COX-derived prostaglandin metabolites were increased not only in CPB upper and lower lobe but also in mechanically ventilated control lower lobe, suggesting an additional, separate mechanism from CPB/DCHA.

Seawater-buffered diagenesis, destruction of carbon isotope excursions, and the composition of DIC in Neoproterozoic oceans.

Carbonate sediments of nonglacial Cryogenian (659 to 649 Ma) and early Ediacaran (635 to 590 Ma) age exhibit large positive and negative δ13Ccarb excursions in a shallow-water marine platform in northern Namibia. The same excursions are recorded in fringing deep-sea fans and in carbonate platforms on other paleocontinents. However, coeval carbonates in the upper foreslope of the Namibian platform, and to a lesser extent in the outermost platform, have relatively uniform δ13Ccarb compositions compatible with dissolved inorganic carbon (DIC) in the modern ocean. We attribute the uniform values to fluid-buffered diagenesis that occurred where seawater invaded the sediment in response to geothermal porewater convection. This attribution, which is testable with paired Ca and Mg isotopes, implies that large δ13Ccarb excursions observed in Neoproterozoic platforms, while sedimentary in origin, do not reflect the composition of ancient open-ocean DIC.

A distinctive epigenetic ageing profile in human granulosa cells.

STUDY QUESTION: Does women's age affect the DNA methylation (DNAm) profile differently in mural granulosa cells (MGCs) from other somatic cells? SUMMARY ANSWER: Accumulation of epimutations by age and a higher number of age-related differentially methylated regions (DMR) in MGCs were found compared to leukocytes from the same woman, suggesting that the MGCs have a distinctive epigenetic profile. WHAT IS KNOWN ALREADY: The mechanisms underlying the decline in women's fertility from the mid-30s remain to be fully elucidated. The DNAm age of many healthy tissues changes predictably with and follows chronological age, but DNAm age in some reproductive tissues has been shown to depart from chronological age (older: endometrium; younger: cumulus cells, spermatozoa). STUDY DESIGN, SIZE, DURATION: This study is a multicenter cohort study based on retrospective analysis of prospectively collected data and material derived from healthy women undergoing IVF or ICSI treatment following ovarian stimulation with antagonist protocol. One hundred and nineteen women were included from September 2016 to June 2018 from four clinics in Denmark and Sweden. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples were obtained from 118 healthy women with varying ovarian reserve status. MGCs were collected from 63 of the 119 women by isolation from pooled follicles immediately after oocyte retrieval. DNA from leukocytes and MGCs was extracted and analysed with a genome-wide methylation array. Data from the methylation array were processed using the ENmix package. Subsequently, DNAm age was calculated using established and tailored age predictors and DMRs were analysed with the DMRcate package. MAIN RESULTS AND ROLE OF CHANCE: Using established age predictors, DNAm age in MGCs was found to be considerable younger and constant (average: 2.7 years) compared to chronological age (average: 33.9 years). A Granulosa Cell clock able to predict the age of both MGCs (average: 32.4 years) and leukocytes (average: 38.8 years) was successfully developed. MGCs differed from leukocytes in having a higher number of epimutations (P = 0.003) but predicted telomere lengths unaffected by age (Pearson's correlation coefficient = -0.1, P = 0.47). DMRs associated with age (age-DMRs) were identified in MGCs (n = 335) and in leukocytes (n = 1) with a significant enrichment in MGCs for genes involved in RNA processing (45 genes, P = 3.96 × 10-08) and gene expression (152 genes, P = 2.3 × 10-06). The top age-DMRs included the metastable epiallele VTRNA2-1, the DNAm regulator ZFP57 and the anti-Müllerian hormone (AMH) gene. The apparent discordance between different epigenetic measures of age in MGCs suggests that they reflect difference stages in the MGC life cycle. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: No gene expression data were available to associate with the epigenetic findings. The MGCs are collected during ovarian stimulation, which may influence DNAm; however, no correlation between FSH dose and number of epimutations was found. WIDER IMPLICATIONS OF THE FINDINGS: Our findings underline that the somatic compartment of the follicle follows a different methylation trajectory with age than other somatic cells. The higher number of epimutations and age-DMRs in MGCs suggest that their function is affected by age. STUDY FUNDING/COMPETING INTEREST(S): This project is part of ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS, the Danish National Research Foundation and the European Research Council. The authors declare no conflict of interest.

DNA methylation and mRNA expression of imprinted genes in blastocysts derived from an improved in vitro maturation method for oocytes from small antral follicles in polycystic ovary syndrome patients.

STUDY QUESTION: Does imprinted DNA methylation or imprinted gene expression differ between human blastocysts from conventional ovarian stimulation (COS) and an optimized two-step IVM method (CAPA-IVM) in age-matched polycystic ovary syndrome (PCOS) patients? SUMMARY ANSWER: No significant differences in imprinted DNA methylation and gene expression were detected between COS and CAPA-IVM blastocysts. WHAT IS KNOWN ALREADY: Animal models have revealed alterations in DNA methylation maintenance at imprinted germline differentially methylated regions (gDMRs) after use of ARTs. This effect increases as more ART interventions are applied to oocytes or embryos. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for patients. CAPA-IVM is an improved IVM system that includes a pre-maturation step (CAPA), followed by an IVM step, both in the presence of physiological compounds that promote oocyte developmental capacity. STUDY DESIGN, SIZE, DURATION: For DNA methylation analysis 20 CAPA-IVM blastocysts were compared to 12 COS blastocysts. For RNA-Seq analysis a separate set of 15 CAPA-IVM blastocysts were compared to 5 COS blastocysts. PARTICIPANTS/MATERIALS, SETTING, METHODS: COS embryos originated from 12 patients with PCOS (according to Rotterdam criteria) who underwent conventional ovarian stimulation. For CAPA-IVM 23 women were treated for 3-5 days with highly purified hMG (HP-hMG) and no hCG trigger was given before oocyte retrieval. Oocytes were first cultured in pre-maturation medium (CAPA for 24 h containing C-type natriuretic peptide), followed by an IVM step (30 h) in medium containing FSH and Amphiregulin. After ICSI, Day 5 or 6 embryos in both groups were vitrified and used for post-bisulphite adaptor tagging (PBAT) DNA methylation analysis or RNA-seq gene expression analysis of individual embryos. Data from specific genes and gDMRs were extracted from the PABT and RNA-seq datasets. MAIN RESULTS AND THE ROLE OF CHANCE: CAPA-IVM blastocysts showed similar rates of methylation and gene expression at gDMRs compared to COS embryos. In addition, expression of major epigenetic regulators was similar between the groups. LIMITATIONS, REASONS FOR CAUTION: The embryos from the COS group were generated in a range of culture media. The CAPA-IVM embryos were all generated using the same sperm donor. The DNA methylation level of gDMRs in purely in vivo-derived human blastocysts is not known. WIDER IMPLICATIONS OF THE FINDINGS: A follow-up of children born after CAPA-IVM is important as it is for other new ARTs, which are generally introduced into clinical practice without prior epigenetic safety studies on human blastocysts. CAPA-IVM opens new perspectives for patient-friendly ART in PCOS. STUDY FUNDING/COMPETING INTEREST(S): IVM research at the Vrije Universiteit Brussel has been supported by grants from the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680), the Fund for Research Flanders (Fonds voor Wetenschappelijk Onderzoek-Vlaanderen-FWO-AL 679 project, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69Ref Nr 2016-119) and the Vrije Universiteit Brussel (IOF Project 4R-ART Nr 2042). Work in G.K.'s laboratory is supported by the UK Biotechnology and Biological Sciences Research Council and Medical Research Council. The authors have no conflicts of interest.

Disentangling the Representation of Identity from Head View Along the Human Face Processing Pathway.

Neural models of a distributed system for face perception implicate a network of regions in the ventral visual stream for recognition of identity. Here, we report a functional magnetic resonance imaging (fMRI) neural decoding study in humans that shows that this pathway culminates in the right inferior frontal cortex face area (rIFFA) with a representation of individual identities that has been disentangled from variable visual features in different images of the same person. At earlier stages in the pathway, processing begins in early visual cortex and the occipital face area with representations of head view that are invariant across identities, and proceeds to an intermediate level of representation in the fusiform face area in which identity is emerging but still entangled with head view. Three-dimensional, view-invariant representation of identities in the rIFFA may be the critical link to the extended system for face perception, affording activation of person knowledge and emotional responses to familiar faces.

The specification of imprints in mammals.

At the heart of genomic imprinting in mammals are imprinting control regions (ICRs), which are the discrete genetic elements that confer imprinted monoallelic expression to several genes in imprinted gene clusters. A characteristic of the known ICRs is that they acquire different epigenetic states, exemplified by differences in DNA methylation, in the sperm and egg, and these imprint marks remain on the sperm- and oocyte-derived alleles into the next generation as a lifelong memory of parental origin. Although there has been much focus on gametic marking of ICRs as the point of imprint specification, recent mechanistic studies and genome-wide DNA methylation profiling do not support the existence of a specific imprinting machinery in germ cells. Rather, ICRs are part of more widespread methylation events that occur during gametogenesis. Instead, a decisive component in the specification of imprints is the choice of which sites of gamete-derived methylation to maintain in the zygote and preimplantation embryo at a time when much of the remainder of the genome is being demethylated. Among the factors involved in this selection, the zinc-finger protein Zfp57 can be regarded as an imprint-specific, sequence-specific DNA binding factor responsible for maintaining methylation at most ICRs. The recent insights into the balance of gametic and zygotic contributions to imprint specification should help understand mechanistic opportunities and constraints on the evolution of imprinting in mammals.

The relationship between post-traumatic stress disorder and gastrointestinal disease in United States Military Veterans.

Background: This study examined the relationship between gastrointestinal disease and post-traumatic stress disorder in U.S. military Veterans. Based on literature and clinical practice data sources from the U.S. Veterans Administration, gastrointestinal disease and post-traumatic stress disorder were hypothesized to be positively correlated in Veterans. Objectives: This study aimed to determine the frequency with which gastrointestinal disease and post-traumatic stress disorder are diagnosed comorbidities, a diagnosis of gastrointestinal disease accompanies a diagnosis of post-traumatic stress disorder, and a diagnosis of post-traumatic stress disorder accompanies a diagnosis of a gastrointestinal disease. Methods: The methodology was a retrospective, correlational design using data collected from the U.S. Department of Veterans Affairs patient database. Results: The results were that post-traumatic stress disorder is bi-directionally correlated with the gastrointestinal diseases of gastroesophageal reflux disease, peptic ulcer disease, functional dyspepsia, Crohn's disease, diverticular disease, irritable bowel syndrome, and the symptoms of constipation and nausea/vomiting within Veterans who served during wartime periods. The study also found that post-traumatic stress disorder is not correlated with ulcerative colitis in Veterans. Conclusions: The conclusions are that clinicians who see a presentation of post-traumatic stress disorder should be screening for gastrointestinal disease, while primary care and gastroenterology providers treating gastrointestinal disease should be screening for a history of trauma, as improved diagnosis rates may lead to improved treatment.

A Pro-Regenerative Supramolecular Prodrug Protects Against and Repairs Colon Damage in Experimental Colitis.

Structural repair of the intestinal epithelium is strongly correlated with disease remission in inflammatory bowel disease (IBD); however, ulcer healing is not addressed by existing therapies. To address this need, this study reports the use of a small molecule prolyl hydroxylase (PHD) inhibitor (DPCA) to upregulate hypoxia-inducible factor one-alpha (HIF-1α) and induce mammalian regeneration. Sustained delivery of DPCA is achieved through subcutaneous injections of a supramolecular hydrogel, formed through the self-assembly of PEG-DPCA conjugates. Pre-treatment of mice with PEG-DPCA is shown to protect mice from epithelial erosion and symptoms of dextran sodium sulfate (DSS)-induced colitis. Surprisingly, a single subcutaneous dose of PEG-DPCA, administered after disease onset, leads to accelerated weight gain and complete restoration of healthy tissue architecture in colitic mice. Rapid DPCA-induced restoration of the intestinal barrier is likely orchestrated by increased expression of HIF-1α and associated targets leading to an epithelial-to-mesenchymal transition. Further investigation of DPCA as a potential adjunctive or stand-alone restorative treatment to combat active IBD is warranted.

Cannabinoid CB2 receptors in primary sensory neurons are implicated in CB2 agonist-mediated suppression of paclitaxel-induced neuropathic nociception and sexually-dimorphic sparing of morphine tolerance.

Cannabinoid CB2 agonists show therapeutic efficacy without unwanted CB1-mediated side effects. The G protein-biased CB2 receptor agonist LY2828360 attenuates the maintenance of chemotherapy-induced neuropathic nociception in male mice and blocks development of morphine tolerance in this model. However, the cell types involved in this phenomenon are unknown and whether this therapeutic profile is observed in female mice has never been investigated. We used conditional deletion of CB2 receptors to determine the cell population(s) mediating the anti-allodynic and morphine-sparing effects of CB2 agonists. Anti-allodynic effects of structurally distinct CB2 agonists (LY2828360 and AM1710) were present in paclitaxel-treated CB2f/f mice and in mice lacking CB2 receptors in CX3CR1 expressing microglia/macrophages (CX3CR1CRE/+; CB2f/f), but were absent in mice lacking CB2 receptors in peripheral sensory neurons (AdvillinCRE/+; CB2f/f). The morphine-sparing effect of LY28282360 occurred in a sexually-dimorphic manner, being present in male, but not female, mice. LY2828360 treatment (3 mg/kg per day i.p. x 12 days) blocked the development of morphine tolerance in male CB2f/f and CX3CR1CRE/+; CB2f/f mice with established paclitaxel-induced neuropathy but was absent in male (or female) AdvillinCRE/+; CB2f/f mice. Co-administration of morphine with a low dose of LY2828360 (0.1 mg/kg per day i.p. x 6 days) reversed morphine tolerance in paclitaxel-treated male CB2f/f mice, but not AdvillinCRE/+; CB2f/f mice of either sex. LY2828360 (3 mg/kg per day i.p. x 8 days) delayed, but did not prevent, the development of paclitaxel-induced mechanical or cold allodynia in either CB2f/f or CX3CR1CRE/+; CB2f/f mice of either sex. Our findings have potential clinical implications.

Cannabinoid CB 2 receptors in primary sensory neurons are implicated in CB 2 agonist-mediated suppression of paclitaxel-induced neuropathic nociception and sexually-dimorphic sparing of morphine tolerance.

Cannabinoid CB 2 agonists show therapeutic efficacy without the unwanted side effects commonly associated with direct activation of CB 1 receptors. The G protein-biased CB 2 receptor agonist LY2828360 attenuates the maintenance of chemotherapy-induced neuropathic nociception in male mice and blocks the development of morphine tolerance in this model. However, the specific cell types involved in this phenomenon have never been investigated and whether this therapeutic profile is observed in female mice remains poorly understood. We used conditional deletion of CB 2 receptors from specific cell populations to determine the population(s) mediating the anti-allodynic and morphine-sparing effects of CB 2 agonists. Anti-allodynic effects of structurally distinct CB 2 agonists (LY2828360 and AM1710) were present in paclitaxel-treated CB 2 f/f mice of either sex. The anti-allodynic effect of the CB 2 agonists were absent in conditional knockout (KO) mice lacking CB 2 receptors in peripheral sensory neurons (Advillin CRE/+ ; CB 2 f/f ) but preserved in mice lacking CB 2 receptors in CX3CR1 expressing microglia/macrophages (CX3CR1 CRE/+ ; CB 2 f/f ). The morphine-sparing effect of LY28282360 occurred in a sexually-dimorphic manner, being present in male mice but absent in female mice of any genotype. In mice with established paclitaxel-induced neuropathy, prior LY2828360 treatment (3 mg/kg per day i.p. x 12 days) blocked the subsequent development of morphine tolerance in male CB 2 f/f mice but was absent in male (or female) Advillin CRE/+ ; CB 2 f/f mice. LY2828360-induced sparing of morphine tolerance was preserved in male CX3CR1 CRE/+ ; CB 2 f/f mice, but this effect was not observed in female CX3CR1 CRE/+ ; CB 2 f/f mice. Similarly, co-administration of morphine with a low dose of LY2828360 (0.1 mg/kg per day i.p. x 6 days) reversed tolerance to the anti-allodynic efficacy of morphine in paclitaxel-treated male CB 2 f/f mice, but this effect was absent in female CB 2 f/f mice and Advillin CRE/+ ; CB 2 f/f mice of either sex. Additionally, LY2828360 (3 mg/kg per day i.p. x 8 days) delayed, but did not prevent, the development of paclitaxel-induced mechanical and cold allodynia in either CB 2 f/f or CX3CR1 CRE/+ ; CB 2 f/f mice of either sex. Our studies reveal that CB 2 receptors in primary sensory neurons are required for the anti-allodynic effects of CB 2 agonists in a mouse model of paclitaxel-induced neuropathic nociception. We also find that CB 2 agonists acting on primary sensory neurons produce a sexually-dimorphic sparing of morphine tolerance in males, but not female, paclitaxel-treated mice.

ACR Appropriateness Criteria® Hydronephrosis on Prior Imaging-Unknown Cause.

Initial imaging evaluation of hydronephrosis of unknown etiology is a complex subject and is dependent on clinical context. In asymptomatic patients, it is often best conducted via CT urography (CTU) without and with contrast, MR urography (MRU) without and with contrast, or scintigraphic evaluation with mercaptoacetyltriglycine (MAG3) imaging. For symptomatic patients, CTU without and with contrast, MRU without and with contrast, MAG3 scintigraphy, or ultrasound of the kidneys and bladder with Doppler imaging are all viable initial imaging studies. In asymptomatic pregnant patients, nonionizing imaging with US of the kidneys and bladder with Doppler imaging is preferred. Similarly, in symptomatic pregnant patients, US of the kidneys and bladder with Doppler imaging or MRU without contrast is the imaging study of choice, as both ionizing radiation and gadolinium contrast are avoided in pregnancy. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision process support the systematic analysis of the medical literature from peer reviewed journals. Established methodology principles such as Grading of Recommendations Assessment, Development, and Evaluation or GRADE are adapted to evaluate the evidence. The RAND/UCLA Appropriateness Method User Manual provides the methodology to determine the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where peer reviewed literature is lacking or equivocal, experts may be the primary evidentiary source available to formulate a recommendation.

Inhibition of nuclear factor kappa B activation in early-stage chronic lymphocytic leukemia by omega-3 fatty acids.

Targeting the nuclear factor kappa B (NFκB) pathway is proposed as therapy for chronic lymphocytic leukemia (CLL). We hypothesized that an omega-3 fatty acids (n-3) supplement would suppress NFκB activation in lymphocytes of Rai Stage 0-1 CLL patients. The initial dose of 2.4 g n-3/day was gradually increased to 7.2 g n-3/day. After n-3 consumption: 1) plasma n-3 increased; 2) NFκB activation was suppressed in lymphocytes; 3) in vitro sensitivity of lymphocytes to doxorubicin was increased; and 4) expression of 32 genes in lymphocytes was significantly decreased.

Disseminated toxoplasmosis after allogeneic stem cell transplantation in a seronegative recipient.

Toxoplasmosis is increasingly diagnosed after hematopoietic stem cell transplantation (HSCT) and is associated with considerable morbidity and mortality. In the majority of cases, reactivation of latent disease secondary to impaired cellular and humoral immunity after HSCT is believed to be the main pathogenetic mechanism. Hence, primary toxoplasmosis is rarely considered in the differential diagnosis of infections after HSCT in a recipient who is seronegative for Toxoplasma gondii pre-transplant. We herein report a seronegative patient with acute T-cell lymphoblastic leukemia, who developed primary disseminated toxoplasmosis 5 months after HSCT from a seronegative unrelated donor. A review of all reported cases of primary toxoplasmosis after HSCT revealed significantly increased morbidity and mortality. Patients with negative pre-transplant Toxoplasma serology should therefore be considered at risk for toxoplasmosis after allogeneic HSCT. Possible prevention and monitoring strategies for seronegative recipients are reviewed and discussed in detail.

Deletion of a silencer element in Igf2 results in loss of imprinting independent of H19.

Igf2 and H19 are closely linked, reciprocally imprinted genes on mouse distal chromosome 7. The paternally expressed Igf2 encodes a potent fetal growth factor and the maternally expressed H19 encodes a non-coding RNA (refs 1,2). Shared endoderm-specific enhancers 3' to H19 are necessary for transcription of the maternal copy of H19 and the paternal copy of Igf2 (ref. 3), a chromatin boundary upstream of H19 preventing access of the enhancers to the maternal Igf2 promoters. Mesoderm-specific control elements have not been identified, and the role of differentially methylated regions (DMRs) in Igf2 has not been addressed. Two DMRs in Igf2 are methylated on the active paternal allele, suggesting that they contain silencers. Here we have deleted the DMR1 region in Igf2. Maternal transmission of the deletion results in biallelic expression of Igf2 in most mesodermally derived tissues without altering H19 imprinting or expression. Paternal or maternal transmission leads to continued postnatal transcription of Igf2, in contrast to the wild-type allele, which is silenced soon after birth. These results reveal a mesodermal silencer, which may be regulated by methylation and which has a major role in H19-independent expression and imprinting control of Igf2. Our results establish a new mechanistic principle for imprinted genes whereby epigenetically regulated silencers interact with enhancers to control expression, and suggest a new mechanism for loss of imprinting (LOI) of Igf2, which may be important in a number of diseases.

Measuring motivation to change in community mental health: A Program Evaluation.

A program evaluation of a community outpatient mental health program was conducted as part of a required course in Spalding University's School of Professional Psychology. Specifically, the program evaluation examined how the client-specific factors of motivation to change were related to attendance and participation in services. Motivation to change was assessed by surveying program participants utilizing the University of Rhode Island Change Assessment (URICA). Students hypothesized stage of change would be significantly correlated with attendance rates. Results indicate stage of change was related to attendance, with pre-contemplation scores negatively associated with attendance although most of the specific analyses were not statistically significant. Students' experiences and feedback regarding conducting the program evaluation are discussed.

Scalable and Chromatography-Free Synthesis of Efflux Pump Inhibitor Phenylalanine Arginine ß-Naphthylamide for Its Validation in Wild-Type Bacterial Strains.

Phenylalanine arginine ß-naphthylamine, or PAßN, is a C-terminus capped dipeptide discovered in 1999 as an RND-type efflux pump inhibitor (EPI). Since then, PAßN has become a standard tool compound in EPI research and development. Despite this, PAßN lacks a detailed or efficient synthesis, and standard parameters for its use in wild-type bacterial strains are inconsistent or non-existent. Therefore, a scalable and chromatography-free synthesis of PAßN was developed using streamlined traditional solution-phase peptide coupling chemistry. With this procedure, gram scale quantities of PAßN were synthesized alongside analogues and stereoisomers to build a focused library to evaluate simple structure activity relationships. While most analogues were less active than the broadly utilized L,L-PAßN itself, we identified that its enantiomer, D,D-PAßN, also provided 8- to 16-fold potentiation of the antibiotic levofloxacin at 40 to 50 µg/mL concentrations of EPI in various wild-type Pseudomonas aeruginosa strains. Additionally, D,D-PAßN was shown to be significantly more hydrolytically stable than L,L-PAßN, indicating that it may be a useful, and now readily synthesized, tool compound facilitating future EPI research.

Conditional deletion of CB2 cannabinoid receptors from peripheral sensory neurons eliminates CB2-mediated antinociceptive efficacy in a mouse model of carrageenan-induced inflammatory pain.

CB2 cannabinoid receptor agonists suppress pathological pain in animal models and lack unwanted side effects commonly associated with direct activation of CB1 receptors. However, the types of pain most responsive to CB2 agonists are incompletely understood and cell types which underlie CB2-mediated therapeutic efficacy remain largely unknown. We previously reported that the CB2 receptor agonist LY2828360 reduced neuropathic nociception induced by toxic challenge with chemotherapeutic and anti-retroviral agents in mice. Whether these findings generalize to models of inflammatory pain is not known. Here we show that LY2828360 (10 mg/kg i.p.) reversed the maintenance of carrageenan-induced mechanical allodynia in female mice. Anti-allodynic efficacy was fully preserved in global CB1 knock out (KO) mice but absent in CB2 KO mice. The anti-allodynic efficacy of LY2828360 was absent in conditional KO (cKO) mice lacking CB2 receptors in peripheral sensory neurons (AdvillinCRE/+; CB2f/f) and preserved in cKO mice lacking CB2 receptors in microglia/macrophages expressing C-X3-C Motif Chemokine Receptor 1 (CX3CR1CRE/+; CB2f/f). Intraplantar administration of LY2828360 (30 µg i.pl.) reversed carrageenan-induced mechanical allodynia in CB2f/f but not AdvillinCRE/+; CB2f/f mice of both sexes. Thus, CB2 receptors in peripheral sensory neurons likely underlie the therapeutic effects of LY2828360 injection in the paw. Lastly, qRT-PCR analyses revealed that LY2828360 reduced carrageenan-induced increases in IL-1ß and IL-10 mRNA in paw skin. Our results suggest that LY2828360 suppresses inflammatory nociception in mice through a neuronal CB2-dependent mechanism that requires peripheral sensory neuron CB2 receptors and suggest that the clinical applications of LY2828360 as an anti-hyperalgesic agent should be re-evaluated.

Assessing the Effect of Augmented Reality on Procedural Outcomes During Ultrasound-Guided Vascular Access.

OBJECTIVE: Augmented reality devices are increasingly accepted in health care, though most applications involve education and pre-operative planning. A novel augmented reality ultrasound application, HoloUS, was developed for the Microsoft HoloLens 2 to project real-time ultrasound images directly into the user's field of view. In this work, we assessed the effect of using HoloUS on vascular access procedural outcomes. METHODS: A single-center user study was completed with participants with (N = 22) and without (N = 12) experience performing ultrasound-guided vascular access. Users completed a venipuncture and aspiration task a total of four times: three times on study day 1, and once on study day 2 between 2 and 4 weeks later. Users were randomized to use conventional ultrasound during either their first or second task and the HoloUS application at all other times. Task completion time, numbers of needle re-directions, head adjustments and needle visualization rates were recorded. RESULTS: For expert users, task completion time was significantly faster using HoloUS (11.5 s, interquartile range [IQR] = 6.5-23.5 s vs. 18.5 s, IQR = 11.0-36.5 s; p = 0.04). The number of head adjustments was significantly lower using the HoloUS app (1.0, IQR = 0.0-1.0 vs. 3.0, IQR = 1.0-5.0; p < 0.0001). No significant differences were identified in other measured outcomes. CONCLUSION: This is the first investigation of augmented reality-based ultrasound-guided vascular access using the second-generation HoloLens. It demonstrates equivalent procedural efficiency and accuracy, with favorable usability, ergonomics and user independence when compared with traditional ultrasound techniques.