A New Systemic Disease Mouse Model for Glioblastoma Capable of Single-Tumour-Cell Detection.

BACKGROUND: Glioblastoma is characterised by extensive infiltration into the brain parenchyma, leading to inevitable tumor recurrence and therapeutic failure. Future treatments will need to target the specific biology of tumour recurrence, but our current understanding of the underlying mechanisms is limited. Significantly, there is a lack of available methods and models that are tailored to the examination of tumour recurrence. METHODS: NOD-SCID mice were orthotopically implanted with luciferase-labelled donor U87MG or MU20 glioblastoma cells. Four days later, an unlabelled recipient tumor was implanted on the contralateral side. The mice were euthanised at a humane end-point and tissue and blood samples were collected for ex vivo analyses. RESULTS: The ex vivo analyses of the firefly-labelled MU20 tumours displayed extensive invasion at the primary tumour margins, whereas the firefly-labelled U87MG tumours exhibited expansive phenotypes with no evident invasions at the tumour margins. Luciferase signals were detected in the contralateral unlabelled recipient tumours for both the U87MG and MU20 tumours compared to the non-implanted control brain. Remarkably, tumour cells were uniformly detected in all tissue samples of the supratentorial brain region compared to the control tissue, with single tumour cells detected in some tissue samples. Circulating tumour cells were also detected in the blood samples of most of the xenografted mice. Moreover, tumour cells were detected in the lungs of all of the mice, a probable event related to haematogenous dissemination. Similar results were obtained when the U87MG cells were alternatively labelled with gaussian luciferase. CONCLUSIONS: These findings describe a systemic disease model for glioblastoma which can be used to investigate recurrence biology and therapeutic efficacy towards recurrence.

Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer.

Cellobiose dehydrogenase (CDH) is a bioelectrocatalyst that enables direct electron transfer (DET) in biosensors and biofuel cells. The application of this bidomain hemoflavoenzyme for physiological glucose measurements is limited by its acidic pH optimum and slow interdomain electron transfer (IET) at pH 7.5. The reason for this rate-limiting electron transfer step is electrostatic repulsion at the interface between the catalytic dehydrogenase domain and the electron mediating cytochrome domain (CYT). We applied rational interface engineering to accelerate the IET for the pH prevailing in blood or interstitial fluid. Phylogenetic and structural analyses guided the design of 17 variants in which acidic amino acids were mutated at the CYT domain. Five mutations (G71K, D160K, Q174K, D177K, M180K) increased the pH optimum and IET rate. Structure-based analysis of the variants suggested two mechanisms explaining the improvements: electrostatic steering and stabilization of the closed state by hydrogen bonding. Combining the mutations into six combinatorial variants with up to five mutations shifted the pH optimum from 4.5 to 7.0 and increased the IET at pH 7.5 over 12-fold from 0.1 to 1.24 s-1 . While the mutants sustained a high enzymatic activity and even surpassed the IET of the wild-type enzyme, the accumulated positive charges on the CYT domain decreased DET, highlighting the importance of CYT for IET and DET. This study shows that interface engineering is an effective strategy to shift the pH optimum and improve the IET of CDH, but future work needs to maintain the DET of the CYT domain for bioelectronic applications.

Dissecting the conformational complexity and mechanism of a bacterial heme transporter.

Iron-bound cyclic tetrapyrroles (hemes) are redox-active cofactors in bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here, we used cellular, biochemical, structural and computational methods to characterize the structure and function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a heme transporter required for functional maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryogenic-electron microscopy approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to rotate 180°.

Electrochemical alkene azidocyanation via 1,4-nitrile migration.

An electrochemical method for the azidocyanation of alkenes via 1,4-nitrile migration has been developed. This organic oxidant free method is applicable across various alkene containing cyanohydrins, and provides access to a broad range of synthetically useful 1,2-azidonitriles (28 examples). This methodology was extended to an electrochemical alkene sulfonylcyanation procedure, as well as to access a trifunctionalized hexanenitrile from a malononitrile starting material. The orthogonal derivatization of the products was also demonstrated through chemoselective transformations.

Prosthetic Management of Severe Mandibular Deviation in a Hemi-Mandibulectomy Patient Following Osteoradionecrosis.

Rehabilitation of a patient after hemi-mandibulectomy without reconstruction represents a prosthodontic challenge. Indeed, mandibular deviation and decreased occlusal contacts are a common presentation post-surgery. This paper reports on a patient who presented with these challenges and where chronic osteoradionecrosis has resulted in significant mandibular deviation. A maxillary cobalt chrome mandibular deviation device, designed with palatal bite plane and constructed using 3D printing methods, resulted in a successful outcome. The authors aim to show how restorative management of similar patients can be successful using a modern approach.

Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications.

Cellobiose dehydrogenase (CDH) is an attractive oxidoreductase for bioelectrochemical applications. Its two-domain structure allows the flavoheme enzyme to establish direct electron transfer to biosensor and biofuel cell electrodes. Yet, the application of CDH in these devices is impeded by its limited stability under turnover conditions. In this work, we aimed to improve the turnover stability of CDH by semirational, high-throughput enzyme engineering. We screened 13 736 colonies in a 96-well plate setup for improved turnover stability and selected 11 improved variants. Measures were taken to increase the reproducibility and robustness of the screening setup, and the statistical evaluation demonstrates the validity of the procedure. The selected CDH variants were expressed in shaking flasks and characterized in detail by biochemical and electrochemical methods. Two mechanisms contributing to turnover stability were found: (i) replacement of methionine side chains prone to oxidative damage and (ii) the reduction of oxygen reactivity achieved by an improved balance of the individual reaction rates in the two CDH domains. The engineered CDH variants hold promise for the application in continuous biosensors or biofuel cells, while the deduced mechanistic insights serve as a basis for future enzyme engineering approaches addressing the turnover stability of oxidoreductases in general.

Swiss neonatal caregivers express diverging views on parental involvement in shared decision-making for extremely premature infants.

AIM: Due to scarce available national data, this study assessed current attitudes of neonatal caregivers regarding decisions on life-sustaining interventions, and their views on parents' aptitude to express their infant's best interest in shared decision-making. METHODS: Self-administered web-based quantitative empirical survey. All 552 experienced neonatal physicians and nurses from all Swiss NICUs were eligible. RESULTS: There was a high degree of agreement between physicians and nurses (response rates 79% and 70%, respectively) that the ability for social interactions was a minimal criterion for an acceptable quality of life. A majority stated that the parents' interests are as important as the child's best interest in shared decision-making. Only a minority considered the parents as the best judges of what is their child's best interest. Significant differences in attitudes and values emerged between neonatal physicians and nurses. The language area was very strongly associated with the attitudes of neonatal caregivers. CONCLUSION: Despite clear legal requirements and societal expectations for shared decision-making, survey respondents demonstrated a gap between their expressed commitment to shared decision-making and their view on parental aptitude to formulate their infant's best interest. National guidelines need to address these barriers to shared decision-making to promote a more uniform nationwide practice.

Live Cell Imaging of the TGF- ß/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System.

Transforming Growth Factor ß (TGF-ß) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-ß is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-ß/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-ß displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-ß signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-ß signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Exposure to brominated and organophosphate ester flame retardants in U.S. childcare environments: Effect of removal of flame-retarded nap mats on indoor levels.

We assessed exposure to 39 brominated and 16 organophosphate ester flame retardants (FRs) from both dust and indoor air at seven childcare centres in Seattle, USA, and investigated the importance of nap mats as a source of these chemicals. Many childcare centres serving young children use polyurethane foam mats for the children's naptime. Until recently, the vast majority of these mats sold in the United States contained flame-retarded polyurethane foam to meet California Technical Bulletin 117 (TB117) requirements. With the 2013 update of TB117, allowing manufacturers to meet flammability standards without adding FRs to filling materials, FR-free nap mats have become widely available. We conducted an intervention study by actively switching out FR-treated nap mats with FR-free nap mats and measuring FR levels in indoor air and dust before and after the switch-out. The predominant FRs found in dust and indoor air were 2-ethylhexyl tetrabromobenzoate (EHTBB) and tris(1-chloro-2-propyl) phosphate (TCIPP), respectively. Nap mat samples analysed from four of the six centres contained a Firemaster® mixture, while one mat was predominantly treated with tris(1,3-dichloroisopropyl) phosphate (TDCIPP) and the other contained no detectable target FRs. After replacement, there was a significant decrease (p = 0.03-0.09) in median dust concentrations for bis(2-ethylhexyl) tetrabromophthalate (BEHTBP), EHTBB, tris(4-butylphenyl) phosphate (TBPP), and TDCIPP with reductions of 90%, 79%, 65%, and 42%, respectively. These findings suggest that the nap mats were an important source of these FRs to dust in the investigated childcare environments and that a campaign of swapping out flame-retarded mats for FR-free ones would reduce exposure to these chemicals. While calculated exposure estimates to the investigated FRs via inhalation, dust ingestion, and dermal absorption were below established reference dose values, they are likely underestimated when considering the toddlers' direct contact to the mats and personal cloud effects.

Demographic and dietary risk factors in relation to urinary metabolites of organophosphate flame retardants in toddlers.

Organophosphate flame retardants (OPFRs), including Tris (1,3-dichloro-isopropyl) phosphate (TDCPP), triphenyl phosphate (TPP), and isopropylated triphenyl phosphate (ITP), are increasingly used in consumer products because of the recent phase out of polybrominated diphenyl ether (PBDE) flame retardants. OPFRs have been widely detected in adults and have been linked to reproductive and endocrine changes in adult males. Carcinogenicity and damage to immunologic, neurologic and developmental systems have been observed in human cell lines. Young children are especially vulnerable to OPFR exposure, but little is known about exposure levels or exposure risk factors in this population. We examined parent-reported demographic and dietary survey data in relation to OPFR urinary metabolite concentrations in 15- to 18-month old toddlers (n = 41). OPFR metabolites were detected in 100% of subjects. The metabolite of TPP, diphenyl phosphate (DPP) was detected most commonly (100%), with TDCPP metabolite, bis(1,3-dichloro-2-propyl) phosphate (BDCPP), detected in 85-95% of samples, and ITP metabolite, monoisopropylphenyl phenyl phosphate (ip-DPP), detected in 81% of samples (n = 21). Toddlers of mothers earning <$10,000 annually had geometric mean DPP concentrations 66% higher (p = 0.05) than toddlers of mothers earning >$10,000/year (7.8 ng/mL, 95% CI 5.03, 12.11 and 4.69 ng/mL, 95% CI 3.65-6.04, respectively). While no dietary factors were significantly associated with OPFR metabolite concentrations, results suggested meat and fish consumption may be associated with higher DPP and BDCPP levels while increased dairy and fresh food consumption may be associated with lower DPP, BDCPP, and ip-DPP levels. Research with larger sample sizes and more detailed dietary data is required to confirm these preliminary findings.

Implant retention systems for implant-retained overdentures.

Implant retained overdentures are being increasingly utilised in both general and specialist practice to rehabilitate patients with missing teeth, particularly those that are edentate. This article aims to inform the reader of a variety of retention systems that are available to retain an implant overdenture and to understand how these systems work, their advantages and disadvantages and to outline some of the clinical and treatment planning considerations involved in selecting the most appropriate retention system for patients.

A practitioner's guide to gutta-percha removal during endodontic retreatment.

Endodontic retreatment can be a challenging task that can result in many complications if not approached cautiously. Many of these difficulties revolve around regaining access to the pulp chamber through extensive coronal restorations and removing residual root filling material, the commonest being gutta-percha (GP), from within obturated canals. This can often be an untidy, time consuming process that places teeth at a greater risk of iatrogenic injury and inhibits the operator achieving the necessary chemical disinfection required to eliminate the persistent apical disease. Therefore the following article aims to aid practitioners, particularly those who are unfamiliar, with accessing and removing GP from endodontically treated teeth. The outlined systematic approach is accessible in general practice, where the vast majority of endodontic treatment is conducted, requires basic equipment and with the correct experience can be applied to both straight and curved canals. By overcoming this initial stage of retreatment, subsequent chemical disinfection, which is critical to success, can be carried out to a higher standard reducing risks of re-infection.

A survey of dental practitioners in Wales to evaluate the management of deep carious lesions with vital pulp therapy in permanent teeth.

Objective To evaluate the management of deep carious lesions with vital pulp therapy in permanent teeth by dental practitioners within Wales.Design Postal questionnaire.Setting General practitioners (GDS), community (CDS) and hospital-based dentists (HDS) in Wales.Methods Community and hospital dental services with a remit for provision of restorative dentistry (CDS = 71; HDS = 46) and general dental practitioners (N = 510) were approached regarding their management of deep carious lesions with vital pulp therapy in permanent teeth. The postal questionnaire took the form of an anonymous survey. Questions covered usage parameters, training issues and reasons for material choice.Results The response rate was 29%. The majority of HDS (89%) used MTA or Biodentine for vital pulp therapy in contrast to GDS (41%) and CDS (32%). The main reasons cited for avoiding the use of MTA or Biodentine included cost, lack of training and difficulty in material handling.Conclusion Usage of MTA or Biodentine for vital pulp therapies is low in the general dental and community dental settings. Cost and lack of training are the main barriers for the uptake of these materials. Postgraduate training may be useful in addressing these barriers. Increasing their adoption would be advantageous as they have been shown to produce a more predictable outcome compared to traditional materials (for example, calcium hydroxide).

The restorative management of microdontia.

Microdontia is a dental abnormality that will often present to the dental practitioner due to the aesthetic concerns of the patient. Treatment is therefore aimed at addressing the aesthetics issue of the patient and this can present a number of challenges which may require a multidisciplinary approach in its management. This article presents the restorative management of localised and generalised microdontia.

Phase I study of PF-03446962, a fully human monoclonal antibody against activin receptor-like kinase-1, in patients with hepatocellular carcinoma.

BACKGROUND: This expansion cohort of a multicenter, dose-escalation, phase I study (NCT00557856) evaluated safety, tolerability, antitumor activity, pharmacokinetics, and pharmacodynamic effects of the anti-activin receptor-like kinase-1 (ALK-1) monoclonal antibody PF-03446962 in advanced hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Patients with HCC and disease progression after prior antiangiogenic therapy or intolerance to treatment received PF-03446962 7 mg/kg intravenously biweekly, as recommended in the dose-escalation part of the study. RESULTS: Twenty-four patients received PF-03446962. The most frequent treatment-related adverse events (AEs) were thrombocytopenia (33.3%), asthenia (29.2), and chills (16.7%). Two patients experienced treatment-related telangiectasia, suggesting an in vivo knockout of ALK-1 function through ALK-1 pathway inhibition. Overall, treatment-related grade 3-4 AEs were reported in eight patients (33.3%). Treatment-related grade 3-4 thrombocytopenia was noted in four patients. No complete or partial responses were reported. Twelve (50%) patients achieved stable disease, which lasted ≥12 weeks in seven (29.2%) patients. The median time to progression was 3 months. Biomarker analyses showed higher mean tumor expression of c-tumor mesenchymal-epithelial transition factor and higher mean serum levels of bone morphogenetic protein-9 in patients with disease control (DC) for ≥12 weeks versus patients with disease progression. Conversely, lower mean serum transforming growth factor-ß and vascular endothelial growth factor receptor-3 levels were detected in patients with DC versus patients with progression. CONCLUSIONS: The observed safety, tolerability, pharmacokinetic profile, and clinical activity support further evaluation of PF-03446962 in patients with HCC and other solid malignancies, as single agent or in combination with other antiangiogenic, chemotherapeutic, or immunotherapeutic agents. TRIAL REGISTRATION NUMBER: NCT00557856.

Malaria transmission potential could be reduced with current and future climate change.

Several studies suggest the potential for climate change to increase malaria incidence in cooler, marginal transmission environments. However, the effect of increasing temperature in warmer regions where conditions currently support endemic transmission has received less attention. We investigate how increases in temperature from optimal conditions (27 °C to 30 °C and 33 °C) interact with realistic diurnal temperature ranges (DTR: ± 0 °C, 3 °C, and 4.5 °C) to affect the ability of key vector species from Africa and Asia (Anopheles gambiae and An. stephensi) to transmit the human malaria parasite, Plasmodium falciparum. The effects of increasing temperature and DTR on parasite prevalence, parasite intensity, and mosquito mortality decreased overall vectorial capacity for both mosquito species. Increases of 3 °C from 27 °C reduced vectorial capacity by 51-89% depending on species and DTR, with increases in DTR alone potentially halving transmission. At 33 °C, transmission potential was further reduced for An. stephensi and blocked completely in An. gambiae. These results suggest that small shifts in temperature could play a substantial role in malaria transmission dynamics, yet few empirical or modeling studies consider such effects. They further suggest that rather than increase risk, current and future warming could reduce transmission potential in existing high transmission settings.

Inhibition of glioblastoma cell proliferation, migration and invasion by the proteasome antagonist carfilzomib.

Glioblastoma multiforme is the most aggressive and lethal tumor of the central nervous system with limited treatment strategies on offer, and as such the identification of effective novel therapeutic agents is paramount. To examine the efficacy of proteasome inhibitors, we tested bortezomib, carfilzomib, nafamostat mesylate, gabexate mesylate and acetylsalicylic acid on glioblastoma cell viability, migration and invasion. Both bortezomib and carfilzomib produced significant reduction of cell viability, while nafamostat mesylate, gabexate mesylate and acetylsalicylic acid did not. Subsequent testing showed that carfilzomib significantly reduced cell viability at nM concentrations. Carfilzomib also reduced cell migration, secretion and activation of MMP2 and also cell invasion of all four glioblastoma cells tested. In summary, carfilzomib represents a novel, yet FDA-approved agent for the treatment of glioblastoma multiforme.

Mouse models of glioma.

Gliomas are the most common primary tumour in the central nervous system in adults. The pathological hallmark of gliomas is their propensity for extensive infiltration into the surrounding brain parenchyma which results in tumour recurrence. Despite the use of optimal surgical removal and adjuvant therapies the most aggressive of these tumours, glioblastoma multiforme, has a poor patient prognosis, with median survival of less than 15 months. In this review, we discuss mouse glioma models that have been utilised to advance our basic knowledge of the processes involved in gliomagenesis and their use in the testing of novel therapies and treatment regimens in the preclinical setting.

Implications of temperature variation for malaria parasite development across Africa.

Temperature is an important determinant of malaria transmission. Recent work has shown that mosquito and parasite biology are influenced not only by average temperature, but also by the extent of the daily temperature variation. Here we examine how parasite development within the mosquito (Extrinsic Incubation Period) is expected to vary over time and space depending on the diurnal temperature range and baseline mean temperature in Kenya and across Africa. Our results show that under cool conditions, the typical approach of using mean monthly temperatures alone to characterize the transmission environment will underestimate parasite development. In contrast, under warmer conditions, the use of mean temperatures will overestimate development. Qualitatively similar patterns hold using both outdoor and indoor temperatures. These findings have important implications for defining malaria risk. Furthermore, understanding the influence of daily temperature dynamics could provide new insights into ectotherm ecology both now and in response to future climate change.