Application of an alchemical free energy method for the prediction of thermostable DuraPETase variants.

Non-equilibrium (NEQ) alchemical free energy calculations are an emerging tool for accurately predicting changes in protein folding free energy resulting from amino acid mutations. In this study, this method in combination with the Rosetta ddg monomer tool was applied to predict more thermostable variants of the polyethylene terephthalate (PET) degrading enzyme DuraPETase. The Rosetta ddg monomer tool efficiently enriched promising mutations prior to more accurate prediction by NEQ alchemical free energy calculations. The relative change in folding free energy of 96 single amino acid mutations was calculated by NEQ alchemical free energy calculation. Experimental validation of ten of the highest scoring variants identified two mutations (DuraPETaseS61M and DuraPETaseS223Y) that increased the melting temperature (Tm) of the enzyme by up to 1 °C. The calculated relative change in folding free energy showed an excellent correlation with experimentally determined Tm resulting in a Pearson's correlation coefficient of r = - 0.84. Limitations in the prediction of strongly stabilizing mutations were, however, encountered and are discussed. Despite these challenges, this study demonstrates the practical applicability of NEQ alchemical free energy calculations in prospective enzyme engineering projects. KEY POINTS: • Rosetta ddg monomer enriches stabilizing mutations in a library of DuraPETase variants • NEQ free energy calculations accurately predict changes in Tm of DuraPETase • The DuraPETase variants S223Y, S42M, and S61M have increased Tm.

Split-GFP complementation at the bacterial cell surface for antibody-free labeling and quantification of heterologous protein display.

The split-GFP system is a versatile tool with numerous applications, but it has been underutilized for the labeling of heterologous surface-displayed proteins. By inserting the 16 amino acid sequence of the GFP11-tag between a protein of interest and an autotransporter protein, it is possible to present a protein at the outer membrane of gram-negative bacteria and to fluorescently label it by complementation with externally added GFP1-10. The labeled cells could be clearly discerned from cells without the protein of interest using flow cytometry and the insertion of the GFP11-tag caused no significant alteration of the catalytic activity for the tested model enzyme CsBglA. Furthermore, the amount of the protein of interest on the cells could be quantified by comparing the green fluorescence resulting from the complementation to that of standards with known concentrations. This allows a precise characterization of whole-cell biocatalysts, which is difficult with existing methods. The split-GFP complementation approach was shown to be specific, in a similar manner as commercial antibodies. It is cost-efficient, minimizes the possibility of adverse effects on protein expression or solubility, and can be performed at high throughput.

Analyzing the interactome of human CK2ß in prostate carcinoma cells reveals HSP70-1 and Rho guanin nucleotide exchange factor 12 as novel interaction partners.

CK2ß is the non-catalytic modulating part of the S/T-protein kinase CK2. However, the overall function of CK2ß is poorly understood. Here, we report on the identification of 38 new interaction partners of the human CK2ß from lysates of DU145 prostate cancer cells using photo-crosslinking and mass spectrometry, whereby HSP70-1 was identified with high abundance. The KD value of its interaction with CK2ß was determined as 0.57 µM by microscale thermophoresis, this being the first time, to our knowledge, that a KD value of CK2ß with another protein than CK2α or CK2α' was quantified. Phosphorylation studies excluded HSP70-1 as a substrate or activity modulator of CK2, suggesting a CK2 activity independent interaction of HSP70-1 with CK2ß. Co-immunoprecipitation experiments in three different cancer cell lines confirmed the interaction of HSP70-1 with CK2ß in vivo. A second identified CK2ß interaction partner was Rho guanin nucleotide exchange factor 12, indicating an involvement of CK2ß in the Rho-GTPase signal pathway, described here for the first time to our knowledge. This points to a role of CK2ß in the interaction network affecting the organization of the cytoskeleton.

Anti-Inflammatory Properties of the SGLT2 Inhibitor Empagliflozin in Activated Primary Microglia.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors, including empagliflozin, are routinely used as antidiabetic drugs. Recent studies indicate that beside its beneficial effects on blood glucose level, empagliflozin may also exert vascular anti-inflammatory and neuroprotective properties. In the brain, microglia are crucial mediators of inflammation, and neuroinflammation plays a key role in neurodegenerative disorders. Dampening microglia-mediated inflammation may slow down disease progression. In this context, we investigated the immunomodulatory effect of empagliflozin on activated primary microglia. As a validated experimental model, rat primary microglial cells were activated into a pro-inflammatory state by stimulation with LPS. The influence of empagliflozin on the expression of pro-inflammatory mediators (NO, Nos2, IL6, TNF, IL1B) and on the anti-inflammatory mediator IL10 was assessed using quantitative PCR and ELISA. Further, we investigated changes in the activation of the ERK1/2 cascade by Western blot and NFkB translocation by immunostaining. We observed that empagliflozin reduces the expression of pro- and anti-inflammatory mediators in LPS-activated primary microglia. These effects might be mediated by NHE-1, rather than by SGLT2, and by the further inhibition of the ERK1/2 and NFkB pathways. Our results support putative anti-inflammatory effects of empagliflozin on microglia and suggest that SGLT2 inhibitors may exert beneficial effects in neurodegenerative disorders.

Enzyme cascade converting cyclohexanol into ε-caprolactone coupled with NADPH recycling using surface displayed alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli.

The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo-, regio- and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram-negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole-cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity VMAX(app) for the oxidation of cyclohexanol with the ADH whole-cell biocatalysts was determined as 59.9 mU ml-1 . For the oxidation of cyclohexanone with the CHMO whole-cell biocatalysts a VMAX(app) of 491 mU ml-1 was obtained. A direct conversion of cyclohexanol to ε-caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole-cell biocatalysts. Gas chromatography was applied to quantify the yield of ε-caprolactone. 1.12 mM ε-caprolactone was produced using ADH and CHMO displaying whole-cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD578nm 10. Furthermore, the reaction cascade as applied provided a self-sufficient regeneration of NADPH for CHMO by the ADH whole-cell biocatalyst.

The Impact of Patient Support Programs on Adherence to Disease-Modifying Therapies of Patients with Relapsing-Remitting Multiple Sclerosis in Germany: A Non-Interventional, Prospective Study.

INTRODUCTION: Disease-modifying therapies (DMTs) in multiple sclerosis (MS) are chronic therapies, and patients are likely to face challenges in adhering to DMT dosing regimens over time. DMT manufacturers offer patient support programs (PSPs) to increase adherence. PSPs are managed offerings typically encompassing nurse services, phone services, online resources, or mobile offerings. This study evaluated whether PSPs have a positive impact on adherence to DMTs among patients with mild-to-moderate relapsing-remitting multiple sclerosis (RRMS) in Germany, independent of the treatment duration on DMT. METHODS: This was a non-interventional, prospective, cross-sectional, multi-center study with patient-reported outcomes. Patients reported their DMT adherence using patient adherence questionnaires at four visits during an observation period of 24 weeks; PSP participation for this period was reported at the last visit. The primary objective was to evaluate the impact of PSPs on adherence across different DMTs by comparing patients with PSP participation versus no participation; adherence was defined as not missing a single dose of DMT. RESULTS: One hundred eighty-four patients were analyzed (mean age: 44.6 years; 73.4% female; mean time on DMT: 7.2 years). Adherence across DMTs was significantly higher for PSP participants (92.9%) compared with non-participants (61.8%) (P = 0.0197). The observed rate of PSP participation (7.6%) was significantly lower than reported in earlier studies (P < 0.0001); PSP awareness among patients analyzed was low (22.3%). CONCLUSION: We consider this study to have shown that PSPs have a positive impact on adherence to DMTs in MS, independent of the treatment duration on DMT. The majority of PSP participants also believe in this positive effect. PSP participation and patient awareness were low, and real-world adherence levels were found to be higher with self-injectable DMTs than with oral DMTs. In summary, physicians should actively advise patients with MS to participate in PSPs and, together with their patients, consider achievable real-world adherence under different DMTs when deciding MS treatment strategies.

3D reconstruction of MR-visible Fe3 O4 -mesh implants: Pelvic mesh measurement techniques and preliminary findings.

AIMS: To develop MR-based measurement technique to evaluate the postoperative dimension and location of implanted magnetic resonance (MR)-visible meshes. METHODS: This technique development study reports findings of six patients (A-F) with cystoceles treated with anterior vaginal MR-visible Fe3 O4 -polypropylene implants. Implanted meshes were reconstructed from 3 months and/or 1 year postsurgical MR-images using 3D Slicer®. Measurements including mesh length, distance to the ischial spines, pudendal, and obturator neurovascular bundles and urethra were obtained using software Rhino® and a custom Matlab® program. The range of implanted mesh length and their placements were reported and compared with mesh design and implantation recommendations. With the anterior/posterior-mesh-segment-ratio mesh shrinkage localization was evaluated. RESULTS: Examinations were possible for patients A-D 3 months and for A, C, E, and F 1 year postsurgical. The mesh was at least 40% shorter in all patients 3 months and/or 1 year postoperatively. A, B showed shrinkage in the anterior segment, D, E in the posterior segment (Patients C, F not applicable due to intraoperative mesh trimming). Patient E presented pain in the area of mesh shrinkage. In Patient C posterior mesh fixations were placed in the iliococcygeal muscle rather than sacrospinous ligaments. Arm placement less than 20 mm from the pudendal neurovascular bundles was seen in all cases. The portion of the urethra having mesh underneath it ranged from 19% to 55%. CONCLUSIONS: MRI-based measurement techniques have been developed to quantify implanted mesh location and dimension. Mesh placement variations possibly correlating with postoperative complications can be illustrated.

Single Molecule Molecular Inversion Probes for High Throughput Germline Screenings in Dystonia.

Background: This study's aim was to investigate a large cohort of dystonia patients for pathogenic and rare variants in the ATM gene, making use of a new, cost-efficient enrichment technology for NGS-based screening. Methods: Single molecule Molecular Inversion Probes (smMIPs) were used for targeted enrichment and sequencing of all protein coding exons and exon-intron boundaries of the ATM gene in 373 dystonia patients and six positive controls with known ATM variants. Additionally, a rare-variant association study was performed. Results: One patient (0.3%) was compound heterozygous and 21 others were carriers of variants of unknown significance (VUS) in the ATM gene. Although mutations in sporadic dystonia patients are not common, exclusion of pathogenic variants is crucial to recognize a potential tumor predisposition syndrome. SmMIPs produced similar results as routinely used NGS-based approaches. Conclusion: Our results underline the importance of implementing ATM in the routine genetic testing of dystonia patients and confirm the reliability of smMIPs and their usability for germline screenings in rare neurodegenerative conditions.

Five-year outcome after pelvic floor reconstructive surgery: evaluation using dynamic magnetic resonance imaging compared to clinical examination and quality-of-life questionnaire.

Background Dynamic magnetic resonance imaging (dMRI) captures the entire pelvis during Valsalva maneuver and helps diagnosing pelvic floor changes after reconstructive surgery. Purpose To evaluate therapeutic outcome five years after reconstructive surgery using clinical examination, dMRI, and quality-of-life (QOL) questionnaire. Material and Methods Clinical examination, dMRI, and QOL questionnaire were conducted before surgery and in the follow-ups at 12 weeks, one year, and five years in women with pelvic organ prolapse (POP) stage ≥2. dMRI was performed at 1.5-T using a predefined protocol including sagittal T2-weighted (T2W) sequence at rest and sagittal T2W true-FISP sequence at maximum strain for metric POP measurements (reference points = bladder, cervix, pouch, rectum). Pelvic organ mobility (POM) was defined as the difference of the metric measurement at maximum strain and at rest. Results Twenty-six women with 104 MRI examinations were available for analysis. dMRI results mostly differ to clinical examination regarding the overall five-year outcome and the posterior compartment in particular. dMRI diagnosed substantially more patients with recurrent or de novo POP in the posterior compartment (n = 17) compared to clinical examination (n = 4). POM after five years aligns to preoperative status except for the bladder. POM reflects best the QOL results regarding defecation disorders. Conclusion A tendency for recurrent and de novo POP was seen in all diagnostic modalities applied. dMRI objectively visualizes the interaction of the pelvic organs and the pelvic floor after reconstructive surgery and POM correlated best with the women's personal impression on pelvic floor complaints.

MRI visible Fe3O4 polypropylene mesh: 3D reconstruction of spatial relation to bony pelvis and neurovascular structures.

INTRODUCTION AND HYPOTHESIS: To demonstrate mesh magnetic resonance imaging (MRI) visibility in living women, the feasibility of reconstructing the full mesh course in 3D, and to document its spatial relationship to pelvic anatomical structures. METHODS: This is a proof of concept study of three patients from a prospective multi-center trial evaluating women with anterior vaginal mesh repair using a MRI-visible Fe3O4 polypropylene implant for pelvic floor reconstruction. High-resolution sagittal T2-weighted (T2w) sequences, transverse T1-weighted (T1w) FLASH 2D, and transverse T1w FLASH 3D sequences were performed to evaluate Fe3O4 polypropylene mesh MRI visibility and overall post-surgical pelvic anatomy 3 months after reconstructive surgery. Full mesh course in addition to important pelvic structures were reconstructed using the 3D Slicer® software program based on T1w and T2w MRI. RESULTS: Three women with POP-Q grade III cystoceles were successfully treated with a partially absorbable MRI-visible anterior vaginal mesh with six fixation arms and showed no recurrent cystocele at the 3-month follow-up examination. The course of mesh in the pelvis was visible on MRI in all three women. The mesh body and arms could be reconstructed allowing visualization of the full course of the mesh in relationship to important pelvic structures such as the obturator or pudendal vessel nerve bundles in 3D. CONCLUSIONS: The use of MRI-visible Fe3O4 polypropylene meshes in combination with post-surgical 3D reconstruction of the mesh and adjacent structures is feasible suggesting that it might be a useful tool for evaluating mesh complications more precisely and a valuable interactive feedback tool for surgeons and mesh design engineers.

A comprehensive quality control workflow for paired tumor-normal NGS experiments.

SUMMARY: Quality control (QC) is an important part of all NGS data analysis stages. Many available tools calculate QC metrics from different analysis steps of single sample experiments (raw reads, mapped reads and variant lists). Multi-sample experiments, as sequencing of tumor-normal pairs, require additional QC metrics to ensure validity of results. These multi-sample QC metrics still lack standardization. We therefore suggest a new workflow for QC of DNA sequencing of tumor-normal pairs. With this workflow well-known single-sample QC metrics and additional metrics specific for tumor-normal pairs can be calculated. The segmentation into different tools offers a high flexibility and allows reuse for other purposes. All tools produce qcML, a generic XML format for QC of -omics experiments. qcML uses quality metrics defined in an ontology, which was adapted for NGS. AVAILABILITY AND IMPLEMENTATION: All QC tools are implemented in C ++ and run both under Linux and Windows. Plotting requires python 2.7 and matplotlib. The software is available under the 'GNU General Public License version 2' as part of the ngs-bits project: https://github.com/imgag/ngs-bits. CONTACT: christopher.schroeder@med.uni-tuebingen.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Single-Incision Mini-Slings: Obturator Complex Pull-Out-Force Measurements.

AIM: To evaluate the different pull-out-force out of the obturator membrane and the obturator internus muscle of 2 mini-sling systems for stress urinary incontinence (SUI) treatment. MATERIAL AND METHODS: In a prospective study, the pull-out-force measurements were performed on 8 female cadaver pelvises, both on the obturator membrane and internal muscle, using the force measurement device PCE-FM50 (PCE Instruments). Tested were the hooks of 2 on the market available mini-sling kits (MiniArc by American Medical System; Ajust by BARD Inc.). Connected to the force measurement device, the hooks were placed into the obturator membrane and the obturator internus muscle. Mean value and SD were documented. RESULTS: On the complete obturator complex, Ajust showed a significantly higher pull-out-force (2,561.75 ± 638.8 g) than did the MiniArc (1,780.69 ± 442.3 g), p = 0.025. No significant differences were performed on the obturator membrane. CONCLUSIONS: The Ajust mini-sling hook is, given the objective results, more stably attached to the obturator complex than the MiniArc. Yet, both systems are sufficiently attachable to the evaluated anatomic structures, fulfilling the needed requirements for their use in the treatment of SUI.

Proof of concept for the simplified breakdown of cellulose by combining Pseudomonas putida strains with surface displayed thermophilic endocellulase, exocellulase and ß-glucosidase.

BACKGROUND: The production and employment of cellulases still represents an economic bottleneck in the conversion of lignocellulosic biomass to biofuels and other biocommodities. This process could be simplified by displaying the necessary enzymes on a microbial cell surface. Such an approach, however, requires an appropriate host organism which on the one hand can withstand the rough environment coming along with lignocellulose hydrolysis, and on the other hand does not consume the generated glucose so that it remains available for subsequent fermentation steps. RESULTS: The robust soil bacterium Pseudomonas putida showed a strongly reduced uptake of glucose above a temperature of 50 °C, while remaining structurally intact hence recyclable, which makes it suitable for cellulose hydrolysis at elevated temperatures. Consequently, three complementary, thermophilic cellulases from Ruminiclostridium thermocellum were displayed on the surface of the bacterium. All three enzymes retained their activity on the cell surface. A mixture of three strains displaying each one of these enzymes was able to synergistically hydrolyze filter paper at 55 °C, producing 20 µg glucose per mL cell suspension in 24 h. CONCLUSION: We could establish Pseudomonas putida as host for the surface display of cellulases, and provided proof-of-concept for a fast and simple cellulose breakdown process at elevated temperatures. This study opens up new perspectives for the application of P. putida in the production of biofuels and other biotechnological products.

Magnetic resonance-visible polypropylene mesh for pelvic organ prolapse repair.

AIM: To develop a magnetic resonance (MR)-visible mesh using iron oxides and prove visibility. METHODS: In a phantom study, a suitable iron oxide, Fe3O4 [iron(II,III) oxide] and FeOOH [iron(III) oxide-hydroxide], concentration was determined using relaxometric MR measurements of the transverse relaxation rates R2 and R2*. Next, a nonabsorbable mesh was designed from the MR-visible threads woven into a polypropylene mesh. The mesh was implanted into a fresh female cadaver via the transobturator route, and MR visibility was assessed with various MR pulse sequences in a clinical 3-tesla system. RESULTS: Optimal contrast was achieved with Fe3O4 at 0.2 weight-% in all imaging sequences, and the optimal contrast was achieved in a 3D spoiled gradient-echo (fast low-angle shot) acquisition. In this concentration range the apparent transverse relaxation rate R2* is below 10 ms. The mesh was visible in the cadaver on T1-weighted 3D spoiled gradient-echo images and T1-weighted fast spin-echo images. CONCLUSION: Mesh materials can be manufactured to be visible on MR with a negative contrast. Fe3O4 meshes could simplify follow-up examinations and help diagnose origins of postsurgical lesions after urogynecological procedures with mesh material.

CD4(+) T cell lineage integrity is controlled by the histone deacetylases HDAC1 and HDAC2.

Molecular mechanisms that maintain lineage integrity of helper T cells are largely unknown. Here we show histone deacetylases 1 and 2 (HDAC1 and HDAC2) as crucial regulators of this process. Loss of HDAC1 and HDAC2 during late T cell development led to the appearance of major histocompatibility complex (MHC) class II-selected CD4(+) helper T cells that expressed CD8-lineage genes such as Cd8a and Cd8b1. HDAC1 and HDAC2-deficient T helper type 0 (TH0) and TH1 cells further upregulated CD8-lineage genes and acquired a CD8(+) effector T cell program in a manner dependent on Runx-CBFß complexes, whereas TH2 cells repressed features of the CD8(+) lineage independently of HDAC1 and HDAC2. These results demonstrate that HDAC1 and HDAC2 maintain integrity of the CD4 lineage by repressing Runx-CBFß complexes that otherwise induce a CD8(+) effector T cell-like program in CD4(+) T cells.

MTSS1 is a metastasis driver in a subset of human melanomas.

In cancers with a highly altered genome, distinct genetic alterations drive subsets rather than the majority of individual tumours. Here we use a sequential search across human tumour samples for transcript outlier data points with associated gene copy number variations that correlate with patient's survival to identify genes with pro-invasive functionality. Employing loss and gain of function approaches in vitro and in vivo, we show that one such gene, MTSS1, promotes the ability of melanocytic cells to metastasize and engages actin dynamics via Rho-GTPases and cofilin in this process. Indeed, high MTSS1 expression defines a subgroup of primary melanomas with unfavourable prognosis. These data underscore the biological, clinical and potential therapeutic implications of molecular subsets within genetically complex cancers.

MRI findings before and after prolapse surgery.

BACKGROUND: Therapeutical outcome after prolapse surgery is evaluated using a standardized grading system based on maximum prolapse extent, which might not provide the full picture of the patient's subjective outcome. We therefore applied an evaluation method, which is detached from a grading system. PURPOSE: To evaluate the impact of pelvic organ mobility in dynamic magnetic resonance imaging (MRI) before and after mesh-repair surgery in patients with symptomatic pelvic organ prolapse. MATERIAL AND METHODS: To obtain measurements, we performed parasagittal T2-weighted turbo spin echo sequence at rest (TR, 3460 ms; TE, 85 ms; matrix, 512; slice thickness [ST], 5 mm), parasagittal T2-weighted true fast imaging with steady-state precession (TrueFISP) single-shot sequence during straining (TR, 397.4 ms; TE, 1.5 ms; matrix, 256; ST, 8 mm), and parasagittal T2-weighted TrueFISP sequence at maximum strain (TR, 4.3 ms; TE, 2.15 ms; matrix, 256; ST, 5 mm) at 1.5 T MRI. Pelvic organ prolapse (anatomical landmarks: bladder, cervix, pouch, rectum) was measured perpendicularly with reference to the pubococcygeal and the midpubic line. Pelvic organ mobility was defined as the difference between the measured distance at rest and at maximum strain for each anatomical landmark. All patients underwent mesh-repair procedure. Eighty patients could be included in this short-term follow-up study. Due to the physical diagnosis of pelvic organ prolapse, 51 underwent anterior mesh repair, 16 underwent posterior mesh repair, and 13 underwent total mesh repair. Surgery was performed by one surgeon, using mesh implants from several manufacturers. RESULTS: Median values of maximum organ prolapse for bladder, cervix, pouch, and rectum preoperatively were 2.54 cm, 0.33 cm, 2.47 cm, and 0.32 cm, respectively, and 12 weeks postoperatively 0.87 cm, -1.79 cm, 1.49 cm, and 0.49 cm, respectively. Highly significant improvement (P < 0.001) of pelvic organ mobility was observed in the treated compartment at 4- and 12-week follow-up. Physical evaluation 12 weeks after mesh-repair showed an asymptomatic POP-Q stage I, if any. CONCLUSION: Dynamic MRI is useful in visualizing the maximum extent of pelvic organ prolapse, as the evaluation of pelvic organ mobility documents the intraindividual therapeutic outcome detached from a grading system based on maximal prolapse values.

An attempt at a molecular prediction of metastasis in patients with primary cutaneous melanoma.

BACKGROUND: Current prognostic clinical and morphological parameters are insufficient to accurately predict metastasis in individual melanoma patients. Several studies have described gene expression signatures to predict survival or metastasis of primary melanoma patients, however the reproducibility among these studies is disappointingly low. METHODOLOGY/PRINCIPAL FINDINGS: We followed extended REMARK/Gould Rothberg criteria to identify gene sets predictive for metastasis in patients with primary cutaneous melanoma. For class comparison, gene expression data from 116 patients with clinical stage I/II (no metastasis) and 72 with III/IV primary melanoma (with metastasis) at time of first diagnosis were used. Significance analysis of microarrays identified the top 50 differentially expressed genes. In an independent data set from a second cohort of 28 primary melanoma patients, these genes were analyzed by multivariate Cox regression analysis and leave-one-out cross validation for association with development of metastatic disease. In a multivariate Cox regression analysis, expression of the genes Ena/vasodilator-stimulated phosphoprotein-like (EVL) and CD24 antigen gave the best predictive value (p = 0.001; p = 0.017, respectively). A multivariate Cox proportional hazards model revealed these genes as a potential independent predictor, which may possibly add (both p = 0.01) to the predictive value of the most important morphological indicator, Breslow depth. CONCLUSION/SIGNIFICANCE: Combination of molecular with morphological information may potentially enable an improved prediction of metastasis in primary melanoma patients. A strength of the gene expression set is the small number of genes, which should allow easy reevaluation in independent data sets and adequately designed clinical trials.

The PACOVAR-trial: a phase I/II study of pazopanib (GW786034) and cyclophosphamide in patients with platinum-resistant recurrent, pre-treated ovarian cancer.

BACKGROUND: The prognosis of patients with recurrent, platinum-resistant epithelial ovarian cancer (EOC) is poor. There is no standard treatment available. Emerging evidence suggests a major role for antiangiogenic treatment modalities in EOC, in particular in combination with the metronomic application of low dose chemotherapy. The novel, investigational oral antiangiogenic agent pazopanib targeting vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and c-kit is currently being studied in different tumour types and is already used as first line therapy in recurrent renal cell carcinoma. A combined therapy consisting of pazopanib and metronomic oral cyclophosphamide may offer a well-tolerable treatment option to patients with recurrent, pretreated EOC. METHODS/DESIGN: This study is designed as a multicenter phase I/II trial evaluating the optimal dose for pazopanib (phase I) as well as activity and tolerability of a combination regimen consisting of pazopanib and metronomic cyclophosphamide in the palliative treatment of patients with recurrent, platinum-resistant, pre-treated ovarian cancer (phase II). The patient population includes patients with histologically or cytologically confirmed diagnosis of EOC, cancer of the fallopian tube or peritoneal cancer which is platinumresistant or -refractory. Patients must have measurable disease according to RECIST criteria and must have failed available standard chemotherapy. Primary objectives are determination of the optimal doses for pazopanib (phase I) and the overall response rate according to RECIST criteria (phase II). Secondary objectives are time to progression, overall survival, safety and tolerability. The treatment duration is until disease progression or intolerability of study drug regimen (with a maximum of 13 cycles up to 52 weeks per subject). DISCUSSION: The current phase I/II trial shall clarify the potential of the multitargeting antiangiogenic tyrosinkinaseinhibitor GW 786034 (pazopanib) in combination with oral cyclophosphamide as salvage treatment in patients with recurrent, pretreated ovarian cancer.

Short-range clinical, dynamic magnetic resonance imaging and P-QOL questionnaire results after mesh repair in female pelvic organ prolapse.

OBJECTIVE: To evaluate clinical, quality-of-life (QoL) and dynamic magnetic resonance imaging (dMRI) results in patients with pelvic organ prolapse (POP) preoperatively, and 4 and 12 weeks after anterior and/or posterior mesh repair. STUDY DESIGN: Thirty-six patients (mean age 65 years) with symptomatic pelvic floor descent underwent mesh repair. The prolapse was quantified using the POP-Q system. Before surgery as well as 4 and 12 weeks after surgery, the pelvic organ positions were measured on dynamic magnetic resonance imaging during Valsalva manoeuvre in relation to the pubococcygeal and mid-pubic lines to assess surgery outcome. Patients also completed the P-QOL questionnaire to evaluate subjective changes at each visit. RESULTS: Four and 12 weeks after surgery patients showed improvement of the POP on clinical examination and on dynamic MRI. The latter demonstrated high significance (p<0.001) especially in bladder and vaginal cuff/cervix positions during maximal straining. All quality-of-life domains and some symptom questions of the P-QOL questionnaire significantly improved (p<0.05) 12 weeks after surgery. CONCLUSION: Significant anatomical and quality-of-life improvement was demonstrated after anterior and/or posterior mesh repair for POP using dynamic MRI and the P-QOL questionnaire.