Multiple effects of electroporation on the adhesive behaviour of breast cancer cells and fibroblasts

<p>Abstract</p> <p>Background</p> <p>Recently electroporation using biphasic pulses was successfully applied in clinical developments for treating tumours in humans and animals. We evaluated the effects of electrical treatment on cell adhesion behaviour of breast cancer cells and fibroblasts. By applying bipolar electrical pulses we studied short- and long-lived effects on cell adhesion and survival, actin cytoskeleton and cell adhesion contacts in adherent cancer cells and fibroblasts.</p> <p>Methods</p> <p>Two cancer cell lines (MDA-MB-231 and MCF-7) and one fibroblast cell line 3T3 were used. Cells were exposed to high field intensity (200 - 1000 V/cm). Cell adhesion and survival after electrical exposure were studied by crystal violet assay and MTS assay. Cytoskeleton rearrangement and cell adhesion contacts were visualized by actin staining and fluorescent microscope.</p> <p>Results</p> <p>The degree of electropermeabilization of the adherent cells elevated steadily with the increasing of the field intensity. Adhesion behaviour of fibroblasts and MCF-7 was not significantly affected by electrotreatment. Interestingly, treating the loosely adhesive cancer cell line MDA-MB-231 with 200 V/cm and 500 V/cm resulted in increased cell adhesion. Cell replication of both studied cancer cell lines was disturbed after electropermeabilization. Electroporation influenced the actin cytoskeleton in cancer cells and fibroblasts in different ways. Since it disturbed temporarily the actin cytoskeleton in 3T3 cells, in cancer cells treated with lower and middle field intensity actin cytoskeleton was well presented in stress fibers, filopodia and lamellipodia. The electrotreatment for cancer cells provoked preferentially cell-cell adhesion contacts for MCF-7 and cell-ECM contacts for MDA-MB- 231.</p> <p>Conclusions</p> <p>Cell adhesion and survival as well as the type of cell adhesion (cell-ECM or cell-cell adhesion) induced by the electroporation process is cell specific. The application of suitable electric pulses can provoke changes in the cytoskeleton organization and cell adhesiveness, which could contribute to the restriction of tumour invasion and thus leads to the amplification of anti-tumour effect of electroporation-based tumour therapy.</p

ON THE INCIDENCE OF THE DIFFERENT FORMS OF PRIMARY GLAUCOMA

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Challenges of diagnostic exome sequencing in an inbred founder population

Exome sequencing was used as a diagnostic tool in a Roma/Gypsy family with three subjects (one deceased) affected by lissencephaly with cerebellar hypoplasia (LCH), a clinically and genetically heterogeneous diagnostic category. Data analysis identified high levels of unreported inbreeding, with multiple rare/novel "deleterious" variants occurring in the homozygous state in the affected individuals. Step‐wise filtering was facilitated by the inclusion of parental samples in the analysis and the availability of ethnically matched control exome data. We identified a novel mutation, p.Asp487Tyr, in the VLDLR gene involved in the Reelin developmental pathway and associated with a rare form of LCH, the Dysequilibrium Syndrome. p.Asp487Tyr is the third reported missense mutation in this gene and the first example of a change affecting directly the functionally crucial β‐propeller domain. An unexpected additional finding was a second unique mutation (p.Asn494His) with high scores of predicted pathogenicity in KCNV2, a gene implicated in a rare eye disorder, retinal cone dystrophy type 3B. This result raised diagnostic and counseling challenges that could be resolved through mutation screening of a large panel of healthy population controls. The strategy and findings of this study may inform the search for new disease mutations in the largest European genetic isolate

Research Communication Deregulated LAP2a Expression in Cervical Cancer Associates with Aberrant E2F and p53 Activities

Summary Lamina-associated polypeptide 2 alpha (LAP2a) plays a role in maintaining nuclear structure, in nuclear assembly/disassembly, and in transcriptional regulation. Elevated LAP2a mRNA expression has been previously reported to associate with certain cancer types. The aim of this study was to investigate LAP2a expression in cervical cancer and transformed cells and to identify factors that associate with its differential expression. LAP2a expression was found to be elevated in cervical cancer tissue by microarray, qRT-PCR, and immunofluorescence analyses. LAP2a also showed elevated expression in cervical cancer cell lines and in transformed fibroblasts compared with normal cells. To determine factors associated with elevated LAP2a in cervical cancer, the effect of inhibiting HPV E7 and E6 oncoproteins was investigated. E7 inhibition resulted in a decrease in phosphorylated Rb and an associated decrease in LAP2a, suggesting a role for E2F in regulating LAP2a expression. This finding was confirmed by inhibiting DP1, a coactivator of E2F, which resulted in decreased LAP2a levels. Inhibition of E6 resulted in elevated p53 and an associated decrease in LAP2a, suggesting that p53 associates with the negative regulation of LAP2a expression. This hypothesis was tested by inhibiting p53 in normal cells, and a resultant increase in LAP2a expression was observed. In conclusion, this study provides evidence for elevated LAP2a expression in cervical cancer and suggests that E2F and p53 activities associate with the positive and negative regulation of LAP2a expression, respectively. 2011 IUBMB IUBMB Life, 00: 000-000, 201

Stroke in urban and rural populations in north-east Bulgaria: incidence and case fatality findings from a 'hot pursuit' study

BACKGROUND: Bulgaria's official stroke mortality rates are higher for rural than urban areas. Official mortality data has indicated that these rates are amongst the highest in Europe. There has been a lack of studies measuring stroke incidence in urban and rural populations. METHODS: We established intensive notification networks covering 37791 residents in Varna city and 18656 residents (55% of them village-dwellers), all aged 45 to 84, in 2 rural districts. From May 1, 2000 to April 30, 2001 frequent contact was maintained with notifiers and death registrations were scanned regularly. Suspected incident strokes were assessed by study neurologists within a median of 8 days from onset. RESULTS: 742 events were referred for neurological assessment and 351 of these, which met the WHO criteria for stroke, were in persons aged 45 to 84 and were first ever in a lifetime. Incidence rates, standardised using the world standard weights for ages 45 to 84, were 909 (/100000/year) (95% CI 712–1105) and 597 (482–712) for rural and urban males and 667 (515–818) and 322 (248–395) for rural and urban females. Less than half were admitted to hospital (15% among rural females over 65). Twenty-eight day case fatality was 35% (123/351) overall and 48% (46/96) in village residents. The excess case fatality in the villages could not be explained by age or severity. CONCLUSIONS: Rural incidence rates were over twice those reported for western populations but the rate for urban females was similar to other western rates. The high level and marked heterogeneity in both stroke incidence and case fatality merit further investigation

GDF15 linked to maternal risk of nausea and vomiting during pregnancy

GDF15, a hormone acting on the brainstem, has been implicated in the nausea and vomiting of pregnancy (NVP) including its most severe form, Hyperemesis Gravidarum (HG), but a full mechanistic understanding is lacking [1-4]. Here we report that fetal production of GDF15, and maternal sensitivity to it, both contribute substantially to the risk of HG. We confirmed that higher GDF15 levels in maternal blood are associated with vomiting in pregnancy and HG. Using mass spectrometry to detect a naturally-labelled GDF15 variant we demonstrate that the vast majority of GDF15 in the maternal plasma is derived from the feto-placental unit. By studying carriers of rare and common genetic variants we found that low levels of GDF15 in the non-pregnant state increase the risk of developing HG. Conversely, women with beta-thalassemia, a condition where GDF15 levels are chronically high [5], report very low levels of NVP. In mice, the acute food intake response to a bolus of GDF15 is influenced bi-directionally by prior levels of circulating GDF15 in a manner suggesting that this system is susceptible to desensitization. Our findings support a putative causal role for fetally-derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by pre-pregnancy exposure to the hormone, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.</p

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background: Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods: The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results: A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P &lt; 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion: Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)

Investigation of an IEC 61850 standard-based process bus implementation of a protection and control scheme for parallelly connected transformers

Purpose This paper aims to focus on the implementation of the International Electrotechnical Commission (IEC) 61850–9-2 standard based process bus with merging units (MUs) and sampled values (SV) to improve the protection and control systems. The digital process interface is important to be included on the process bus level. Design/methodology/approach The IEC 61850–9-2 process bus standard is not extensively used in regard to SV when the IEC 61850 standard is implemented by power utilities. Many protection and control intelligent electronic devices (IEDs) are connected to a substation communication network, routers and switches using fibre-optic linked Ethernet. However, inductive current transformers (CTs) and voltage transformers (VTs) secondary circuits are still hardwired to the IEDs. The paper highlight issues with the copper wires for currents signals and how these issues can be eliminated by using the MUs and the SV protocol. The voltage regulator control IED of each transformer is required to regulate the voltage level of the secondary side bus bar it is connected to. All the regulating IEDs of parallel-connected transformers are required to communicate with each other to share information. They collectively control the bus bar voltage depending on the switching configuration of the parallel transformers. Findings It is shown that process bus information such as the high voltage switchgear status information of primary plant in the yard, can be used to improve the substation protection and control systems. The power transformer protection and voltage regulator control are focused on. Research limitations/implications The deliverables of the research work can be applied in: The Centre for Substation Automation and Energy Management systems of the Department of Electrical Engineering, power utilities and other establishments using power systems and digital substations in the electrical supply industry. The research work on the thesis led to the development of a laboratory test-bench where students can learn and understand the basics of the IEC 61850–9-2 SVs principles. The test-bench components such as the IEDs, real-time digital simulator, standalone MUs and Ethernet equipment can be used for future research applications. The test-bench can be used to demonstrate during course work for students at the University, the basics of digital substations using a process bus network with IEDs, MUs and Ethernet equipment. Practical implications The research work showed where lab equipment is getting outdated and future equipment will be required for research work in IEC 61850–9-2 process bus. Originality/value Power utilities can benefit from implementing the IEC 61850 part 9–2 of the standard and by using MUs and other process interface information in substations. A cost reduction in high voltage equipment, substation installation and commissioning costs and better performance of protection and control system can be achieved. </jats:sec

Development of a nonlinear linearizing controller using Input-Output / Input-State linearization for implementation in Programmable Logic Controller (PLC)

This paper describes the design of a nonlinear linearizing controller based on feedback linearizing method to stabilize a nonlinear magnetic levitation system. The linearized model states are selected through input-state linearization, and the mathematical expression of the linearizing controller is obtained. An additional innovative integral LQR is designed to make the closed-loop system more robust. The closed-loop system is implemented in real-time on an IEC 61499 standardbased programmable logic controller using a new model transformation technique. The closed-loop system is simulated in MATLAB/SIMULINK and TwinCAT 3 real-time environment, and the simulation results obtained on both environments are compared