Search

Cell culture has long been essential for preclinical modeling of human development and disease. However, conventional two-dimensional (2D) cell culture fails to faithfully model the complexity found in vivo, and novel drug candidates that show promising results in 2D models often do not translate to the clinic. More recently, three-dimensional (3D) cell culture models have gained popularity owing to their greater physiological relevance to in vivo biology. In particular, 3D spheroid models are becoming widely used due to their ability to mimic solid tumors, both in architecture and gradation of nutrients distributed from the outer, proliferative layers into the inner, quiescent layers of cells. Similar to in vivo tumors, cell lines grown in 3D spheroid models tend to be more resistant to antitumor drug treatments than their 2D cultured counterparts, though distinct signaling pathways and gene targets conferring this resistance have yet to be fully explored. RNA interference (RNAi) is an effective tool to elucidate gene function and discover novel druggable targets in 2D models; however, only a few studies have successfully performed RNAi in complex 3D models to date. Here, we demonstrate efficient RNAi-mediated knockdown using "transfection-free" Dharmacon Accell siRNAs in three spheroid culture models, in the presence or absence of the extracellular matrix. This methodology has the potential to be scaled up for complex arrayed screening experiments, which may aid in the identification of novel druggable targets with greater clinical relevance than those identified in 2D experiments. © 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 3D spheroids in matrix-free ULA plates Alternate Protocol 1: Generation of Matrigel matrix-embedded 3D spheroids Alternate Protocol 2: Generation of GrowDex hydrogel-embedded 3D spheroids Basic Protocol 2: Delivery of siRNA and collection of matrix-free 3D spheroids Alternate Protocol 3: Delivery of siRNA and collection of matrix-embedded spheroids Basic Protocol 3: RNA and protein extraction from spheroids for characterization of gene knockdown., (© 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC.)

Background & Aims: Early detection of pancreatic cancer (PaC) can drastically improve survival rates. Approximately 25% of subjects with PaC have type 2 diabetes diagnosed within 3 years prior to the PaC diagnosis, suggesting that subjects with type 2 diabetes are at high risk of occult PaC. We have developed an early-detection PaC test, based on changes in 5-hydroxymethylcytosine (5hmC) signals in cell-free DNA from plasma., Methods: Blood was collected from 132 subjects with PaC and 528 noncancer subjects to generate epigenomic and genomic feature sets yielding a predictive PaC signal algorithm. The algorithm was validated in a blinded cohort composed of 102 subjects with PaC, 2048 noncancer subjects, and 1524 subjects with non-PaCs., Results: 5hmC differential profiling and additional genomic features enabled the development of a machine learning algorithm capable of distinguishing subjects with PaC from noncancer subjects with high specificity and sensitivity. The algorithm was validated with a sensitivity for early-stage (stage I/II) PaC of 68.3% (95% confidence interval [CI], 51.9%-81.9%) and an overall specificity of 96.9% (95% CI, 96.1%-97.7%)., Conclusions: The PaC detection test showed robust early-stage detection of PaC signal in the studied cohorts with varying type 2 diabetes status. This assay merits further clinical validation for the early detection of PaC in high-risk individuals., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: A novel bi-exponential method has emerged to estimate critical speed (CS) and D-prime ( D ') from a 3-min all-out test (3MT)., Objectives: To compare CS analysis methods to determine whether parameter estimations were interchangeable. Reference values and relationships with key soccer match-play variables were explored., Methods: Thirteen elite male youth (14-15 years old) players completed a 30 m shuttle run 3MT to estimate CS, D ', rate of speed decline time constant, maximal speed ( S max ), time to S max ( t max ), and fatigue index (FI), using the traditional method and bi-exponential model on average (Bi-Exp Average ) and max speed settings (Bi-Exp Max-Speed ). High-speed running (HSR) and sprinting distances and counts, and the number of accelerations were collected from two matches. Magnitude-based inferences ( p < 0.05) with smallest worthwhile change of 0.2 effect sizes were used to analyse differences. Pearson's and Spearman's correlation coefficients were used to measure associations between CS model variables and match-play parameters., Results: There were significant differences between the traditional method and both bi-exponential models for CS and D ', as well as between the bi-exponential models for all variables except t max . Using the Bi-Exp Average model, strong correlations ( r = 0.70-0.73; p < 0.05) were observed for D ' and FI with the number of standardised and individualised HSRs, respectively. With the Bi-Exp Max-Speed model, there were strong correlations ( r /ρ = 0.64-0.68; p < 0.05) between D ' and the number of standardised HSRs and sprints, and the number of individualised sprints., Conclusion: There is a lack of interchangeability between analysis methods. It appears that D ' and FI from the bi-exponential models could be associated with high-intensity actions in soccer match-play., Competing Interests: Conflict of interest and source of funding: The authors declare no conflict of interest and no source of funding., (Copyright © 2021 South African Journal of Sports Medicine.)

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

Background: Chronic obstructive pulmonary disease (COPD) is an incurable and debilitating chronic disease characterized by progressive airflow limitation associated with abnormal levels of tissue inflammation. Therefore, stem cell-based approaches to tackle the condition are currently a focus of regenerative therapies for COPD. Extracellular vesicles (EVs) released by all cell types are crucially involved in paracrine, extracellular communication. Recent advances in the field suggest that stem cell-derived EVs possess a therapeutic potential which is comparable to the cells of their origin., Methods: In this study, we assessed the potential anti-inflammatory effects of human umbilical cord mesenchymal stem cell (hUC-MSC)-derived EVs in a rat model of COPD. EVs were isolated from hUC-MSCs and characterized by the transmission electron microscope, western blotting, and nanoparticle tracking analysis. As a model of COPD, male Sprague-Dawley rats were exposed to cigarette smoke for up to 12 weeks, followed by transplantation of hUC-MSCs or application of hUC-MSC-derived EVs. Lung tissue was subjected to histological analysis using haematoxylin and eosin staining, Alcian blue-periodic acid-Schiff (AB-PAS) staining, and immunofluorescence staining. Gene expression in the lung tissue was assessed using microarray analysis. Statistical analyses were performed using GraphPad Prism 7 version 7.0 (GraphPad Software, USA). Student's t test was used to compare between 2 groups. Comparison among more than 2 groups was done using one-way analysis of variance (ANOVA). Data presented as median ± standard deviation (SD)., Results: Both transplantation of hUC-MSCs and application of EVs resulted in a reduction of peribronchial and perivascular inflammation, alveolar septal thickening associated with mononuclear inflammation, and a decreased number of goblet cells. Moreover, hUC-MSCs and EVs ameliorated the loss of alveolar septa in the emphysematous lung of COPD rats and reduced the levels of NF-κB subunit p65 in the tissue. Subsequent microarray analysis revealed that both hUC-MSCs and EVs significantly regulate multiple pathways known to be associated with COPD., Conclusions: In conclusion, we show that hUC-MSC-derived EVs effectively ameliorate by COPD-induced inflammation. Thus, EVs could serve as a new cell-free-based therapy for the treatment of COPD.

Due to the ageing population, there is a steadily increasing incidence of osteoporosis and osteoporotic fractures. As conventional pharmacological therapy options for osteoporosis are often associated with severe side effects, bone grafts are still considered the clinical gold standard. However, the availability of viable, autologous bone grafts is limited making alternative cell-based strategies a promising therapeutic alternative. Adipose-derived stem cells (ASCs) are a readily available population of mesenchymal stem/stromal cells (MSCs) that can be isolated within minimally invasive surgery. This ease of availability and their ability to undergo osteogenic differentiation makes ASCs promising candidates for cell-based therapies for bone fractures. Recent studies have suggested that both exposure to electrical fields and cultivation in 3D can positively affect osteogenic potential of MSCs. To elucidate the osteoinductive potential of a combination of these biophysical cues on ASCs, cells were embedded within anionic nanofibrillar cellulose (aNFC) hydrogels and exposed to electrical stimulation (ES) for up to 21 days. ES was applied to ASCs in 2D and 3D at a voltage of 0.1 V/cm with a duration of 0.04 ms, and a frequency of 10 Hz for 30 min per day. Exposure of ASCs to ES in 3D resulted in high alkaline phosphatase (ALP) activity and in an increased mineralisation evidenced by Alizarin Red S staining. Moreover, ES in 3D aNFC led to an increased expression of the osteogenic markers osteopontin and osteocalcin and a rearrangement and alignment of the actin cytoskeleton. Taken together, our data suggest that a combination of ES with 3D cell culture can increase the osteogenic potential of ASCs. Thus, exposure of ASCs to these biophysical cues might improve the clinical outcomes of regenerative therapies in treatment of osteoporotic fractures.

Platelets are small circulating blood cells that play essential roles in the maintenance of haemostasis via blood clotting. However, they also play critical roles in the regulation of innate immune responses. Inflammatory receptors, specifically Toll-like receptor (TLR)-4, have been reported to modify platelet reactivity. A plethora of studies have reported controversial functions of TLR4 in the modulation of platelet function using various chemotypes and preparations of its ligand, lipopolysaccharide (LPS). The method of preparation of LPS may explain these discrepancies however this is not fully understood. Hence, to determine the impact of LPS on platelet activation, we used ultrapure preparations of LPS from Escherichia coli (LPS EC ), Salmonella minnesota (LPS SM ), and Rhodobacter sphaeroides (LPS RS ) and examined their actions under diverse experimental conditions in human platelets. LPS EC did not affect platelet activation markers such as inside-out signalling to integrin α IIb β 3 or P-selectin exposure upon agonist-induced activation in platelet-rich plasma or whole blood whereas LPS SM and LPS RS inhibited platelet activation under specific conditions at supraphysiological concentrations. Overall, our data demonstrate that platelet activation is not largely influenced by any of the ultrapure LPS chemotypes used in this study on their own except under certain conditions.

Objective: To develop a resource of systematically collected, longitudinal clinical data and biospecimens for assisting in the investigation into neuromyelitis optica spectrum disorder (NMOSD) epidemiology, pathogenesis, and treatment., Methods: To illustrate its research-enabling purpose, epidemiologic patterns and disease phenotypes were assessed among enrolled subjects, including age at disease onset, annualized relapse rate (ARR), and time between the first and second attacks., Results: As of December 2017, the Collaborative International Research in Clinical and Longitudinal Experience Study (CIRCLES) had enrolled more than 1,000 participants, of whom 77.5% of the NMOSD cases and 71.7% of the controls continue in active follow-up. Consanguineous relatives of patients with NMOSD represented 43.6% of the control cohort. Of the 599 active cases with complete data, 84% were female, and 76% were anti-AQP4 seropositive. The majority were white/Caucasian (52.6%), whereas blacks/African Americans accounted for 23.5%, Hispanics/Latinos 12.4%, and Asians accounted for 9.0%. The median age at disease onset was 38.4 years, with a median ARR of 0.5. Seropositive cases were older at disease onset, more likely to be black/African American or Hispanic/Latino, and more likely to be female., Conclusions: Collectively, the CIRCLES experience to date demonstrates this study to be a useful and readily accessible resource to facilitate accelerating solutions for patients with NMOSD.

Background: The mechanisms underpinning the regenerative capabilities of mesenchymal stem cells (MSC) were originally thought to reside in their ability to recognise damaged tissue and to differentiate into specific cell types that would replace defective cells. However, recent work has shown that molecules produced by MSCs (secretome), particularly those packaged in extracellular vesicles (EVs), rather than the cells themselves are responsible for tissue repair., Methods: Here we have produced a secretome from adipose-derived mesenchymal stem cells (ADSC) that is free of exogenous molecules by incubation within a saline solution. Various in vitro models were used to evaluate the effects of the secretome on cellular processes that promote tissue regeneration. A cardiotoxin-induced skeletal muscle injury model was used to test the regenerative effects of the whole secretome or isolated extracellular vesicle fraction in vivo. This was followed by bioinformatic analysis of the components of the protein and miRNA content of the secretome and finally compared to a secretome generated from a secondary stem cell source., Results: Here we have demonstrated that the secretome from adipose-derived mesenchymal stem cells shows robust effects on cellular processes that promote tissue regeneration. Furthermore, we show that the whole ADSC secretome is capable of enhancing the rate of skeletal muscle regeneration following acute damage. We assessed the efficacy of the total secretome compared with the extracellular vesicle fraction on a number of assays that inform on tissue regeneration and demonstrate that both fractions affect different aspects of the process in vitro and in vivo. Our in vitro, in vivo, and bioinformatic results show that factors that promote regeneration are distributed both within extracellular vesicles and the soluble fraction of the secretome., Conclusions: Taken together, our study implies that extracellular vesicles and soluble molecules within ADSC secretome act in a synergistic manner to promote muscle generation.

The power of citizen science to contribute to both science and society is gaining increased recognition, particularly in physics and biology. Although there is a long history of public engagement in agriculture and food science, the term 'citizen science' has rarely been applied to these efforts. Similarly, in the emerging field of citizen science, most new citizen science projects do not focus on food or agriculture. Here, we convened thought leaders from a broad range of fields related to citizen science, agriculture, and food science to highlight key opportunities for bridging these overlapping yet disconnected communities/fields and identify ways to leverage their respective strengths. Specifically, we show that (i) citizen science projects are addressing many grand challenges facing our food systems, as outlined by the United States National Institute of Food and Agriculture, as well as broader Sustainable Development Goals set by the United Nations Development Programme, (ii) there exist emerging opportunities and unique challenges for citizen science in agriculture/food research, and (iii) the greatest opportunities for the development of citizen science projects in agriculture and food science will be gained by using the existing infrastructure and tools of Extension programmes and through the engagement of urban communities. Further, we argue there is no better time to foster greater collaboration between these fields given the trend of shrinking Extension programmes, the increasing need to apply innovative solutions to address rising demands on agricultural systems, and the exponential growth of the field of citizen science., (© 2018 The Author(s).)

A rising incidence of oropharyngeal squamous cell carcinoma (OPSCC) incidence has occurred throughout the developed world, where it has been attributed to an increasing impact of human papillomavirus (HPV) on disease etiology. This report presents the findings of a multicenter cross-sectional retrospective study aimed at determining the proportion of HPV-positive and HPV-negative OPSCC within the United Kingdom. Archival tumor tissue blocks from 1,602 patients previously diagnosed with OPSCC (2002-2011) were collated from 11 centers. HPV status was determined with three validated commercial tests to provide valid data for 1,474 cases in total. Corresponding national incidence data from the same decade were obtained from UK Cancer registries. The overall proportion of HPV + OPSCC between 2002 and 2011 was 51.8% [95% confidence interval (CI), 49.3-54.4], and this remained unchanged throughout the decade [unadjusted RR = 1.00 (95% CI, 0.99-1.02)]. However, over the same period, the incidence of OPSCC in the broader UK population underwent a 2-fold increase [age-standardized rate 2002: 2.1 (95% CI, 1.9-2.2); 2011: 4.1 (95% CI, 4.0-4.3)]. Although the number of OPSCCs diagnosed within the United Kingdom from 2002 to 2011 nearly doubled, the proportion of HPV + cases remained static at approximately 50%. Our results argue that the rapidly increasing incidence of OPSCC in the United Kingdom cannot be solely attributable to the influence of HPV. The parallel increase in HPV + and HPV - cases we documented warrants further investigation, so that appropriate future prevention strategies for both types of disease can be implemented. Cancer Res; 76(22); 6598-606. ©2016 AACR., (©2016 American Association for Cancer Research.)

Objective: The rapid worldwide rise in incidence of human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) has generated studies confirming this disease as an entity distinct from traditional OPSCC. Based on pathology, surgical studies have revealed prognosticators specific to HPV-positive OPSCC. The current AJCC/UICC staging and pathologic nodal (pN)-classification do not differentiate for survival, demonstrating the need for new, HPV-specific OPSCC staging. The objective of this study was to define a pathologic staging system specific to HPV-positive OPSCC., Methods: Data were assembled from a surgically-managed, p16-positive OPSCC cohort (any T, any N, M0) of 704 patients from five cancer centers. Analysis was performed for (a) the AJCC/UICC pathologic staging, (b) newly published clinical staging for non-surgically managed HPV-positive OPSCC, and (c) a novel, pathology-based, "HPVpath" staging system that combines features of the primary tumor and nodal metastases., Results: A combination of AJCC/UICC pT-classification and pathology-confirmed metastatic node count (⩽4 versus ⩾5) yielded three groups: stages I (pT1-T2, ⩽4 nodes), II (pT1-T2, ⩾5 nodes; pT3-T4, ⩽4 nodes), and III (pT3-T4, ⩾5 nodes), with incrementally worse prognosis (Kaplan-Meier overall survival of 90%, 84% and 48% respectively). Existing AJCC/UICC pathologic staging lacked prognostic definition. Newly published HPV-specific clinical stagings from non-surgically managed patients, although prognostic, showed lower precision for this surgically managed cohort., Conclusions: Three loco-regional "HPVpath" stages are identifiable for HPV-positive OPSCC, based on a combination of AJCC/UICC primary tumor pT-classification and metastatic node count. A workable, pathologic staging system is feasible to establish prognosis and guide adjuvant therapy decisions in surgically-managed HPV-positive OPSCC., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mesenchymal stem cells (MSCs) have been used in cell replacement therapies for connective tissue damage, but also can stimulate wound healing through paracrine activity. In order to further understand the potential use of MSCs to treat dogs with neurological disorders, this study examined the paracrine action of adipose-derived canine MSCs on neuronal and endothelial cell models. The culture-expanded MSCs exhibited a MSC phenotype according to plastic adherence, cell morphology, CD profiling and differentiation potential along mesenchymal lineages. Treating the SH-SY5Y neuronal cell line with serum-free MSC culture-conditioned medium (MSC CM) significantly increased SH-SY5Y cell proliferation (P <0.01), neurite outgrowth (P = 0.0055) and immunopositivity for the neuronal marker βIII-tubulin (P = 0.0002). Treatment of the EA.hy926 endothelial cell line with MSC CM significantly increased the rate of wound closure in endothelial cell scratch wound assays (P = 0.0409), which was associated with significantly increased endothelial cell proliferation (P <0.05) and migration (P = 0.0001). Furthermore, canine MSC CM induced endothelial tubule formation in EA.hy926 cells in a soluble basement membrane matrix. Hence, this study has demonstrated that adipose-derived canine MSC CM stimulated neuronal and endothelial cells probably through the paracrine activity of MSC-secreted factors. This supports the use of canine MSC transplants or their secreted products in the clinical treatment of dogs with neurological disorders and provides some insight into possible mechanisms of action., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: The contemporary treatment of oropharyngeal squamous cell carcinoma (SCC) is an area of debate. We report outcomes of a minimally invasive approach involving transoral laser microsurgery (TLM)., Methods: A consecutive series of patients (n = 153) undergoing primary TLM for oropharyngeal SCC from 2006 to 2013 was studied. Human papillomavirus (HPV) status was determined by p16 immunohistochemistry and high-risk HPV DNA in situ hybridization. Survival analyses were evaluated using Kaplan-Meier statistics., Results: Tumor subsites included tonsil (n = 94; 61.5%), tongue base (n = 38; 24.8%), and soft palate (n = 21; 13.7%), with the majority being American Joint Committee on Cancer (AJCC) stage III/IVa (n = 124; 81.0%) and HPV-positive (n = 101; 66.0%). Three-year overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS) were 84.5%, 91.7%, and 78.2%, respectively. HPV-positivity portended favorable oncologic outcomes. One-year gastrostomy tube (G-tube) dependency was 1.3%., Conclusion: To the best of our knowledge, this is the largest single-center TLM oropharyngeal SCC series to date. Our data suggest that TLM +/- postoperative radiotherapy (PORT) results in at least as good oncologic outcomes as chemoradiotherapy (CRT), while conferring swallowing function advantages. © 2016 Wiley Periodicals, Inc. Head Neck , 2016 © 2016 Wiley Periodicals, Inc. Head Neck 38:1263-1270, 2016., (© 2016 Wiley Periodicals, Inc.)

Colorectal cancer (CRC) is one of the top three cancer-related causes of death worldwide. FBXW7 is a known tumor-suppressor gene, commonly mutated in CRC and in a variety of other epithelial tumors. Low expression of FBXW7 is also associated with poor prognosis. Loss of FBXW7 sensitizes cancer cells to certain drugs, while making them more resistant to other types of chemotherapies. However, is not fully understood how epithelial cells within normal gut and primary tumors respond to potential cancer therapeutics. We have studied genetically engineered mice in which the fbxw7 gene is conditionally knocked-out in the intestine (fbxw7(∆G)). To further investigate the mechanism of Fbxw7-action, we grew intestinal crypts from floxed-fbxw7 (fbxw7(fl/fl)) and fbxw7(ΔG) mice, in a Matrigel-based organoid (mini-gut) culture. The fbxw7(ΔG) organoids exhibited rapid budding events in the crypt region. Furthermore, to test organoids for drug response, we exposed day 3 intestinal organoids from fbxw7(fl/fl) and fbxw7(∆G) mice, to various concentrations of 5-fluorouracil (5-FU) for 72 hours. 5-FU triggers phenotypic differences in organoids including changing shape, survival, resistance, and death. 5-FU however, rescues the drug-resistance phenotype of fbxw7(ΔG) through the induction of terminal differentiation. Our results support the hypothesis that a differentiating therapy successfully targets FBXW7-mutated CRC cells.

Objective: To examine the impact of efforts to improve nutrition on the Anangu Pitjantjatjara Yankunytjatjara (APY) Lands from 1986, especially in Mai Wiru (good food) stores., Methods: Multiple methods were employed. Literature was searched systematically. In 2012, the store-turnover method quantified dietary intake of the five APY communities that have a Mai Wiru store. The price of a standard market basket of basic foods, implementation of store nutrition policy requirements and healthy food checklists were also assessed in all seven APY community stores at intervals from 2012. Results were compared with available data from 1986., Results: Despite concerted efforts and marked achievements, including decreased intake of sugar, increased availability and affordability of healthy foods (particularly fruit and vegetables) and consequent improvement in some nutrient intakes, the overall effect has been a decrease in total diet quality since 1986. This is characterised by increased supply and intake of discretionary foods high in saturated fat, added sugar and salt, particularly sugar sweetened beverages, convenience meals and take-away foods., Conclusions: The documented improvements confirm that residing in these communities can help Aboriginal residents exert control over key aspects of their food supply. However, the overall findings reflect broader changes to the general Australian food supply, and reinforce the notion that, in the absence of supportive regulation and market intervention, adequate and sustained resources are required to improve nutrition and prevent diet-related chronic disease on the APY Lands., Implications: This study also provides insights into food supply/security issues affecting other remote communities and wider Australia., (© 2015 The Authors.)

Background. Mobile health and digital medicine technologies are becoming increasingly used by individuals with common, chronic diseases to monitor their health. Numerous devices, sensors, and apps are available to patients and consumers-some of which have been shown to lead to improved health management and health outcomes. However, no randomized controlled trials have been conducted which examine health care costs, and most have failed to provide study participants with a truly comprehensive monitoring system. Methods. We conducted a prospective randomized controlled trial of adults who had submitted a 2012 health insurance claim associated with hypertension, diabetes, and/or cardiac arrhythmia. The intervention involved receipt of one or more mobile devices that corresponded to their condition(s) (hypertension: Withings Blood Pressure Monitor; diabetes: Sanofi iBGStar Blood Glucose Meter; arrhythmia: AliveCor Mobile ECG) and an iPhone with linked tracking applications for a period of 6 months; the control group received a standard disease management program. Moreover, intervention study participants received access to an online health management system which provided participants detailed device tracking information over the course of the study. This was a monitoring system designed by leveraging collaborations with device manufacturers, a connected health leader, health care provider, and employee wellness program-making it both unique and inclusive. We hypothesized that health resource utilization with respect to health insurance claims may be influenced by the monitoring intervention. We also examined health-self management. Results & Conclusions. There was little evidence of differences in health care costs or utilization as a result of the intervention. Furthermore, we found evidence that the control and intervention groups were equivalent with respect to most health care utilization outcomes. This result suggests there are not large short-term increases or decreases in health care costs or utilization associated with monitoring chronic health conditions using mobile health or digital medicine technologies. Among secondary outcomes there was some evidence of improvement in health self-management which was characterized by a decrease in the propensity to view health status as due to chance factors in the intervention group.

Objectives/hypothesis: The incidence of human papillomavirus (HPV)-driven disease beyond the oropharynx varies greatly in the reported literature., Study Design: Case series., Methods: Two hundred twenty-one samples were strictly classified to the subsites of oral cavity, larynx, or hypopharynx at the time of primary surgery. Formalin-fixed paraffin-embedded samples were subjected to a validated, tiered, diagnostic algorithm of p16 immunohistochemistry, high-risk HPV in situ hybridization, and quantitative polymerase chain reaction for HPV E6 DNA. An additional 60 oropharyngeal cases acted as an internal biological control., Results: An incidence of 4% of HPV-driven cases was observed across the subsites outside the oropharynx compared to 70% of tumors confined within it., Conclusions: This is the first reporting of a broad range of nonoropharyngeal HPV rates using this validated diagnostic algorithm. It remains unclear whether patients with HPV-driven disease originating outside the oropharynx enjoy the same survival advantage apparent in those patients with oropharyngeal squamous cell carcinomas., Level of Evidence: 4 Laryngoscope, 124:2739-2744, 2014., (© 2014 The American Laryngological, Rhinological and Otological Society, Inc.)

The subcellular localization of WT1 is controversial and has received little attention in the epithelial tumors of salivary glands. Paraffin-embedded, surgical specimens from 80 salivary tumors were investigated by immunohistochemistry using a monoclonal, anti-WT1 antibody (6F-H2, Dako). Immunostaining was seen in 14/14 pleomorphic adenomas (PAs), 6/6 myoepitheliomas, 4/4 basal cell adenomas, 4/4 canalicular adenomas, 0/7 Warthin tumors, 0/1 oncocytoma, 1/6 acinic cell carcinomas (Cas), 0/11 mucoepidemoid Cas, 1/11 adenoid cystic Cas, 11/12 polymorphous low-grade adenocarcinomas (PLGAs), 1/2 Ca ex PA, 0/1 salivary duct Ca and 0/1 clear cell adenocarcinoma. Stained-cell subpopulations up to 90% were not uncommon in the benign tumors. Up to 80% of cells in PLGA could be stained. Staining was weak to intense and confined to the cytoplasm of preferentially non-luminal or adjacent to stroma cells. One adenoid cystic Ca showed nuclear staining. The results suggest that WT1 is often highly expressed in benign non-oncocytic salivary tumors whereas the malignant tumors show decreased expression, the exception being PLGA. The expression is usually cytoplasmic and associated with non-luminal cells. PLGA immunoreactivities could be useful in histological differential diagnosis., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Background: Cardiac arrhythmias are remarkably common and routinely go undiagnosed because they are often transient and asymptomatic. Effective diagnosis and treatment can substantially reduce the morbidity and mortality associated with cardiac arrhythmias. The Zio Patch (iRhythm Technologies, Inc, San Francisco, Calif) is a novel, single-lead electrocardiographic (ECG), lightweight, Food and Drug Administration-cleared, continuously recording ambulatory adhesive patch monitor suitable for detecting cardiac arrhythmias in patients referred for ambulatory ECG monitoring., Methods: A total of 146 patients referred for evaluation of cardiac arrhythmia underwent simultaneous ambulatory ECG recording with a conventional 24-hour Holter monitor and a 14-day adhesive patch monitor. The primary outcome of the study was to compare the detection arrhythmia events over total wear time for both devices. Arrhythmia events were defined as detection of any 1 of 6 arrhythmias, including supraventricular tachycardia, atrial fibrillation/flutter, pause greater than 3 seconds, atrioventricular block, ventricular tachycardia, or polymorphic ventricular tachycardia/ventricular fibrillation. McNemar's tests were used to compare the matched pairs of data from the Holter and the adhesive patch monitor., Results: Over the total wear time of both devices, the adhesive patch monitor detected 96 arrhythmia events compared with 61 arrhythmia events by the Holter monitor (P < .001)., Conclusions: Over the total wear time of both devices, the adhesive patch monitor detected more events than the Holter monitor. Prolonged duration monitoring for detection of arrhythmia events using single-lead, less-obtrusive, adhesive-patch monitoring platforms could replace conventional Holter monitoring in patients referred for ambulatory ECG monitoring., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: People with Parkinson's disease (PD) are at higher risk of malnutrition due to PD symptoms and pharmacotherapy side effects. When pharmacotherapy is no longer effective for symptom control, deep-brain stimulation (DBS) surgery may be considered. The aim of this study was to assess the nutritional status of people with PD who may be at higher risk of malnutrition related to unsatisfactory symptom management with optimised medical therapy., Design: This was an observational study using a convenience sample., Setting: Participants were seen during their hospital admission for their deep brain stimulation surgery., Participants: People with PD scheduled for DBS surgery were recruited from a Brisbane neurological clinic (n=15)., Measurements: The Patient-Generated Subjective Global Assessment (PG-SGA), weight, height and body composition were assessed to determine nutritional status., Results: Six participants (40%) were classified as moderately malnourished (SGA-B). Eight participants (53%) reported previous unintentional weight loss (average loss of 13%). On average, participants classified as well-nourished (SGA-A) were younger, had shorter disease durations, lower PG-SGA scores, higher body mass (BMI) and fat free mass indices (FFMI) when compared to malnourished participants (SGA-B). Five participants had previously received dietetic advice but only one in relation to unintentional weight loss., Conclusion: Malnutrition remains unrecognised and untreated in this group despite unintentional weight loss and presence of nutrition impact symptoms. Improving nutritional status prior to surgery may improve surgical outcomes.

The exceptional high mortality of lung cancer can be instigated to a high degree by late diagnosis. Despite the plethora of studies on potential molecular biomarkers for lung cancer diagnosis, very few have reached clinical implementation. In this study, we developed a panel of DNA methylation biomarkers and validated their diagnostic efficiency in bronchial washings from a large retrospective cohort. Candidate targets from previous high-throughput approaches were examined by pyrosequencing in an independent set of 48 lung tumor/normal paired. Ten promoters were selected and quantitative methylation-specific PCR (qMSP) assays were developed and used to screen 655 bronchial washings from the Liverpool Lung Project (LLP) subjects divided into training (194 cases and 214 controls) and validation (139 cases and 109 controls) sets. Three statistical models were used to select the optimal panel of markers and to evaluate the performance of the discriminatory algorithms. The final logit regression model incorporated hypermethylation at p16, TERT, WT1, and RASSF1. The performance of this 4-gene methylation signature in the validation set showed 82% sensitivity and 91% specificity. In comparison, cytology alone in this set provided 43% sensitivity at 100% specificity. The diagnostic efficiency of the panel did not show any biases with age, gender, smoking, and the presence of a nonlung neoplasm. However, sensitivity was predictably higher in central (squamous and small cell) than peripheral (adenocarcinomas) tumors, as well as in stage 2 or greater tumors. These findings clearly show the impact of DNA methylation-based assays in the diagnosis of cytologically occult lung neoplasms. A prospective trial is currently imminent in the LLP study to provide data on the enhancement of diagnostic accuracy in a clinical setting, including by additional markers., (©2012 AACR.)

Objectives: We examined the accuracy of magnetic resonance imaging in assessing thyroid cartilage and thyroid gland invasion in patients undergoing total laryngectomy for squamous cell carcinoma, by comparing histopathology results with imaging findings., Study Design: A retrospective study reviewed histology and magnetic resonance scan results for all total laryngectomies performed between 1998-2008 at University Hospital Aintree, Liverpool., Methods: Pre-operative magnetic resonance images were reviewed independently by two consultant head and neck radiologists masked to the histology; their opinions were then compared with histology findings., Results: Eighty-one magnetic resonance scans were reviewed. There were 22 laryngectomy patients with histologically verified thyroid cartilage invasion and one patient with thyroid gland invasion. There were 31 patients with apparent radiological thyroid cartilage invasion pre-operatively (with 17 false positives), giving sensitivity, specificity, and positive and negative predictive values of 64, 71, 45 and 84 per cent, respectively. On assessing thyroid gland invasion, there were nine false positive scans and no false negative scans, giving sensitivity, specificity, and positive and negative predictive values of 100, 89, 10 and 100 per cent, respectively., Conclusion: Magnetic resonance scanning over-predicts thyroid cartilage and gland invasion in patients undergoing total laryngectomy. Magnetic resonance scans have limited effectiveness in predicting thyroid cartilage invasion by squamous cell carcinoma in laryngectomy patients.

Purpose: Human papillomavirus-16 (HPV16) is the causative agent in a biologically distinct subset of oropharyngeal squamous cell carcinoma (OPSCC) with highly favorable prognosis. In clinical trials, HPV16 status is an essential inclusion or stratification parameter, highlighting the importance of accurate testing., Experimental Design: Fixed and fresh-frozen tissue from 108 OPSCC cases were subject to eight possible assay/assay combinations: p16 immunohistochemistry (p16 IHC); in situ hybridization for high-risk HPV (HR HPV ISH); quantitative PCR (qPCR) for both viral E6 RNA (RNA qPCR) and DNA (DNA qPCR); and combinations of the above., Results: HPV16-positive OPSCC presented in younger patients (mean 7.5 years younger, P = 0.003) who smoked less than HPV-negative patients (P = 0.007). The proportion of HPV16-positive cases increased from 15% to 57% (P = 0.001) between 1988 and 2009. A combination of p16 IHC/DNA qPCR showed acceptable sensitivity (97%) and specificity (94%) compared with the RNA qPCR "gold standard", as well as being the best discriminator of favorable outcome (overall survival P = 0.002). p16 IHC/HR HPV ISH also had acceptable specificity (90%) but the substantial reduction in its sensitivity (88%) impacted upon its prognostic value (P = 0.02). p16 IHC, HR HPV ISH, or DNA qPCR was not sufficiently specific to recommend in clinical trials when used in isolation., Conclusions: Caution must be exercised in applying HPV16 diagnostic tests because of significant disparities in accuracy and prognostic value in previously published techniques., (©2011 AACR)

This article outlines a research study that sought staff views on whether they needed further training and guidance on the Mental Capacity Act 2005, and if so, on which aspects they needed more guidance. As a result, a new publication, Guidance for Adult Community Services Staff on the Mental Capacity Act 2005, was produced to give practical, easy-to-use advice. The guidance is summarised here.

Background: Fine-needle aspiration cytology (FNAC) is used to diagnose masses presenting in the head and neck region. No systematic review of FNAC in this group has yet been performed., Methods: A systematic review of the published literature and meta-analysis of data extracted from the included studies were compared with a 10-year review of head and neck FNAC from our institution., Results: Systematic review identified 30 studies; 3459 FNAC aspirates from all head and neck sites were included. Overall results were as follows: sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) were 89.6%, 96.5%, 93.1%, 96.2%, and 90.3%, respectively. Two thousand seven hundred two head and neck aspirates were included in our institutional review. Sensitivity, specificity, PPV, NPV, and accuracy were 89.5%, 98.5%, 97.3%, 94.0%, and 95.1%, respectively., Conclusion: Meta-analysis and comparative systematic review confirm that FNAC is highly effective in the diagnosis of head and neck masses, with some limitations.

Introduction: Blind percutaneous pleural biopsy has traditionally been performed to investigate the etiology of exudative pleural effusion in which the initial thoracentesis has been nondiagnostic. In view of the increasing use of image-guided and thoracoscopic pleural biopsies, this study examines the role of blind Abrams pleural biopsy in the investigation of pleural effusion in a large urban hospital., Method: Patients undergoing blind Abrams needle biopsy between January 1997 and 2003 were identified from the hospital pathology database. The case notes and pathology records of these patients were analyzed retrospectively. All patients had presented to respiratory teams with an exudative pleural effusion and had initial nondiagnostic thoracentesis., Results: Seventy-five patients undergoing blind biopsy were identified. Pleural tissue was obtained in 59 biopsies (79%), with no statistically significant difference in pleural yield between respiratory specialist registrars (equivalent to pulmonary fellows in training) and senior house officers/preregistration house officers (equivalent to junior residents and interns, respectively) performing the biopsy (chi(2) test, p = 0.43). When up to three samples were obtained per episode, sufficient pleural tissue was obtained in 18 of 25 patients (72%) compared to 80% (32 of 40 patients) in whom four to six samples were taken (chi(2) test, p = 0.55 [not significant]). For all diagnoses, blind biopsy had a sensitivity of 38%, which rose to 43% when reviewing patients in whom sufficient pleural tissue was obtained (for malignant diagnosis alone, sensitivity values were 43% and 51%, respectively; specificity, 100%; negative and positive predictive values, 51%). No fatalities were reported, and pneumothorax was seen in eight patients (11%), with only two patients requiring specific intervention., Conclusions: Blind Abrams needle biopsy obtaining pleural tissue was diagnostic in approximately 50% of patients presenting with malignant effusion in the sample, and can be performed safely by all grades of medical staff with due attention to technique and supervision. The data support the continued use of the Abrams needle in the investigation of malignant pleural disease.

To study the effects of grapefruit and grapefruit products on body weight and metabolic syndrome, 91 obese patients were randomized to either placebo capsules and 7 ounces (207 mL) of apple juice, grapefruit capsules with 7 ounces (207 mL) of apple juice, 8 ounces (237 mL) of grapefruit juice with placebo capsule, or half of a fresh grapefruit with a placebo capsule three times a day before each meal. Metabolic syndrome parameters were measured at the beginning and end of 12 weeks. After 12 weeks, the fresh grapefruit group had lost 1.6 kg, the grapefruit juice group had lost 1.5 kg, the grapefruit capsule group had lost 1.1 kg, and the placebo group had lost 0.3 kg. The fresh grapefruit group lost significantly more weight than the placebo group (P < .05). A secondary analysis of those with the metabolic syndrome in the four treatment groups demonstrated a significantly greater weight loss in the grapefruit, grapefruit capsule, and grapefruit juice groups compared with placebo (P < .02). There was also a significant reduction in 2-hour post-glucose insulin level in the grapefruit group compared with placebo. Half of a fresh grapefruit eaten before meals was associated with significant weight loss. In metabolic syndrome patients the effect was also seen with grapefruit products. Insulin resistance was improved with fresh grapefruit. Although the mechanism of this weight loss is unknown it would appear reasonable to include grapefruit in a weight reduction diet.

Purpose: To report our surgical technique for LASIK enhancement by ablating the underside of the flap., Methods: Surgical case report of clinical outcomes in three eyes of two patients who had an initial laser ablation in the bed with subsequent enhancements on the underside of the flap., Results: Following LASIK enhancement on the back of the flap, two eyes gained one line of best spectacle-corrected visual acuity (BSCVA) after treatment compared to preoperatively, going from 20/15 to 20/12, and one eye maintained BSCVA at 20/20., Conclusions: Excimer laser ablation of the underside of the flap is a viable alternative to treat corneas with minimal residual tissue whether primary or enhancement procedures are needed. A surgical learning curve exists with this technique.

A 41-year-old man presented to his general practitioner (GP) with a wheeze and dyspnoea on exercise. Asthma was diagnosed and treatment with inhaled corticosteroid and a beta(2)-agonist commenced. Despite this, his condition deteriorated over three weeks culminating in stridor, requiring emergency admission to hospital. Nasendoscopy revealed a polypoidal lesion in the upper trachea, acting like a ball valve. A local anaesthetic tracheostomy, secured his airway. Subsequent direct laryngoscopy allowed avulsion of the lesion. Alternative methods of airway management are discussed. Histology revealed a paraganglioma. The aetiology of paragangliomas is described and a literature review of tracheal paraganglioma is presented. Post-operative recovery was unremarkable. However, tumour recurrence occurred at nine months. A subsequent revision tracheostomy and laser resection has ensured disease-free survival, one year later. We recommend that acute onset wheeze, refractive to appropriate therapy, should be referred for urgent examination of the upper aerodigestive tract.

The effectiveness of multidisciplinary stroke rehabilitation may be enhanced by nurses and therapists adopting a single consistent approach to the positioning and mobilizing of patients. As patients can spend as little as 4% of the waking day receiving 'therapy' there is considerable potential for a more dynamic nursing intervention, which may contribute to improving patient care. We aimed to investigate whether physiotherapists could step back from direct patient treatment in order to participate in a structured training programme for nurses involved with patients recovering from stroke on an established elderly care rehabilitation ward in a district general hospital. Qualitative methods were used within a participatory action research framework to describe the development process and content of the training programme. Nursing staff, physiotherapists and their respective managers were interviewed to identify perceived training needs. This informed the structure and content of the training course and allowed insight into interprofessional working.

Aim: to assess the effects of a physiotherapist-led stroke training programme for nurses working in a rehabilitation ward on clinical practice and patient outcome., Method: before and after group comparison with outcome assessment by observational and quantitative methods. Non-participant observation before and after the 5-month training programme recorded patient position, transfers and contact with nursing staff. Quantitative assessments of disability, satisfaction and mood were made at baseline, discharge and 4 months after stroke onset. We also noted selected stroke complications, rehabilitation ward length of stay and discharge destination., Results: there was a significant improvement in the number of observed 'good' transfers of patients undertaken by nurses (chi2 = 9.13, df = 1, P = 0.003) but the training programme had no impact on the time the patients spent in 'poor' positions. There was no significant difference between the two groups for Barthel index scores at discharge and at 4 months. Neither was there any significant difference in the Hospital Anxiety and Depression scale, occurrence of secondary complications, length of stay or the Patient and Carer Satisfaction Questionnaires., Conclusion: within the limitations of the research design adopted, some improvements in clinical practice were reported but there were no significant differences in patient outcome. The training programme required no additional resources and should be replicable in most district general hospitals.

Background: A multidisciplinary project team developed a five-month physiotherapist-led training programme designed to enhance nurses' rehabilitation skills., Aims: Investigation of the effects of the training programme on nurses attitudes to patients with stroke., Method: An attitude questionnaire was completed by the participating nurses before and after the training programme. In a complementary qualitative evaluation the nurses' expectations and views of the training programme were explored., Results: Analysis of the attitude questionnaire indicated that the programme was successful in positively influencing the nurses' attitudes (median of the change in score 2, p = 0.005). The qualitative interviews reflected a similar outcome., Conclusions: Quantitative and qualitative evaluation indicates that the training programme had some effect in changing nurses' attitudes to treating patients after a stroke. Such a training programme could be a useful contribution to post-stroke care.

Background: When hormones are detected in the serum of patients with bronchial carcinoma they are generally considered to originate from the tumour, but this may be not the only explanation. Pulmonary endocrine cells proliferate in lungs affected by non-neoplastic disease and their products are often demonstrable in the serum. The aim of this study was to examine the pulmonary endocrine systems of a series of tumour-bearing lungs to see whether any changes in them could possibly account for raised levels of pulmonary peptides in the blood., Methods: The morphology, number, distribution, and content of pulmonary endocrine cells in 30 pairs of tumour-bearing lungs from patients coming to necropsy with bronchial carcinoma were examined. These features were related to the pathology of the tumour and to other pathological changes present in the lungs, and compared with pulmonary endocrine cells in 10 pairs of control lungs from patients without pulmonary disease., Results: Increased numbers of endocrine cells, often in the form of large abnormal aggregates, were present in 17 pairs of tumour-bearing lungs where they were associated not with the tumour but with non-tumoral pathology, especially inflammation and changes associated with cardiac failure. Appropriate and inappropriate peptides were identified within them., Conclusion: The possibility is raised that, in some subjects with bronchial carcinoma who have raised serum hormone levels, the source of these substances might be the endocrine cells in the diseased lung around the tumour.

Natural background activity and food chain transfer of the uranium decay products, 210Po and 210Pb, were examined in the lichen-caribou-wolf food chain at two locations in the Northwest Territories of Canada. 210Po and 210Pb activities in lichens differed with species and location. Both 210Po and 210Pb were markedly higher in caribou bone than in wolf bone. 210Po activities in liver, kidney, and muscle were similar in both species. Caribou fetuses had lower activities of 210Po but higher activities of 210Pb than maternal muscle and placenta, suggesting greater placental transport of 210Pb than 210Po. Concentration ratios (CR = Bq kg-1 in consumer/Bq kg-1 in its food source) and ff values (ff in d kg-1 = Bq kg-1 in muscle/Bq d-1 ingested) showed that wolves retain more 210Po and less 210Pb from their diet than do caribou. 210Po CRs averaged 0.38 for caribou/lichens, 0.26 for caribou/rumen contents, and 0.40 for wolves/caribou. 210Pb CRs averaged 0.36 for caribou/lichens, 0.57 for caribou/rumen contents, and 0.13 for wolves/caribou.

We describe a case of cardiac amyloidosis, which is an uncommon cause of heart failure. This case is unusual, as the patient presented with symptoms of angina in the presence of normal coronary arteries and subsequently developed heart failure. Amyloidosis was secondary to myeloma with Bence-Jones proteinuria alone, which is rare.

A single induced sputum sample using hypertonic saline and a ward nebulizer was obtained from 24/26 (92%) patients prior to fibreoptic bronchoscopy for suspected bronchial carcinoma. In 11 of the 19 patients later found to have bronchial carcinoma, positive cytology was found in the induced sputum sample (58% sensitivity), with no false positives (100% specificity). These results are higher than those reported for ordinary sputum samples. They suggest a possible role for this technique in diagnosing bronchial carcinoma in elderly patients in whom bronchoscopy may be considered hazardous, or where this facility is not readily available.

Five patients over the age of 40 with malignant mesothelioma of the pleura presented with a spontaneous pneumothorax in the course of five years. The diagnosis of malignant mesothelioma was not suspected at surgery but was made by histological examination of the pleurectomy specimens. During this time 91 pleurectomies for recurrent pneumothorax were performed, 45 in patients over the age of 40; malignant mesothelioma therefore accounted for 11% of spontaneous pneumothorax requiring pleurectomy in this age group. The association of spontaneous pneumothorax and malignant mesothelioma is not emphasised in current publications. These five cases highlight the need for all pleurectomy specimens in cases of spontaneous pneumothorax to be sent for histological examination and for a full occupational history to be taken, especially in older patients.