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Background Iontophoresis passes electrical charge through skin to deliver drugs or reduce excessive sweating. Treatments can be performed by patients at home following initial instruction. A limitation of the technique is that patients are not permitted to have metal implants. These are hypothesized to increase the risk of electric shock, cause localized heating and/or corrosion. Objectives To investigate whether metallic materials (titanium, stainless steel and copper) placed in the iontophoresis circuit would lead to an unfavourable outcome regarding corrosion or local heating of the metallic object. Methods This was carried out using mass loss and temperature change experiments, together with atomic force microscopy for stainless steel, to assess any surface roughness changes. The investigations were carried out under accelerated conditions (70 V compared with standard use 20–30 V). Results No changes in mass or clinically significant changes in temperature of any of the metallic objects (or surface roughness for stainless steel) were observed. Conclusions This study suggests that patients with these metallic implants can safely undergo iontophoresis treatment. Further work is needed to review the impact on metallic implants with repeated exposure to the iontophoresis system to represent real-world evidence.

Background and Aims Outcomes in ANCA vasculitis remain difficult to predict and therapeutic decision-making can be challenging. We aimed to establish if a renal risk score (RRS) could predict outcomes in this population. Method The Scottish Renal Biopsy Registry is a complete national dataset of all renal biopsies performed in Scotland. Those who had a first renal biopsy between 01/01/2014 and 31/12/2017 with evidence of ANCA vasculitis were included. Demographic data, treatment regimens, episodes of relapse and patient and kidney survival were recorded, retrospectively. The RRS was calculated using the system proposed by Brix et al (1). Each patient was categorised according to % of normal glomeruli (N0 >25%, N1 10 to 25%, N2 25%) and eGFR (CKD-EPI) at time of biopsy (eGFR: G0 >15 mL/min/1.73 m2, G1 ≤15 mL/min/1.73 m2). Individual scores were summated and patients defined as low, medium or high risk. Cox proportional hazard models were created for survival to ESKD, relapse and death, stratified by risk category. Analyses were conducted using R statistical software. Results Two-hundred and forty-six patients with biopsy proven ANCA vasculitis were identified. Fifty percent (n=123), 46% (n=112) and 5% (n=11) were stratified as low, medium and high risk respectively. Fifty-two percent (n=129) were male and mean age at biopsy was 66.7±12.2 years. This was similar across the risk categories. Mean eGFR was lower in the high-risk category (High risk 8.6±6.1 ‘v’ Low risk 45.7±26.0 ml/min/1.73m2, p Those categorised as medium or high risk were more likely to receive plasma exchange and/or haemodialysis at presentation (p Conclusion A simple RRS, using routinely reported data, in patients with renal biopsy proven ANCA vasculitis can help to predict development of ESKD. It may also be predictive of future relapse in those with a lower RRS, most likely explained by reduced irreversible damage in this group. The RRS could inform monitoring and treatment decisions. Whilst the numbers are small, a unique strength of this data is that it is based on a complete national dataset making it less susceptible to bias from regional variations in diagnostic and therapeutic practice.

Ureides are the N-rich products of N-fixation that are transported from soybean nodules to the shoot. Ureides are known to accumulate in leaves in response to water-deficit stress, and this has been used to identify genotypes with reduced N-fixation sensitivity to drought. Our objectives in this research were to determine shoot ureide concentrations in 374 Maturity Group IV soybean accessions and to identify genomic regions associated with shoot ureide concentration. The accessions were grown at two locations (Columbia, MO, and Stuttgart, AR) in 2 yr (2009 and 2010) and characterized for ureide concentration at beginning flowering to full bloom. Average shoot ureide concentrations across all four environments (two locations and two years) and 374 accessions ranged from 12.4 to 33.1 µmol g−1 and were comparable to previously reported values. SNP–ureide associations within and across the four environments were assessed using 33,957 SNPs with a MAF ≥0.03. In total, 53 putative loci on 18 chromosomes were identified as associated with ureide concentration. Two of the putative loci were located near previously reported QTL associated with ureide concentration and 30 loci were located near genes associated with ureide metabolism. The remaining putative loci were not near chromosomal regions previously associated with shoot ureide concentration and may mark new genes involved in ureide metabolism. Ultimately, confirmation of these putative loci will provide new sources of variation for use in soybean breeding programs.

Background CD15, which is overexpressed on various cancers, has been reported as a cell adhesion molecule that plays a key role in non-CNS metastasis. However, the role of CD15 in brain metastasis is largely unexplored. This study provides a better understanding of CD15/CD62E interaction, enhanced by tumor necrosis factor-α (TNF-α), and its correlation with brain metastasis in non–small cell lung cancer (NSCLC). Methods CD15 and E-selectin (CD62E) expression was demonstrated in both human primary and metastatic NSCLC cells using flow cytometry, immunofluorescence, and Western blotting. The role of CD15 was investigated using an adhesion assay under static and physiological flow live-cell conditions. Human tissue sections were examined using immunohistochemistry. Results CD15, which was weakly expressed on hCMEC/D3 human brain endothelial cells, was expressed at high levels on metastatic NSCLC cells (NCI-H1299, SEBTA-001, and SEBTA-005) and at lower levels on primary NSCLC (COR-L105 and A549) cells (P < .001). The highest expression of CD62E was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC cells followed by primary NSCLC cells. Metastatic NSCLC cells adhered most strongly to hCMEC/D3 compared with primary NSCLC cells. CD15 immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (P < .0001), confirming a correlation between CD15 and cerebral metastasis. Both CD15 and CD62E expression were detected in lung metastatic brain biopsies. Conclusion This study enhances the understanding of cancer cell-brain endothelial adhesion and confirms that CD15 plays a crucial role in adhesion in concert with TNF-α activation of its binding partner, CD62E.

Summary There is a need for physical standards (reference materials) to ensure both reproducibility and consistency in the production of somatic cell types from human pluripotent stem cell (hPSC) sources. We have outlined the need for reference materials (RMs) in relation to the unique properties and concerns surrounding hPSC-derived products and suggest in-house approaches to RM generation relevant to basic research, drug screening, and therapeutic applications. hPSCs have an unparalleled potential as a source of somatic cells for drug screening, disease modeling, and therapeutic application. Undefined variation and product variability after differentiation to the lineage or cell type of interest impede efficient translation and can obscure the evaluation of clinical safety and efficacy. Moreover, in the absence of a consistent population, data generated from in vitro studies could be unreliable and irreproducible. Efforts to devise approaches and tools that facilitate improved consistency of hPSC-derived products, both as development tools and therapeutic products, will aid translation. Standards exist in both written and physical form; however, because many unknown factors persist in the field, premature written standards could inhibit rather than promote innovation and translation. We focused on the derivation of physical standard RMs. We outline the need for RMs and assess the approaches to in-house RM generation for hPSC-derived products, a critical tool for the analysis and control of product variation that can be applied by researchers and developers. We then explore potential routes for the generation of RMs, including both cellular and noncellular materials and novel methods that might provide valuable tools to measure and account for variation. Multiparametric techniques to identify “signatures” for therapeutically relevant cell types, such as neurons and cardiomyocytes that can be derived from hPSCs, would be of significant utility, although physical RMs will be required for clinical purposes.

Background Oseltamivir, a specific influenza neuraminidase inhibitor, is an effective treatment for seasonal influenza. Emergence of drug-resistant influenza viruses after treatment has been reported, particularly in children in Japan, where the dosing schedule is different from that used throughout the rest of the world. We investigated the emergence of drug-resistant infection in children treated with a tiered weight-based dosing regimen. Methods We analyzed sequential clinical nasopharyngeal samples, obtained before and after tiered weight-based oseltamivir therapy, from children with acute influenza during 2005-2007. We isolated viruses, tested for drug resistance with use of a fluorescence-based neuraminidase inhibition assay, performed neuraminidase gene sequencing, and determined quantitative viral loads. Results Sixty-four children (34 with influenza A subtype H3N2, 11 with influenza A subtype H1N1, and 19 with influenza B virus) aged 1-12 years (median age, 3 years, 1 month) were enrolled. By days 4-7 after initiation of treatment, of 64 samples tested, 47 (73.4%) and 26 (40.6%) had virus detectable by reverse-transcriptase polymerase chain reaction and culture, respectively. By days 8-12 after initiation of treatment, of 53 samples tested, 18 (33.9%) and 1 (1.8%) had virus detectable by reverse-transcriptase polymerase chain reaction and culture, respectively. We found no statistically significant differences in the reduction of viral shedding or time to clearance of virus between viral subtypes. Antiviral-resistant viruses were recovered from 3 (27.3%) of 11 children with influenza A subtype H1N1, 1 (2.9%) of 34 children with influenza A subtype H3N2, and 0 (0%) of 19 children with influenza B virus, all of whom were treated with oseltamivir (P = .004). There was no evidence of prolonged illness in children infected with drug-resistant virus. Conclusions Drug resistance emerges at a higher rate in influenza A subtype H1N1 virus than in influenza A subtype H3N2 or influenza B virus after tiered weight-based oseltamivir therapy. Virological surveillance for patterns of drug resistance is essential for determination of antiviral treatment strategies and for composition of pandemic preparedness stockpiles.

Metformin (N,N-dimethylbiguanide) is the most widely used anti-diabetic drug to treat type II diabetes. It exhibits blood glucose lowering action by regulating glucose formation in the liver (gluconeogenesis), increases glucose utilisation in muscles and reduces glucose absorption in the intestine. Clinical studies have shown that metformin, and other biguanides (buformin and phenformin), have lowered the risk of different kinds of cancer, such as breast, lung, colon, pancreas, and liver cancers. The purpose of this project is to investigate the efficacy of metformin and other biguanides to treat glioblastoma multiforme (GBM). The first step was to produce these compounds in sufficient quantities and purity, to enable all the necessary in vitro experiments to be carried out. The work presented in this study, reports the synthesis and purification of metformin. Literature methods have been vague and/or purification methods have not been disclosed, partly for commercial reasons. Metformin hydrochloride has been prepared by refluxing a reaction mixture of dimethylamine hydrochloride and dicyandiamide using different molar ratios, different reaction solvents (water, water/methanol, ethanol, toluene, xylene, and butanol), various reaction times (2 to 48 h) and using different recrystallisation solvents. In some cases, solvent extraction was used for further purification. Solid products were analysed using a range of techniques, such as melting points, Fourier Transform Infra-red (FTIR) spectroscopy, 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography (HPLC). Impurities associated with starting materials and other known side-products, e.g., melamine, were identified. The conditions for the synthesis of pure metformin in good yield were also identified. The project is now moving on to testing the synthesised metformin on various GBM cell lines. Other biguanides, including buformin and phenformin, will also be synthesised, purified and their anti-neoplastic properties towards GBM investigated.

We examined the in vitro proliferative potential of 669 cell cultures established from skin biopsies of members of the Baltimore Longitudinal Study of Aging. The colony size distribution was used to estimate the proliferative life span of the cultures. A significant decline in proliferative potential with donor age was observed for female but not male donors. For both male and female donors, the proliferative potential was significantly greater for donors under the age of 30 years compared with all donors over the age of 30 years. In an attempt to reduce genetic heterogeneity, we examined the proliferative potential of cultures derived at different ages from the same donor. These studies revealed a trend (approaching statistical significance) toward low proliferative potential as donors aged. Interestingly, samples obtained from donors who had a history of skin cancer at the time of biopsy had a significantly lower doubling potential than those from donors who did not. The implications of these results for the use of cells derived from donors of different ages for aging research are discussed.

INTRODUCTION: CD15, a cell adhesion molecule which is overexpressed in various cancers, is thought to be a key player in non-central nervous system (CNS) metastasis. However, the role of CD15 in brain metastasis is largely unexplored. CD15, a trisaccharide adhesion molecule, is over-expressed in non-CNS metastatic cancers. The role of CD15/CD62E interaction in lung to brain metastasis is still unexplored. The aim of this study is to therefore provide a better understanding of CD15/CD62E interaction and its role in adhesion in brain metastasis. METHOD: Expression of CD62E and CD15 were evaluated in brain microvascular endothelial cells (hCMEC/D3), primary (A549 and COR-L105) and metastatic NSCLC cells (NCI-H1299, SEBTA-001 and SEBTA-005) using flow cytometry (FC), immunocytochemistry (ICC) and Western blotting (WB). Adhesion assays under static conditions and physiological flow (shear stress 2.5 dyn/cm2) conditions were conducted. Immunoblocking of CD15 was performed to investigate its role in adhesion. RESULTS: Low levels of CD15 was seen on hCMEC/D3 (19.69%) compared to metastatic NSCLC cells (NCI-H1299: 79%, SEBTA-001: 54% and SEBTA-005: 39%) and primary NSCLC (COR-L105: 31% and A549: 23%) cells (P < 0.001). CD62E expression was high in hCMEC/D3 cells activated with TNF-α. Metastatic NSCLC adhered most strongly to hCMEC/D3 compared to primary NSCLC cells. CD15 immunoblocking blunted this adhesion under static and shear stress conditions (p < 0.001), confirming the correlation between CD15 and cerebral metastasis. CONCLUSION: This study enhances understanding of lung cancer cell-brain endothelial adhesion and suggests that CD15 may play a role in adhesion in concert with TNF-α activation of its binding partner CD62E.

INTRODUCTION: CD44 is important in cellular adhesion and invasion, and is upregulated in glioma cells. Here, we extend our previous CD44-antibody blocking and gene silencing experiments to examine, the downstream effects of CD44 knockdown (kd) on the expression of cytoskeletal proteins involved in cellular migration. Atomic force microscope (AFM) was used to measure the stiffness (Young's Modulus, E) of wild type (wt) and CD44-kd glioma cells. METHOD: SNB-19 cells were transfected with either CD44 siRNA, scrambled siRNA, or a non-related gene siRNA. Human non-neoplastic astrocytes were used as a control. Western Blotting (WB), immunocytochemistry (ICC) and flow cytometric analysis were used to analyse changes in cytoskeletal proteins. AFM (CellHesion 200, JPK Instruments) was used to measure E values obtained from force vs. distance plots, using Hertzian mechanics models, by indenting a microsphere (R = 5.6 µm), attached to a AFM cantilever, into cells (10% of cell thickness, nuclear and cytoplasm regions) maintained at 37°C in culture media. RESULTS: Vimentin, GFAP, and microtubule protein expression was associated with lower CD44 protein levels in CD44-kd cells compared to cells with scrambled siRNA. Functionally, CD44-kd cells were less migratory compared to controls. AFM E-values were found to be log-normally distributed, with no differences observed between nuclear and cytoplasm regions. Knockdown CD44 SNB-19 cells (E = 359 ± 180 Pa) were less stiff than wt cells (E = 1099 ± 991 Pa; p = 0.023). The stiffness of wt SNB-19 cells was also greater than control astrocytes (E = 444 ± 311 Pa; p < 0.001). CONCLUSION: The combined results suggest gene silencing of CD44 may be an important therapeutic strategy for reducing adhesion, migration and invasion of glioma cells.

Human diploid fibroblasts were separated on the basis of size by sedimentation at unit gravity in a 5 to 30% fetal bovine serum gradient. The larger cells sedimented faster and were comprised of a larger proportion of cells that failed to incorporate tritiated thymidine during the 48 hours prior to sedimentation. However, the large cells and small cells had the same long-term replicative ability as determined both by ability to form large clones and by the total number of population doublings accrued in citro. Even after refractionation the larger and smaller cells had the same long-term proliferation ability. These results indicate that, although old cultures contain a high proportion of large cells and cells not able to synthesize DNA during a 24 to 48 hour period, these parameters cannot be used to select subpopulations of cells with different proliferative potentials from young or late passage cultures.