Role of protein arginine methyltransferase 5 in group 3 (MYC-driven) Medulloblastoma.

BACKGROUND: MYC amplification or overexpression is common in Group 3 medulloblastoma and is associated with the worst prognosis. Recently, protein arginine methyl transferase (PRMT) 5 expression has been closely associated with aberrant MYC function in various cancers, including brain tumors such as glioblastoma. However, the role of PRMT5 and its association with MYC in medulloblastoma have not been explored. Here, we report the role of PRMT5 as a novel regulator of MYC and implicate PRMT5 as a potential therapeutic target in MYC-driven medulloblastoma. METHODS: Expression and association between PRMT5 and MYC in primary medulloblastoma tumors were investigated using publicly available databases. Expression levels of PRMT5 protein were also examined using medulloblastoma cell lines and primary tumors by western blotting and immunohistochemistry, respectively. Using MYC-driven medulloblastoma cells, we examined the physical interaction between PRMT5 and MYC by co-immunoprecipitation and co-localization experiments. To determine the functional role of PRMT5 in MYC-driven medulloblastoma, PRMT5 was knocked-down in MYC-amplified cells using siRNA and the consequences of knockdown on cell growth and MYC expression/stability were investigated. In vitro therapeutic potential of PRMT5 in medulloblastoma was also evaluated using a small molecule inhibitor, EPZ015666. RESULTS: We observed overexpression of PRMT5 in MYC-driven primary medulloblastoma tumors and cell lines compared to non-MYC medulloblastoma tumors and adjacent normal tissues. We also found that high expression of PRMT5 is inversely correlated with patient survival. Knockdown of PRMT5 using siRNA in MYC-driven medulloblastoma cells significantly decreased cell growth and MYC expression. Mechanistically, we found that PRMT5 physically associated with MYC by direct protein-protein interaction. In addition, a cycloheximide chase experiment showed that PRMT5 post-translationally regulated MYC stability. In the context of therapeutics, we observed dose-dependent efficacy of PRMT5 inhibitor EPZ015666 in suppressing cell growth and inducing apoptosis in MYC-driven medulloblastoma cells. Further, the expression levels of PRMT5 and MYC protein were downregulated upon EPZ015666 treatment. We also observed a superior efficacy of this inhibitor against MYC-amplified medulloblastoma cells compared to non-MYC-amplified medulloblastoma cells, indicating specificity. CONCLUSION: Our results reveal the regulation of MYC oncoprotein by PRMT5 and suggest that targeting PRMT5 could be a potential therapeutic strategy for MYC-driven medulloblastoma.

Street-level diplomacy and local enforcement for meat safety in northern Tanzania: knowledge, pragmatism and trust.

BACKGROUND: With increasing demand for red meat in Tanzania comes heightened potential for zoonotic infections in animals and humans that disproportionately affect poor communities. A range of frontline government employees work to protect public health, providing services for people engaged in animal-based livelihoods (livestock owners and butchers), and enforcing meat safety and food premises standards. In contrast to literature which emphasises the inadequacy of extension support and food safety policy implementation in low- and middle-income countries, this paper foregrounds the 'street-level diplomacy' deployed by frontline actors operating in challenging contexts. METHODS: This research is based on semi-structured interviews with 61 government employees, including livestock extension officers/meat inspectors and health officers, across 10 randomly-selected rural and urban wards. RESULTS: Frontline actors combined formal and informal strategies including the leveraging of formal policy texts and relationships with other state employees, remaining flexible and recognising that poverty constrained people's ability to comply with health regulations. They emphasised the need to work with livestock keepers and butchers to build their knowledge to self-regulate and to work collaboratively to ensure meat safety. Remaining adaptive and being hesitant to act punitively unless absolutely necessary cultivated trust and positive relations, making those engaged in animal-based livelihoods more open to learning from and cooperating with extension officers and inspectors. This may result in higher levels of meat safety than might be the case if frontline actors stringently enforced regulations. CONCLUSION: The current tendency to view frontline actors' partial enforcement of meat safety regulations as a failure obscures the creative and proactive ways in which they seek to ensure meat safety in a context of limited resources. Their application of 'street-level diplomacy' enables them to be sensitive to local socio-economic realities, to respect local social norms and expectations and to build support for health safety interventions when necessary. More explicitly acknowledging the role of trust and positive state-society relations and the diplomatic skills deployed by frontline actors as a formal part of their inspection duties offers new perspectives and enhanced understandings on the complicated nature of their work and what might be done to support them.

The modified Memorial Symptom Assessment Scale Short Form: a modified response format and rational scoring rules.

PURPOSE: The Memorial Symptom Assessment Scale Short Form (MSAS-SF) is a widely used symptom assessment instrument. Patients who self-complete the MSAS-SF have difficulty following the two-part response format, resulting in incorrectly completed responses. We describe modifications to the response format to improve useability, and rational scoring rules for incorrectly completed items. METHODS: The modified MSAS-SF was completed by 311 women in our Peer and Nurse support Trial to Assist women in Gynaecological Oncology; the PeNTAGOn study. Descriptive statistics were used to summarise completion of the modified MSAS-SF, and provide symptom statistics before and after applying the rational scoring rules. Spearman's correlations with the Functional Assessment for Cancer Therapy-General (FACT-G) and Hospital Anxiety and Depression Scale (HADS) were assessed. RESULTS: Correct completion of the modified MSAS-SF items ranged from 91.5 to 98.7%. The rational scoring rules increased the percentage of useable responses on average 4% across all symptoms. MSAS-SF item statistics were similar with and without the scoring rules. The pattern of correlations with FACT-G and HADS was compatible with prior research. CONCLUSION: The modified MSAS-SF was useable for self-completion and responses demonstrated validity. The rational scoring rules can minimise loss of data from incorrectly completed responses. Further investigation is recommended.

Examination of the recommended safe and unsafe zone for placement of surgical instruments in thoracentesis and video-assisted thoracic surgery: a cadaveric study.

BACKGROUND: Thoracentesis and video-assisted thoracic surgery procedures can result in haemorrhage as a consequence of severing the collateral branches of the posterior intercostal artery. These branches have been shown to be most common in the 5th intercostal space (ICS). Tortuosity has been shown to be especially prevalent nearer to midline. A group of investigators have recommended the 4th and 7th ICS, 120 mm lateral to midline as a safe zone, least likely to hit branches when cutting into the ICS. The present study aimed to investigate that safe zone as a better entry points for procedures. In addition, investigation of the least safe 5th ICS was also performed. MATERIALS AND METHODS: A total of 56 embalmed human cadavers were selected for the study. With the cadavers laid prone, 2 cm incisions were made at the 4th, 5th and 7th ICS, 120 mm lateral to midline bilaterally. The cadavers were then placed supine and the incisions were dissected. Careful attention was paid to identify if any collateral branches were cut. RESULTS: After thorough dissection of the 4th, 5th and 7th ICS incision sites, it was shown that damage to the 5th intercostal was seen most frequently. CONCLUSIONS: Based on this cadaveric study, a 2 cm incision at the 4th, 5th and 7th ICS 120 mm lateral from midline resulted in the most damage at the level of the 5th ICS. The 4th ICS had the least damage seen. Therefore, it is recommended that insertion should be placed at the level of the 4th ICS bilaterally.

High-frequency acousto-optic effects in Bragg reflectors.

Picosecond acoustic interferometry was used to study the acousto-optic properties of a distributed Bragg reflector (DBR) manufactured from two immiscible polymers (cellulose acetate and polyvinylcarbyzole). Picosecond strain pulses were injected into the structure and changes in its reflectance were monitored as a function of time. The reflectance exhibited single-frequency harmonic oscillations as the strain pulse traversed the DBR. A transfer matrix method was used to model the reflectance of the DBR in response to interface modulation and photo-elastic effects. This work shows that photo-elastic effects can account for the acousto-optic response of DBRs with acoustically matched layers.

Origin of contact line forces during the retraction of dilute polymer solution drops.

The forced dewetting of water and dilute poly(ethylene oxide) solution (PEO) drops is investigated for syringe-driven flow. Comparisons are made with the free dewetting observed during drop impact. We provide strong evidence that during droplet retraction, polymer deposited on the substrate results in a velocity-dependent force at the contact line. These findings are in stark contrast to previous studies which attributed dissipation to bulk viscoelastic effects or normal stress effects at the contact line.

Uncertainty and urban water recharge for managing groundwater availability using decision support.

Quantifying groundwater availability depends upon sound methods and the use of integrated models. To determine availability or sustainable yield, the influence of scientific uncertainty from key sources, such as anthropogenic recharge, must be considered. This study evaluates uncertainty in recharge interpretations on the modeled available water balance for an urban case in Texas, USA. Analyses are completed using the Groundwater Decision Support System, which is a research code-base for an integrated modeling. The case study develops spatially and temporally resolved recharge interpretations based on NEXRAD precipitation and detailed land use data. Results demonstrate the implications of scientific uncertainty as it influences recommendations for policy and urban water management decisions that are based on modeled outputs. Geospatial methods account for spatial and temporal components and can be replicated for other systems. These methods are also useful for resolving uncertainty in relation to the influence of urbanization on recharge through land use change.

Improving secondary ion mass spectrometry C60(n+) sputter depth profiling of challenging polymers with nitric oxide gas dosing.

Organic depth profiling using secondary ion mass spectrometry (SIMS) provides valuable information about the three-dimensional distribution of organic molecules. However, for a range of materials, commonly used cluster ion beams such as C60(n+) do not yield useful depth profiles. A promising solution to this problem is offered by the use of nitric oxide (NO) gas dosing during sputtering to reduce molecular cross-linking. In this study a C60(2+) ion beam is used to depth profile a polystyrene film. By systematically varying NO pressure and sample temperature, we evaluate their combined effect on organic depth profiling. Profiles are also acquired from a multilayered polystyrene and polyvinylpyrrolidone film and from a polystyrene/polymethylmethacrylate bilayer, in the former case by using an optimized set of conditions for C60(2+) and, for comparison, an Ar2000(+) ion beam. Our results show a dramatic improvement for depth profiling with C60(2+) using NO at pressures above 10(-6) mbar and sample temperatures below -75 °C. For the multilayered polymer film, the depth profile acquired using C60(2+) exhibits high signal stability with the exception of an initial signal loss transient and thus allows for successful chemical identification of each of the six layers. The results demonstrate that NO dosing can significantly improve SIMS depth profiling analysis for certain organic materials that are difficult to analyze with C60(n+) sputtering using conventional approaches/conditions. While the analytical capability is not as good as large gas cluster ion beams, NO dosing comprises a useful low-cost alternative for instruments equipped with C60(n+) sputtering.

Genomic variations associated with attenuation in Mycobacterium avium subsp. paratuberculosis vaccine strains.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) whole cell vaccines have been widely used tools in the control of Johne's disease in animals despite being unable to provide complete protection. Current vaccine strains derive from stocks created many decades ago; however their genotypes, underlying mechanisms and relative degree of their attenuation are largely unknown. RESULTS: Using mouse virulence studies we confirm that MAP vaccine strains 316 F, II and 2e have diverse but clearly attenuated survival and persistence characteristics compared with wild type strains. Using a pan genomic microarray we characterise the genomic variations in a panel of vaccine strains sourced from stocks spanning over 40 years of maintenance. We describe multiple genomic variations specific for individual vaccine stocks in both deletion (26-32 Kbp) and tandem duplicated (11-40 Kbp) large variable genomic islands and insertion sequence copy numbers. We show individual differences suitable for diagnostic differentiation between vaccine and wild type genotypes and provide evidence for functionality of some of the deleted MAP-specific genes and their possible relation to attenuation. CONCLUSIONS: This study shows how culture environments have influenced MAP genome diversity resulting in large tandem genomic duplications, deletions and transposable element activity. In combination with classical selective systematic subculture this has led to fixation of specific MAP genomic alterations in some vaccine strain lineages which link the resulting attenuated phenotypes with deficiencies in high reactive oxygen species handling.

Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice.

Targeted mutagenesis in mice, a powerful tool for the analysis of gene function and human disease, makes extensive use of 129 mouse substrains. Although all are named 129, we document that outcrossing of these substrains, both deliberate and accidental, has lead to extensive genetic variability among substrains and embryonic stem cells derived from them. This clearer understanding of 129 substrain variability allows consideration of its negative impact on targeting technology, including: homologous recombination frequencies, preparation of inbred animals, and availability of appropriate controls. Based on these considerations we suggest a number of recommendations for future experimental design.

The neuronal ceroid lipofuscinoses in human EPMR and mnd mutant mice are associated with mutations in CLN8.

The neuronal ceroid lipofuscinoses (NCLs) are a genetically heterogeneous group of progressive neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in various tissues. Progressive epilepsy with mental retardation (EPMR, MIM 600143) was recently recognized as a new NCL subtype (CLN8). It is an autosomal recessive disorder characterized by onset of generalized seizures between 5 and 10 years, and subsequent progressive mental retardation. Here we report the positional cloning of a novel gene, CLN8, which is mutated in EPMR. It encodes a putative transmembrane protein. EPMR patients were homozygous for a missense mutation (70C-->G, R24G) that was not found in homozygosity in 433 controls. We also cloned the mouse Cln8 sequence. It displays 82% nucleotide identity with CLN8, conservation of the codon harbouring the human mutation and is localized to the same region as the motor neuron degeneration mouse, mnd, a naturally occurring mouse NCL (ref. 4). In mnd/mnd mice, we identified a homozygous 1-bp insertion (267-268insC, codon 90) predicting a frameshift and a truncated protein. Our data demonstrate that mutations in these orthologous genes underlie NCL phenotypes in human and mouse, and represent the first description of the molecular basis of a naturally occurring animal model for NCL.

Novel therapy for therapy-resistant mantle cell lymphoma: multipronged approach with targeting of hedgehog signaling.

Mantle cell lymphoma (MCL) is one of the most aggressive B-cell lymphomas with a median patient survival of only 5-7 years. The failure of existing therapies is mainly due to disease relapse when therapy-resistant tumor cells remain after chemotherapy. Therefore, development and testing of novel therapeutic strategies to target these therapy-resistant MCL are needed. Here, we developed an in vivo model of therapy-resistant MCL by transplanting a patient-derived MCL cell line (Granta 519) into NOD/SCID mice followed by treatment with combination chemotherapy. Cytomorphologic, immunophenotypic, in vitro and in vivo growth analyses of these therapy-resistant MCL cells confirm their MCL origin and resistance to chemotherapy. Moreover, quantitative real-time PCR revealed the upregulation of GLI transcription factors, which are mediators of the hedgehog signaling pathway, in these therapy-resistant MCL cells. Therefore, we developed an effective therapeutic strategy for resistant MCL by treating the NOD/SCID mice bearing Granta 519 MCL with CHOP chemotherapy to reduce tumor burden combined with GLI-antisense oligonucleotides or bortezomib, a proteosome inhibitor, to target therapy-resistant MCL cells that remained after chemotherapy. This regimen was followed by treatment with MCL-specific cytotoxic T lymphocytes to eliminate all detectable leftover minimal residual disease. Mice treated with this strategy showed a significantly increased survival and decreased tumor burden compared to the mice in all other groups. Such therapeutic strategies that combine chemotherapy with targeted therapy followed by tumor-specific immunotherapy are effective and have excellent potential for clinical application to provide long-term, disease-free survival in MCL patients.

Functional convergence of microbes associated with temperate marine sponges.

Most marine sponges establish a persistent association with a wide array of phylogenetically and physiologically diverse microbes. To date, the role of these symbiotic microbial communities in the metabolism and nutrient cycles of the sponge-microbe consortium remains largely unknown. We identified and quantified the microbial communities associated with three common Mediterranean sponge species, Dysidea avara, Agelas oroides and Chondrosia reniformis (Demospongiae) that cohabitate coralligenous community. For each sponge we quantified the uptake and release of dissolved organic carbon (DOC) and nitrogen (DON), inorganic nitrogen and phosphate. Low microbial abundance and no evidence for DOC uptake or nitrification were found for D. avara. In contrast A. oroides and C. reniformis showed high microbial abundance (30% and 70% of their tissue occupied by microbes respectively) and both species exhibited high nitrification and high DOC and NH(4) (+) uptake. Surprisingly, these unique metabolic pathways were mediated in each sponge species by a different, and host specific, microbial community. The functional convergence of microbial consortia found in these two sympatric sponge species, suggest that these metabolic processes may be of special relevance to the success of the holobiont.

Concentration dependence of upconversion emission from Er:YAG fibers.

Fibers of Er:YAG have been grown using the laser-heated pedestal growth method. Fibers with Er concentrations ranging from 0.5 to 10 mol. % have been produced and excitation at 965 nm has been used to produce intense upconversion emission at green and red wavelengths. The dependence of ground state absorption and upconversion emission on dopant concentration has been studied and shown to exhibit lower levels of self-absorption than previously reported. At Er concentrations of 5 mol. % and above; however, the variation of upconversion emission intensity with pump power deviates from theoretical predictions, exhibiting saturation behavior consistent with concentration quenching.

Use of CTA Test Dose to Trigger a Low Cardiac Output Protocol Improves Acute Stroke CTP Data Analyzed with RAPID Software.

BACKGROUND AND PURPOSE: Contrast curve truncation in CTP protocols may introduce errors. We sought to identify risk factors and design a protocol to avoid truncation while limiting radiation. MATERIALS AND METHODS: In an initial fixed-timing cohort, patients underwent a 65-second CTP with 2-second delay postcontrast injection. Multivariable analysis identified factors associated with truncation. A later case-specific cohort underwent either the original protocol or a low cardiac output protocol with a 7-second delay and 75-second scanning window, with selection determined by CTA test-dose enhancement upswing delay. Time-density curves were assessed for truncation and compared between the 2 groups, and the radiation dose was evaluated. RESULTS: From September 2017 through May 2018, one hundred fifty-three patients underwent the standard fixed-timing protocol. Age (OR, 1.82/10-year increase; P = .019), reduced left ventricle ejection fraction (OR, 9.23; P = .001), and hypertension (OR, 0.32; P = .06) were independently associated with truncation in an exploratory multivariable model. From May 2018 through April 2019, one hundred fifty-seven patients underwent either the standard (72 patients) or low cardiac output protocol (85 patients). The fixed-timing cohort had 15 truncations (9.8%) versus 4 in the case-specific cohort (2.5%; P = .009). If the low cardiac output protocol were applied to those with >10.6% predicted risk of truncation based on age, left ventricle ejection fraction, and hypertension, the number of truncations would have decreased from 15 to 4 in the fixed-timing cohort. CONCLUSIONS: Older age, left ventricle ejection fraction, and the absence of hypertension increase the risk of time-density curve truncation. However, a CTA test-dose-directed case-specific protocol can reduce truncation to ensure accurate data while mitigating radiation dose increases.

High-concentration Er:YAG single-crystal fibers grown by laser-heated pedestal growth technique.

High-concentration Er:YAG single-crystal fibers have been grown using the laser-heated pedestal growth technique. Instability in the melt and concomitant opacity of fibers were observed at source concentrations higher than 15 mol.%. Spectroscopic examination shows that broadening of the linewidth of the 4I(13/2) → 4I(15/2) transition is strongly dependent on Er³âº concentration.

Effects of substrate constraint on crack pattern formation in thin films of colloidal polystyrene particles.

Crack formation and the evolution of stress in drying films of colloidal particles were studied using optical microscopy and a modified cantilever deflection technique, respectively. Drying experiments were performed using polystyrene particles with diameters of 47 ± 10 nm, 100 ± 16 nm, and 274 ± 44 nm that were suspended in water. As the films dried, cracks with a well-defined spacing were observed to form. The crack spacing was found to be independent of the particle size used, but to increase with the film thickness. The characteristic crack spacing was found to vary between 20 and 300 µm for films with thickness values in the range 3-70 µm. Cantilever deflection measurements revealed that the stresses that develop in the film increase with decreasing film thickness (increasing surface-to-volume ratio). The latter observation was interpreted in terms of the effects of a substrate constraint which causes the build up of stresses in the films. This interpretation was confirmed by crack formation experiments that were performed on liquid mercury surfaces in which removal of the substrate constraint prevented crack formation. Experiments were also performed on compliant elastomer surfaces in which the level of constraint was varied by changing the substrate modulus. The cracking length scale was found to increase with decreasing substrate modulus. A simple theory was also developed to describe the substrate modulus dependence of the cracking length scale. These combined experiments and theory provide convincing evidence that substrate constraints are an important factor in driving crack formation in thin colloidal films.

Referrals to a liaison psychiatry outpatient clinic.

This study aimed to describe the demographic and clinical characteristics of consecutive referrals to a liaison psychiatry outpatient clinic, based within a National Health Service (NHS) general hospital. All outpatient referrals to the service between January 2005 and January 2008 were subjected to retrospective completion of a proforma for post hoc data collection. Data relating to characteristics of the referral, patients, diagnosis, and nature and extent of contact were extracted for scrutiny. The majority (60.2%) of referrals to the service came from physicians based within the general hospital. Where conveyed, depression (26.7%) was the most commonly cited reason for referral. Diagnoses of both mood (affective) disorders (32.9%) and neurotic, stress-related and somatoform disorders (54.7%) were common. Cognitive behaviour therapy (56.1%) was the most commonly used intervention followed by pharmacological therapy (25.4%). In conclusion, the study provides a description of the nature of the work encountered in a liaison psychiatry setting and demonstrates the extent of services provided for medical specialties within the general hospital.

Evaluation of intranasal nafamostat or camostat for SARS-CoV-2 chemoprophylaxis in Syrian golden hamsters.

Successful development of a chemoprophylaxis against SARS-CoV-2 could provide a tool for infection prevention implementable alongside vaccination programmes. Camostat and nafamostat are serine protease inhibitors that inhibit SARS-CoV-2 viral entry in vitro but have not been characterised for chemoprophylaxis in animal models. Clinically, nafamostat is limited to intravenous delivery and while camostat is orally available, both drugs have extremely short plasma half-lives. This study sought to determine whether intranasal dosing at 5 mg/kg twice daily was able to prevent airborne transmission of SARS-CoV-2 from infected to uninfected Syrian golden hamsters. SARS-CoV-2 viral RNA was above the limits of quantification in both saline- and camostat-treated hamsters 5 days after cohabitation with a SARS-CoV-2 inoculated hamster. However, intranasal nafamostat-treated hamsters remained RNA negative for the full 7 days of cohabitation. Changes in body weight over the course of the experiment were supportive of a lack of clinical symptomology in nafamostat-treated but not saline- or camostat-treated animals. These data are strongly supportive of the utility of intranasally delivered nafamostat for prevention of SARS-CoV-2 infection and further studies are underway to confirm absence of pulmonary infection and pathological changes.

Thin film polymer photonics: Spin cast distributed Bragg reflectors and chirped polymer structures.

Polymer based photonic structures were produced by spin coating up to 50 alternating layers of polystyrene (PS) and poly(vinylpyrrolidone) (PVP) from mutually exclusive (orthogonal) solvents. The resulting thin film multi-layer structures were studied using a simple optical reflectivity apparatus and were shown to have narrow (10-20nm wide) reflectance bands in the visible region. The position of the reflectance bands was controlled by varying the spin speed used during production of the multi-layers and peak reflectance values of 55% were obtained for samples containing 50 layers. The results were shown to be in agreement with modified optical transfer matrix method calculations which include the effects of diffuse polymer interfaces. This modelling approach revealed that the width of the polymer/polymer interfaces formed by spin coating was in the range 15-20nm. Data and calculations were also obtained for chirped polymer photonic structures. These results were also shown to be in good agreement. These experiments demonstrate that simple processing methods such as spin coating can be used to produce organic photonic structures with tailored optical properties.