Growth interferences between bacterial strains from raw cow's milk and their impact on growth of Listeria monocytogenes and Staphylococcus aureus.

AIMS: The purpose of this study was to detect growth enhancing or inhibiting activity between bacterial populations from raw milk under different conditions (temperature, medium). METHODS AND RESULTS: The interference of 24 raw milk isolates on growth of each other and on Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus was screened by drop assay and for selected pairs in co-cultivation experiments. By drop assay, antibacterial activity was observed for 40% of the strains. About 30% of the strains showed growth-enhancing activity on other strains. Most of the isolates were well adapted to cold temperatures and showed consistent or even increased inhibiting or enhancing effects on growth of other strains at 10°C. The growth of L. monocytogenes DSM 20600T and S. aureus DSM 1104T was significantly (P < 0·05) reduced in co-cultivation with Pseudomonas protegens JZ R-192. CONCLUSIONS: Growth interferences between bacterial populations have an impact on the structure of raw milk microbiota, especially when it develops under cold storage, and it may have an effect on the prevalence of certain foodborne pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates growth-inhibiting and also growth-enhancing interactions between raw milk bacteria, which must be considered when predicting bacterial growth and spoilage in food. A Ps. protegens strain isolated from raw milk showed an antagonistic effect on growth of L. monocytogenes in refrigerated raw milk.

A seven-Gene Signature assay improves prognostic risk stratification of perioperative chemotherapy treated gastroesophageal cancer patients from the MAGIC trial.

Background: Following neoadjuvant chemotherapy for operable gastroesophageal cancer, lymph node metastasis is the only validated prognostic variable; however, within lymph node groups there is still heterogeneity with risk of relapse. We hypothesized that gene profiles from neoadjuvant chemotherapy treated resection specimens from gastroesophageal cancer patients can be used to define prognostic risk groups to identify patients at risk for relapse. Patients and methods: The Medical Research Council Adjuvant Gastric Infusional Chemotherapy (MAGIC) trial (n = 202 with high quality RNA) samples treated with perioperative chemotherapy were profiled for a custom gastric cancer gene panel using the NanoString platform. Genes associated with overall survival (OS) were identified using penalized and standard Cox regression, followed by generation of risk scores and development of a NanoString biomarker assay to stratify patients into risk groups associated with OS. An independent dataset served as a validation cohort. Results: Regression and clustering analysis of MAGIC patients defined a seven-Gene Signature and two risk groups with different OS [hazard ratio (HR) 5.1; P < 0.0001]. The median OS of high- and low-risk groups were 10.2 [95% confidence interval (CI) of 6.5 and 13.2 months] and 80.9 months (CI: 43.0 months and not assessable), respectively. Risk groups were independently prognostic of lymph node metastasis by multivariate analysis (HR 3.6 in node positive group, P = 0.02; HR 3.6 in high-risk group, P = 0.0002), and not prognostic in surgery only patients (n = 118; log rank P = 0.2). A validation cohort independently confirmed these findings. Conclusions: These results suggest that gene-based risk groups can independently predict prognosis in gastroesophageal cancer patients treated with neoadjuvant chemotherapy. This signature and associated assay may help risk stratify these patients for post-surgery chemotherapy in future perioperative chemotherapy-based clinical trials.

Carbon dating cancer: defining the chronology of metastatic progression in colorectal cancer.

Background: Patients often ask oncologists how long a cancer has been present before causing symptoms or spreading to other organs. The evolutionary trajectory of cancers can be defined using phylogenetic approaches but lack of chronological references makes dating the exact onset of tumours very challenging. Patients and methods: Here, we describe the case of a colorectal cancer (CRC) patient presenting with synchronous lung metastasis and metachronous thyroid, chest wall and urinary tract metastases over the course of 5 years. The chest wall metastasis was caused by needle tract seeding, implying a known time of onset. Using whole genome sequencing data from primary and metastatic sites we inferred the complete chronology of the cancer by exploiting the time of needle tract seeding as an in vivo 'stopwatch'. This approach allowed us to follow the progression of the disease back in time, dating each ancestral node of the phylogenetic tree in the past history of the tumour. We used a Bayesian phylogenomic approach, which accounts for possible dynamic changes in mutational rate, to reconstruct the phylogenetic tree and effectively 'carbon date' the malignant progression. Results: The primary colon cancer emerged between 5 and 8 years before the clinical diagnosis. The primary tumour metastasized to the lung and the thyroid within a year from its onset. The thyroid lesion presented as a tumour-to-tumour deposit within a benign Hurthle adenoma. Despite rapid metastatic progression from the primary tumour, the patient showed an indolent disease course. Primary cancer and metastases were microsatellite stable and displayed low chromosomal instability. Neo-antigen analysis suggested minimal immunogenicity. Conclusion: Our data provide the first in vivo experimental evidence documenting the timing of metastatic progression in CRC and suggest that genomic instability might be more important than the metastatic potential of the primary cancer in dictating CRC fate.

Prognostic role of the LCS6 KRAS variant in locally advanced rectal cancer: results of the EXPERT-C trial.

BACKGROUND: Lethal-7 (let-7) is a tumour suppressor miRNA which acts by down-regulating several oncogenes including KRAS. A single-nucleotide polymorphism (rs61764370, T > G base substitution) in the let-7 complementary site 6 (LCS-6) of KRAS mRNA has been shown to predict prognosis in early-stage colorectal cancer (CRC) and benefit from anti-epidermal growth factor receptor monoclonal antibodies in metastatic CRC. PATIENTS AND METHODS: We analysed rs61764370 in EXPERT-C, a randomised phase II trial of neoadjuvant CAPOX followed by chemoradiotherapy, surgery and adjuvant CAPOX plus or minus cetuximab in locally advanced rectal cancer. DNA was isolated from formalin-fixed paraffin-embedded tumour tissue and genotyped using a PCR-based commercially available assay. Kaplan-Meier method and Cox regression analysis were used to calculate survival estimates and compare treatment arms. RESULTS: A total of 155/164 (94.5%) patients were successfully analysed, of whom 123 (79.4%) and 32 (20.6%) had the LCS-6 TT and LCS-6 TG genotype, respectively. Carriers of the G allele were found to have a statistically significantly higher rate of complete response (CR) after neoadjuvant therapy (28.1% versus 10.6%; P = 0.020) and a trend for better 5-year progression-free survival (PFS) [77.4% versus 64.5%: hazard ratio (HR) 0.56; P = 0.152] and overall survival (OS) rates (80.3% versus 71.9%: HR 0.59; P = 0.234). Both CR and survival outcomes were independent of the use of cetuximab. The negative prognostic effect associated with KRAS mutation appeared to be stronger in patients with the LCS-6 TT genotype (HR PFS 1.70, P = 0.078; HR OS 1.79, P = 0.082) compared with those with the LCS-6 TG genotype (HR PFS 1.33, P = 0.713; HR OS 1.01, P = 0.995). CONCLUSION: This analysis suggests that rs61764370 may be a biomarker of response to neoadjuvant treatment and an indicator of favourable outcome in locally advanced rectal cancer possibly by mitigating the poor prognosis of KRAS mutation. In this setting, however, this polymorphism does not appear to predict cetuximab benefit.

Nuclear magnetic resonance therapy in lumbar disc herniation with lumbar radicular syndrome: effects of the intervention on pain intensity, health-related quality of life, disease-related disability, consumption of pain medication, duration of sick leave and MRI analysis.

PURPOSE: The objective was to assess the effects of therapeutic nuclear magnetic resonance (tNMR) as a conservative treatment for lumbar radicular syndrome (LRS) in patients with lumbar disc herniation. METHODS: The prospective, randomised, double-blind, placebo-controlled trial included 94 patients, aged 20-60 years (44.79 ± 8.83), with LRS caused by lumbar disc herniation confirmed by MRI scans and with clinical signs of a radicular lesion without indication for surgical intervention. Treatment group (TG) and control group (CG) received standard non-surgical therapy. Additionally, the TG had seven sessions with the tNMR device with a magnetic flux density of 2.3 mT and a frequency of 85 kHz; the CG received 7 sham treatments. Outcome parameters were the treatment effect on pain intensity (Visual Analogue Scale-VAS), health-related quality of life (36-item Short Form Health Survey-SF-36), disease-related disability (Roland Morris Disability Questionnaire-RMDQ), pain medication intake, duration of sick leave and morphological changes assessed by MRI scan analysis. RESULTS: VAS scores improved significantly in both groups (p < 0.000). Only in week 4, improvement in the TG significantly surpassed that of the CG (morning pain p = 0.011, evening pain = 0.001). In both groups, SF-36 scores reflected a significant amendment in the physical component score (p < 0.000) and a significant deterioration in the mental component score (p < 0.000). SF-36 scores did not differ significantly between groups. RMDQ showed a significant amelioration in both groups (TG and CG p < 0.000), with a tendency to a superior benefit in the TG (p = 0.083). Patients in the TG recorded significantly fewer days of sick leave in month 3 after treatment (p = 0.026). MRI scan summary scores improved significantly in both groups (L4/5 p < 0.000, L5/S1 p < 0.001) and did not differ significantly between the groups. CONCLUSIONS: This trial was the first to investigate the effects of tNMR as an additional treatment of lumbar disc herniation with LRS. The application of tNMR did not meet MCID criteria. It rendered few statistically significant differences between patient groups. The overall results of this trial make a clinical implementation of tNMR in the treatment of lumbar disc herniation with LRS appear premature. Further research is needed to better understand the mode of action of tNMR on compressed neural tissue and to elucidate the issue of the cost/benefit ratio.

A flexible beamline combining XUV attosecond pulses with few-femtosecond UV and near-infrared pulses for time-resolved experiments.

We describe a beamline where few-femtosecond ultraviolet (UV) pulses are generated and synchronized to few-cycle near-infrared (NIR) and extreme ultraviolet (XUV) attosecond pulses. The UV light is obtained via third-harmonic generation in argon or neon gas when focusing a phase-stabilized NIR driving field inside a glass cell that was designed to support high pressures for enhanced conversion efficiency. A recirculation system allows reducing the large gas consumption required for the nonlinear process. Isolated attosecond pulses are generated using the polarization gating technique, and the photon spectrometer employed to characterize the XUV radiation consists of a new design based on the combination of a spherical varied-line-space grating and a cylindrical mirror. This design allows for compactness while providing a long entrance arm for integrating different experimental chambers. The entire interferometer is built under vacuum to prevent both absorption of the XUV light and dispersion of the UV pulses, and it is actively stabilized to ensure an attosecond delay stability during experiments. This table-top source has been realized with the aim of investigating UV-induced electron dynamics in neutral states of bio-relevant molecules, but it also offers the possibility to implement a manifold of novel time-resolved experiments based on photo-ionization/excitation of gaseous and liquid targets by ultraviolet radiation. UV pump-XUV probe measurements in ethyl-iodide showcase the capabilities of the attosecond beamline.

Studies on the role of osteopontin-1 in endometrial cancer cell lines.

BACKGROUND: Osteopontin-1 is a well characterized protein in many tumour entities. Multiple roles in the processes invasion, metastasis and angiogenesis of tumours are attributed to osteopontin-1. The putative role of osteopontin-1 has not been characterized for endometrial cancer. MATERIAL AND METHODS: We investigated multiple endometrial cancer cell lines for osteopontin-1 mRNA- and protein-expression. Osteopontin-1 dependent effects were analysed in vitro by siRNA inhibition. RESULTS: All endometrial cell lines expressed osteopontin-1. Expression of osteopontin-1 was successfully inhibited by specific siRNA. Cells with reduced osteopontin-1 expression showed decreased migration in the Boyden chamber assay and invasion was reduced in the wound-healing assay. Osteopontin-1 seems to play a role in apoptotic processes of endometrial cancer cells. Inhibition of osteopontin-1 expression was associated with an increased susceptibility for radiation therapy. CONCLUSION: Osteopontin-1 seems to play a role in endometrial cancer. Inhibition of osteopontin-1 expression leads to a higher susceptibility for radiation therapy. Our results suggest that a reduced expression of osteopontin-1 in endometrial cancer could inhibit the development of invasion and metastasis in these cells.

Intratumor morphologic and transcriptomic heterogeneity in V600EBRAF-mutated metastatic colorectal adenocarcinomas.

BACKGROUND: Intratumor heterogeneity (ITH) is described as the presence of various clones within one tumor, each with their own unique features in terms of morphology, inflammation, genetics or transcriptomics. Heterogeneity provides the fuel for drug resistance; therefore, an accurate assessment of tumor heterogeneity is essential for the development of effective therapies. The purpose of this study was to dissect morphologic and molecular ITH in colorectal adenocarcinoma. MATERIALS AND METHODS: A series of 120 V600EBRAF-mutated (V600EBRAFmt) consecutive metastatic colorectal adenocarcinomas was assessed for morphologic heterogeneity. The two heterogeneous components of each specimen underwent a histopathological, immunohistochemical and molecular characterization to evaluate: histologic variant, grading, tumor-infiltrating lymphocytes (TILs), mismatch repair proteins' expression, KRAS/BRAF/NRAS mutations, microsatellite instability (MSI) status and consensus molecular subtype (CMS). RESULTS: Thirty-one out of 120 (25.8%) V600EBRAFmt primary colorectal adenocarcinomas presented a heterogeneous morphology. Among these, eight cases had adequate material for molecular profiling. Five out of the eight (62.5%) cases resulted instable at MSI testing. The majority (62.5%) of the samples showed a CMS4 phenotype based on gene expression profiling. Heterogeneity in CMS classification was observed in four out of eight cases. One out of eight cases presented significant heterogeneity in the number of TILs between the two components of the tumor. CONCLUSIONS: Although the distribution of the immune infiltrate appears relatively conserved among heterogeneous areas of the same tumor, changes in gene expression profile and CMS occur in 50% of V600EBRAFmt adenocarcinoma cases in our small series and might contribute to variability in response to anticancer therapy and clinical outcomes. Assessment of morphological and molecular ITH is needed to improve colorectal cancer classification and to tailor anticancer treatments and should be included in the pathology report.

Long-term results and comparison of the Green-Anderson and multiplier growth prediction methods after permanent epiphysiodesis using Canale's technique.

PURPOSE: The aim of the study was to evaluate the accuracy and radiographic outcomes of Canale's method in patients with idiopathic leg-length discrepancy (LLD) following percutaneous epiphysiodesis. The accuracy of two common growth prediction methods was assessed. METHODS: A total of 18 patients with 26 affected bones (eight distal femur, two proximal tibia, five combined) were clinically and radiologically analyzed after reaching skeletal maturity. We compared the final effect of epiphysiodesis at maturity with the expected effect of epiphysiodesis before surgery; these measures were calculated using the Green-Anderson and multiplier methods, respectively. We furthermore compared pre- and postoperative frontal and lateral plane radiographs. RESULTS: The average LLD was 21.2 mm before surgery and 7.9 mm after epiphysiodesis. The final effect of both methods was not significantly different compared with the expected effect of epiphysiodesis before surgery. However, the prediction by the Green-Anderson method was closer to the definitive epiphysiodesis effect. The frontal plane radiographic deformity parameters did not change significantly after epiphysiodesis. The postoperative sagittal plane radiographic deformity parameters were in the normal range. CONCLUSION: The Canale technique is a reliable method to reduce LLD in children. With regards to growth prediction, the Green-Anderson method using bone age seems to be more accurate than the multiplier method using chronological age. However, a relative over-estimation was observed with both methods in several cases, which might result in an insufficient correction. LEVEL OF EVIDENCE: IV, Therapeutic study.

Relieving phantom limb pain with multimodal sensory-motor training.

OBJECTIVE: The causes for the disabling condition of phantom limb pain (PLP), affecting 85% of amputees, are so far unknown, with few effective treatments available. Sensory feedback based strategies to normalize the motor commands to control the phantom limb offer important targets for new effective treatments as the correlation between phantom limb motor control and sensory feedback from the motor intention has been identified as a possible mechanism for PLP development. APPROACH: Ten upper-limb amputees, suffering from chronic PLP, underwent 16 days of intensive training on phantom-limb movement control. Visual and tactile feedback, driven by muscular activity at the stump, was provided with the aim of reducing PLP intensity. MAIN RESULTS: A 32.1% reduction of PLP intensity was obtained at the follow-up (6 weeks after the end of the training, with an initial 21.6% reduction immediately at the end of the training) reaching clinical effectiveness for chronic pain reduction. Multimodal sensory-motor training on phantom-limb movements with visual and tactile feedback is a new method for PLP reduction. SIGNIFICANCE: The study results revealed a substantial reduction in phantom limb pain intensity, obtained with a new training protocol focused on improving phantom limb motor output using visual and tactile feedback from the stump muscular activity executed to move the phantom limb.

Approaches to optimize focused extracorporeal shockwave therapy (ESWT) based on an observational study of 363 feet with recalcitrant plantar fasciitis.

INTRODUCTION: Extracorporeal shockwave therapy (ESWT) is an established second-line treatment option for plantar fasciitis. Longer term results of focused ESWT are rare in literature. This study assessed the treatment success-rates of single session ESWT compared to repetitive ESWT treatment sessions, the mid-term results as well as treatment- or patient-related factors influencing the outcome of focused ESWT for plantar fasciitis. METHODS: 284 patients (363 feet) received ESWT for plantar fasciitis and answered a questionnaire on socio-demographic and anamnestic data immediately before as well as 19-77 weeks after the first application of ESWT. RESULTS: 76 percent of patients treated only once and 74 percent of all patients reported satisfying pain relief (with up to three treatment sessions). This was consistent in the mid-term and over different physicians as well as independent of assessed patient- or treatment-related factors. DISCUSSION: Applying repeated ESWT in weekly intervals by default may be helpful in reducing healing time for those patients requiring more than one treatment session. Prospective research is needed to find out whether further treatment sessions are justifiable in patients who indicate no improvement after two or three treatment sessions. CONCLUSIONS: In many cases, focused ESWT needs to be applied only once. Further research should focus on the number of treatment sessions as well as the minimum energy flux density needed.

Repression of the human immunodeficiency virus type 1 promoter by the human KRAB domain results in inhibition of virus production.

The Krüppel-associated box (KRAB) domain has been described as a eukaryotic repressor of transcription. We show that fusion of KRAB to DNA-binding-domains provides a novel approach to inhibit expression of a replication-competent human immunodeficiency virus (HIV) genome. The KRAB domain from the human zinc finger protein KOX1 was combined with the DNA binding domain of the Escherichia coli tetracycline repressor (TetR). Constitutive expression of the TetR-KRAB protein in HeLa cells inhibited virus production from an HIV genome encoding TetR target sequences by 80%. The same inhibition was observed with HIV-promoter-driven reporter plasmids. The specificity of inhibition was shown with informative KRAB mutants, plasmids lacking the respective target sequences, and by reversal of the TetR-KRAB-mediated inhibition with tetracycline. Virus production was suppressed by binding of TetR-KRAB at a distance of 6 kbp to the promoter. We therefore conclude that any site of the genuine HIV genome could serve as target of a chimeric KRAB repressor protein. Specific targeting of the KRAB domain by artificially selected binding domains may be generally applicable to control transcription in mammalian cells.

Advanced nitrogen elimination by encapsulated nitrifiers.

By introducing a mixed population of nitrifiers encapsulated in gel lens beads a more selective nitrification process was found in treatment of settled sewage in lab scale at a hydraulic retention time (HRT) of about 30 to 60 minutes. The reaction rates for oxidation of soluble chemical oxygen demand (SCOD) were found to vary between 25 to 150 mg/L x h while nitrification takes place around 50 mg nitrogen per hour and litre reaction volume. However, based on this SCOD removal in the nitrification step, a consequent post-denitrification process without nitrate recycle and dosage of external carbon sources has been proven to reach substantial nitrate elimination of up to 20 mg nitrogen per litre at COD/N-ratios of approx. 6 in settled sewage. At such COD/N-ratios, suitable nitrogen elimination seems to be possible, because the bioflocs of settled sewage, produced so far by SCOD oxidation and entrapment of particulate COD, are passing through the nitrification process having a substantial contribution to the denitrification rate additionally to the remaining SCOD.

Pre-nitrification by encapsulated nitrifiers--a possibility for self-sufficient energy operation of domestic WWTPs.

The overall energy consumption of domestic wastewater treatment plants (WWTPs) increases with treatment efficiency. Approximately 30 to 45 kWh per people equivalent and year is mostly necessary for advanced nitrogen and phosphorus removal, while the aeration contains the main part of approximately 60%. A new process using encapsulated nitrifiers on gel lens beads is introduced to overcome the high energy consumption of aeration. A more selective nitrification process was found at a nitrification rate of between 50 and 60 mg nitrogen per hour and litre reaction volume corresponding to a hydraulic retention time (HRT) of about 30 to 60 minutes while the soluble Chemical Oxygen Demand (COD) removal could be less than 30% depending on operational conditions of the bio-reactor. The latter enables internal use of wastewater's COD for a post denitrification. For the new process the energy consumption as well as total volume of bio-reactor are much less (approximately 30 to 50% for both) than conventional processes due to the low sludge age for COD and nitrate removal and the avoidance of internal wastewater recycle. Therefore, self-sufficient energy operation of domestic WWTPs operating with advanced treatment efficiency could become possible, if energy recovery by anaerobic sludge digestion is included.

Linear and nonlinear regression techniques for simultaneous and proportional myoelectric control.

In recent years the number of active controllable joints in electrically powered hand-prostheses has increased significantly. However, the control strategies for these devices in current clinical use are inadequate as they require separate and sequential control of each degree-of-freedom (DoF). In this study we systematically compare linear and nonlinear regression techniques for an independent, simultaneous and proportional myoelectric control of wrist movements with two DoF. These techniques include linear regression, mixture of linear experts (ME), multilayer-perceptron, and kernel ridge regression (KRR). They are investigated offline with electro-myographic signals acquired from ten able-bodied subjects and one person with congenital upper limb deficiency. The control accuracy is reported as a function of the number of electrodes and the amount and diversity of training data providing guidance for the requirements in clinical practice. The results showed that KRR, a nonparametric statistical learning method, outperformed the other methods. However, simple transformations in the feature space could linearize the problem, so that linear models could achieve similar performance as KRR at much lower computational costs. Especially ME, a physiologically inspired extension of linear regression represents a promising candidate for the next generation of prosthetic devices.

Follow-up of abdominal aortic aneurysm after endovascular aortic repair: comparison of volumetric and diametric measurement.

PURPOSE: To determine the correlation of maximal diameter measurements with volumetric evaluation of size after endovascular aortic repair (EVAR) of abdominal aortic aneurysms (AAA) using computed tomography angiography (CTA) and to survey its applicability for clinical follow-up. MATERIALS AND METHODS: 73 consecutive patients (2 females, 71 males; age 38-84 years; mean age, 69.1 ± 8 years) with AAA were treated with percutaneous EVAR in a single institution. For follow-up, CTA was performed periodically after EVAR. Images were evaluated for maximal diameter in consensus by two experienced radiologists. Using OsirixTM, volumetric measurements were done by one radiologist, including the entire infrarenal abdominal aorta. RESULTS: In 73 patients 220 CTA examinations were performed after EVAR with a mean follow-up of 17.3 months (range, 1.8-42.7 months). The mean postinterventional volume of aneurysm was 165.63 ml ± 93.29 ml (range, 47.94-565.67 ml). The mean maximal postinterventional diameter was 5.91 ± 1.52 cm (range, 3.72-13.82 cm). At large over the entire observation period a slight, non-significant decrease of 1.6% (2.58ml ± 69.05 ml, range 82.82-201.92 ml) in volumes and a 9.3% (mean 0.55 cm ± 1.22 cm, range 2.85-1.93cm) in diameters were observed. For all examinations a high correlation of volume and diameter was calculated (r = 0.813-0.905; α<0.01). CONCLUSION: For follow-up of abdominal EVAR using CTA there is a high correlation between volumetric and diametric measurements of aneurysm. Based on a daily clinical routine setting, measurements of maximal diameters in cross sectional imaging of AAA after EVAR seems to be sufficient to exclude post interventional enlargement.

PI3K inhibitor D-116883 is effective in in vitro models of ovarian cancer.

BACKGROUND: D-116883 (Aeterna Zentaris GmbH, Frankfurt, Germany) is an orally effective drug that acts via inhibition of phosphatidylinositol 3-kinase (PI3K). The PI3K/AKT signal transduction pathway is involved in ovarian cancer tumorigenesis. Phosphatase and Tensin homolog (PTEN) loss and other activating mutations frequently contribute to the activation of this pathway. We tested whether D-116883 exerts cytostatic effects in in vitro models of ovarian cancer and analyzed the induced programmed cell death. MATERIALS AND METHODS: We evaluated the potency of D-116883 in four ovarian carcinoma cell lines with different cellular assays. The effects of D-116883 on cell proliferation was analysed by crystal-violet staining and tetrazolium salt [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT] assay. The capacity for anchorage-independent growth was analyzed in two ovarian carcinoma cell lines without and with D-116883 addition by using the soft agar assay. Fluorescence activated cell sorting (FACS) cell cycle analyses were performed. Cells were incubated with multicaspase inhibitor benzyloxycarbonyl-val-ala-asp(OMe)-fluoromethylketone (zVAD) and inhibitor of necroptosis necrostatin. RESULTS: Growth inhibition occurred in all ovarian carcinoma cell lines studied (A2780, A2780cis, OAW42 and SKOV3) in a micromolar range (IC(50)<1 µM). By using soft agar assay, a reduced capacity for anchorage-independent growth, a hallmark of tumor cells, caused by D-116883 was demonstrated. Cell cycle analyses showed that D-116883 dose-dependently increased apoptotic cells. Multicaspase inhibitor zVAD and inhibitor of necroptosis necrostatin did not abrogate the growth-inhibiting effect of the compound. CONCLUSION: PI3K inhibitor D-116883 showed substantial cytotoxic effects in various in vitro models of ovarian cancer. Our results make D-116883 a good candidate for further ovarian cancer research including in vivo experiments.

Peptidomimetic GnRH antagonist AEZS-115 inhibits the growth of ovarian and endometrial cancer cells.

BACKGROUND: AEZS-115 (Aeterna Zentaris GmbH, Frankfurt/M, Germany) is an orally active peptidomimetic antagonist of gonadotropin-releasing hormone (GnRH). In various tumors, an autocrine growth-promoting loop has been described for GnRH. The current study evaluates the antitumor activity and mechanism of action of AEZS-115 in models of ovarian and endometrial cancer. MATERIALS AND METHODS: Human A2780, Acis2780, OAW-42, Ovcar-3, SKOV-3, Hec1A and Ishikawa cells were analyzed for GnRH receptor expression by reverse transcription polymerase chain reaction (RT-PCR). These cell lines were incubated with AEZS-115 at 1, 10 and 100 µM for 24 h, 48 h, and 72 h and the number of viable cells was determined. Fluorescence activated cell sorting (FACS) cell cycle analyses were performed with increasing concentrations of AEZS-115. Co-treatment experiments of cancer cells with GnRH antagonist cetrorelix and peptidomimetic GnRH antagonist AESZ-115 were carried out. RESULTS: A2780, Acis2780, OAW-42, Ovcar-3, SKOV-3, Hec1A and Ishikawa cells expressed GnRH receptors as demonstrated by RT-PCR. GnRH antagonist AEZS-115 inhibited growth of all cell lines in a dose- and time-dependent manner. Half maximal inhibitory concentration (IC(50)) values at 48 h of incubation were between 7 and 17.5 µM and for 72 h between 4.5 and 12.5 µM. IC(50) values for ovarian and endometrial cancer cells were rather similar. These results were obtained by tetrazolium salt [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT] assay and confirmed by additional crystal violet staining. Cell cycle FACS analysis revealed that AEZS-115 dose-dependently increased the fraction of apoptotic cells. Co-treatment experiments carried out with AEZS-115 and peptidic GnRH-antagonist cetrorelix suggest that the antitumor effect of AEZS-115 is not mediated by blockade of the GnRH receptor. CONCLUSION: GnRH antagonist AEZS-115 exhibited substantial antitumor activity in ovarian as well as endometrial cancer cell lines. However, this antitumor effect was not mediated by the tumoral GnRH receptors. To identify the mechanism of action of this compound, further research is warranted. Its in vitro antitumor activity makes AEZS-115 a promising candidate for in vivo studies of ovarian and endometrial cancer.

Downregulation of AKT reverses platinum resistance of human ovarian cancers in vitro.

Platinum resistance is the most crucial problem for treatment of ovarian cancer. Increasing evidence points towards AKT overexpression as a mechanistic reason for this clinical condition. The present study evaluates the effect of overexpression and downregulation of AKT on the sensitivity to cisplatin in a platinum-resistant human ovarian cancer cell line and the corresponding platinum-sensitive parental cell line. A2780 and A2780cis ovarian cancer cell lines were stably transfected with an AKT-sense and AKT-antisense plasmid. Successful transfection was evaluated by western blot analysis. Cytotoxic effects of cisplatin were evaluated by metabolic (MTT) and clonogenicity assays as well as by FACS analysis. AKT overexpression (confirmed by western blotting) converted platinum-sensitive A2780 into platinum-resistant cells as shown by MTT assay. Importantly, platinum resistance of A2780cis cells could be reversed by downregulation of AKT, as demonstrated by MTT and clonogenicity assays and FACS analysis. Our data provide strong evidence that cisplatin resistance in ovarian cancer is mediated by AKT overexpression and can be overcome by AKT downregulation, thus, providing a rationale for clinical phase II/III studies combining AKT inhibitors with cisplatin.

[Impact of magnetic resonance therapy on sickness absence of patients with nerve root irritation following a lumbar disc problem]. / Effekte der Kernspinresonanztherapie auf Krankenstand bei Patienten mit Nervenwurzelirritation infolge eines lumbalen Bandscheibenvorfalls.

BACKGROUND: The prevalence of spinal symptoms in Western industrialised countries ranges up to 80 %. Back pain ranks second among the most common reasons to seek medical advice. The resulting financial burden on the health-care system is proportional to the subjectively experienced pain. The aim of the present study was to determine whether the use of magnetic resonance therapy alters the duration of sickness absence in patients with discogenic radiculopathy. PATIENTS AND METHOD: In a double-blind prospective randomised study, the use of magnetic resonance therapy for back pain in patients with discogenic radiculopathy was evaluated in the context of health economics. Patients aged 20 to 55 years with lumboischialgia and no indication for surgery were included in the study. The primary variable was the number of days of sickness absence in a study group before and after magnetic field therapy, and in a control group. The number of days of sickness absence was determined on the basis of a pain diary and by telephone inquiry. RESULTS: Patients who were treated with an activated magnetic resonance therapy device had significantly fewer days of sickness absence (p = 0.009) when evaluated by personal telephone calls. The duration of sickness absence before therapy was 14.7 days and that after therapy 5.8 days. In contrast, the days of sickness absence in the control group were 7.6 days before therapy and 13.8 days after therapy. The duration of symptoms was negatively correlated with the days of sickness absence. Patients who reported a burden at work had more days of sickness absence (8.3 days) than those with no burden at work (3.2 days). This correlation does not apply to familial burden. The cost-effectiveness analysis showed different degrees of compensation of the cost of magnetic resonance therapy, depending on the occupational group. Direct and indirect costs of magnetic resonance therapy were compensated by 16.9 fewer days of sickness absence among workers, 11.4 fewer days of sickness absence among employees, and 9.1 fewer days of sickness absence among civil servants. CONCLUSION: Based on the number of days of sickness absence, the study confirmed that a relatively economical alternative technique is able to provide pain relief as well as benefit the health economy. Unemployed patients or patients who have submitted an application for a pension may be problematic because they may not wish to be pronounced healthy by their doctors.