Propafenone analogue with additional H-bond acceptor group shows increased inhibitory activity on P-glycoprotein.

P-glycoprotein (P-gp) is an ATP-dependent efflux pump that has a marked impact on the absorption, distribution, and excretion of therapeutic drugs. As P-gp inhibition can result in drug-drug interactions and altered drug bioavailability, identifying molecular properties that are linked to inhibition is of great interest in drug development. In this study, we combined chemical synthesis, in vitro testing, quantitative structure-activity relationship analysis, and docking studies to investigate the role of hydrogen bond (H-bond) donor/acceptor properties in transporter-ligand interaction. In a previous work, it has been shown that propafenone analogs with a 4-hydroxy-4-piperidine moiety exhibit a generally 10-fold higher P-gp inhibitory activity than expected based on their lipophilicity. Here, we specifically expanded the data set by introducing substituents at position 4 of the 4-phenylpiperidine moiety to assess the importance of H-bond donor/acceptor features in this region. The results suggest that indeed an H-bond acceptor, such as hydroxy and methoxy, increases the affinity by forming a H-bond with Tyr310.

The long zinc finger domain of PRDM9 forms a highly stable and long-lived complex with its DNA recognition sequence.

PR domain containing protein 9 (PRDM9) is a meiosis-specific, multi-domain protein that regulates the location of recombination hotspots by targeting its DNA recognition sequence for double-strand breaks (DSBs). PRDM9 specifically recognizes DNA via its tandem array of zinc fingers (ZnFs), epigenetically marks the local chromatin by its histone methyltransferase activity, and is an important tether that brings the DNA into contact with the recombination initiation machinery. A strong correlation between PRDM9-ZnF variants and specific DNA motifs at recombination hotspots has been reported; however, the binding specificity and kinetics of the ZnF domain are still obscure. Using two in vitro methods, gel mobility shift assays and switchSENSE, a quantitative biophysical approach that measures binding rates in real time, we determined that the PRDM9-ZnF domain forms a highly stable and long-lived complex with its recognition sequence, with a dissociation halftime of many hours. The ZnF domain exhibits an equilibrium dissociation constant (K D) in the nanomolar (nM) range, with polymorphisms in the recognition sequence directly affecting the binding affinity. We also determined that alternative sequences (15-16 nucleotides in length) can be specifically bound by different subsets of the ZnF domain, explaining the binding plasticity of PRDM9 for different sequences. Finally, longer binding targets are preferred than predicted from the numbers of ZnFs contacting the DNA. Functionally, a long-lived complex translates into an enzymatically active PRDM9 at specific DNA-binding sites throughout meiotic prophase I that might be relevant in stabilizing the components of the recombination machinery to a specific DNA target until DSBs are initiated by Spo11.

Effect of optical clearing agents on optical coherence tomography images of cervical epithelium.

Optical coherence tomography (OCT) can be used as an adjunct to colposcopy in order to detect precancerous and cancerous cervical lesions. Optical clearing agents (OCAs) temporarily reduce the optical scattering of biological tissues. The purpose of this study was to investigate their influence on OCT imaging. OCT images were taken from unsuspicious and suspicious areas of fresh conization specimens immediately after resection and 5, 10, and 20 min after application of dimethyl sulfoxide (DMSO) or polyethylene glycol (PEG). Corresponding histologies were obtained from all sites. The images taken 5, 10, and 20 min after application of OCA were compared to the initial images with respect to changes in brightness, contrast, and scanning depth using a standard nonparametric test of differences of proportions. Further, mean intensity backscattering curves were calculated from all OCT images in the histological groups CIN2, CIN3, inflammation, and normal epithelium. Mean difference profiles within each of these groups were determined, reflecting the mean differences between the condition before the application of OCA and the exposure times 5, 10, and 20 min, respectively. The null hypothesis was tested employing the Dicky-Fuller-test, Hotelings-test and run test. The visual analysis of 434 OCT images from 109 different sites of 24 conization specimens showed a statistically significant increase in brightness and contrast for normal and dysplastic epithelium after application of DMSO or PEG. Further, the analysis of mean intensity profiles suggests the existence of an increased backscattering intensity after application of DMSO or PEG. DMSO and PEG contribute substantially to optical clearing in cervical squamous epithelium and therefore influence OCT imaging in a positive way. With further refinement of the OCT technology, the observed changes may be beneficial in interpreting the tissue microstructure and identifying cervical intraepithelial neoplasia.

Vascular incorporation of endothelial colony-forming cells is essential for functional recovery of murine ischemic tissue following cell therapy.

OBJECTIVE: Cord blood-derived human endothelial colony-forming cells (ECFCs) bear a high proliferative capacity and potently enhance tissue neovascularization in vivo. Here, we investigated whether the leading mechanism for the functional improvement relates to their physical vascular incorporation or perivascular paracrine effects and whether the effects can be further enhanced by dual-cell-based therapy, including mesenchymal stem cells (MSCs). METHODS AND RESULTS: ECFCs or MSCs were lentivirally transduced with thymidine kinase suicide gene driven by the endothelial-specific vascular endothelial growth factor 2 (kinase insert domain receptor) promoter and evaluated in a hindlimb ischemia model. ECFCs and MSCs enhanced neovascularization after ischemic events to a similar extent. Dual therapy using ECFCs and MSCs further enhanced neovascularization. Mechanistically, 3 weeks after induction of ischemia followed by cell therapy, ganciclovir-mediated elimination of kinase insert domain receptor(+) cells completely reversed the therapeutic effect of ECFCs but not that of MSCs. Histological analysis revealed that ganciclovir effectively eliminated ECFCs incorporated into the vasculature. CONCLUSIONS: Endothelial-specific suicide gene technology demonstrates distinct mechanisms for ECFCs and MSCs, with complete abolishment of ECFC-mediated effects, whereas MSC-mediated effects remained unaffected. These data strengthen the notion that a dual-cell-based therapy represents a promising approach for vascular regeneration of ischemic tissue.

Psoas muscle index predicts time to rehospitalization in liver cirrhosis: An observational study.

Sarcopenia is frequent in liver cirrhosis (LC) where it is associated with morbidity and mortality. However, prognostic scores such as model for end-stage liver disease (MELD), MELD-sodium (MELD-Na), or Child-Turcotte-Pugh (CTP) do not contain sarcopenia as a variable. For this study, we utilized psoas muscle index (PMI) to objectively determine sarcopenia in hospitalized LC patients, and evaluated it as a predictor of time between discharge and readmission in LC. Abdominal computed tomography and magnetic resonance imaging scans of 65 consecutive LC patients were retrospectively examined to determine PMI. MELD, MELD-Na, and CTP were calculated from clinical data. PMI was then combined with CTP to form an experimental score: CTP sarcopenia (CTPS). For PMI alone and for each score, correlation with time between discharge and readmission for liver-related complications was calculated. PMI was also tested for correlation with sex, body mass index (BMI), MELD, MELD-Na, and CTP. CTPS was most closely correlated with time to readmission (R = 0.730; P < .001), followed by CTP (R = 0.696; P < .001), MELD-Na (R = 0.405; P = .009), and PMI alone (R = 0.388; P = .01). Correlation with MELD (R = 0.354; P = .05) was lowest. Additionally, there were significant differences in PMI between male and female individuals (5.16 vs 4.54 cm2/m2; P = .04) and in BMI between sarcopenic and nonsarcopenic individuals (29.63 vs 25.88 kg/m2; P = .009). Sarcopenia is an independent short-term prognostic factor in LC. By combining data on sarcopenia with CTP, we created an experimental score that predicts time to readmission better than MELD, MELD-Na, or CTP.

Combination of injectable multiple growth factor-releasing scaffolds and cell therapy as an advanced modality to enhance tissue neovascularization.

OBJECTIVE: Vasculogenic progenitor cell therapy for ischemic diseases bears great potential but still requires further optimization for justifying its clinical application. Here, we investigated the effects of in vivo tissue engineering by combining vasculogenic progenitors with injectable scaffolds releasing controlled amounts of proangiogenic growth factors. METHODS AND RESULTS: We produced biodegradable, injectable polylactic coglycolic acid-based scaffolds releasing single factors or combinations of vascular endothelial growth factor, hepatocyte growth factor, and angiopoietin-1. Dual and triple combinations of scaffold-released growth factors were superior to single release. In murine hindlimb ischemia models, scaffolds releasing dual (vascular endothelial growth factor and hepatocyte growth factor) or triple combinations improved effects of cord blood-derived vasculogenic progenitors. Increased migration, homing, and incorporation of vasculogenic progenitors into the vasculature augmented capillary density, translating into improved blood perfusion. Most importantly, scaffold-released triple combinations including the vessel stabilizer angiopoietin-1 enhanced the number of perivascular smooth muscle actin(+) vascular smooth muscle cells, indicating more efficient vessel stabilization. CONCLUSIONS: Vasculogenic progenitor cell therapy is significantly enhanced by in vivo tissue engineering providing a proangiogenic and provasculogenic growth factor-enriched microenvironment. Therefore, combined use of scaffold-released growth factors and cell therapy improves neovascularization in ischemic diseases and may translate into more pronounced clinical effects.

Prostaglandin E positively modulates endothelial progenitor cell homeostasis: an advanced treatment modality for autologous cell therapy.

AIMS: The mobilization of endothelial progenitor cells (EPC) and their functioning in postnatal neovascularization are tightly regulated. To identify new modulators of EPC homeostasis, we screened biologically active prostaglandin E compounds for their effects on EPC production, trafficking and function. METHODS AND RESULTS: We found that EPC are a rich source for prostaglandin E(2) (PGE(2)), stimulating their number and function in an auto- and paracrine manner. In vivo blockade of PGE(2) production by selective cyclooxygenase-2 inhibition virtually abrogated ischemia-induced EPC mobilization demonstrating its crucial role in EPC homeostasis following tissue ischemia. Conversely, ex vivo treatment of isolated EPC with the clinically approved PGE(1) analogue alprostadil enhanced EPC number and function. These effects were mediated by increased expression of the chemokine receptor CXCR4 and were dependent on nitric oxide synthase activity. Most importantly, ex vivo PGE(1) pretreatment of isolated EPC significantly enhanced their neovascularization capacity in a murine model of hind limb ischemia as assessed by laser Doppler analysis, exercise stress test and immunohistochemistry. CONCLUSIONS: The conserved role for PGE in the regulation of EPC homeostasis suggests that ex vivo modulation of the prostaglandin pathway in isolated progenitor cells may represent a novel and safe strategy to facilitate cell-based therapies.

PRDM9 forms a trimer by interactions within the zinc finger array.

PRDM9 is a trans-acting factor directing meiotic recombination to specific DNA-binding sites by its zinc finger (ZnF) array. It was suggested that PRDM9 is a multimer; however, we do not know the stoichiometry or the components inducing PRDM9 multimerization. In this work, we used in vitro binding studies and characterized with electrophoretic mobility shift assays, mass spectrometry, and fluorescence correlation spectroscopy the stoichiometry of the PRDM9 multimer of two different murine PRDM9 alleles carrying different tags and domains produced with different expression systems. Based on the migration distance of the PRDM9-DNA complex, we show that PRDM9 forms a trimer. Moreover, this stoichiometry is adapted already by the free, soluble protein with little exchange between protein monomers. The variable ZnF array of PRDM9 is sufficient for multimerization, and at least five ZnFs form already a functional trimer. Finally, we also show that only one ZnF array within the PRDM9 oligomer binds to the DNA, whereas the remaining two ZnF arrays likely maintain the trimer by ZnF-ZnF interactions.

The consequences of sequence erosion in the evolution of recombination hotspots.

Meiosis is initiated by a double-strand break (DSB) introduced in the DNA by a highly controlled process that is repaired by recombination. In many organisms, recombination occurs at specific and narrow regions of the genome, known as recombination hotspots, which overlap with regions enriched for DSBs. In recent years, it has been demonstrated that conversions and mutations resulting from the repair of DSBs lead to a rapid sequence evolution at recombination hotspots eroding target sites for DSBs. We still do not fully understand the effect of this erosion in the recombination activity, but evidence has shown that the binding of trans-acting factors like PRDM9 is affected. PRDM9 is a meiosis-specific, multi-domain protein that recognizes DNA target motifs by its zinc finger domain and directs DSBs to these target sites. Here we discuss the changes in affinity of PRDM9 to eroded recognition sequences, and explain how these changes in affinity of PRDM9 can affect recombination, leading sometimes to sterility in the context of hybrid crosses. We also present experimental data showing that DNA methylation reduces PRDM9 binding in vitro Finally, we discuss PRDM9-independent hotspots, posing the question how these hotspots evolve and change with sequence erosion.This article is part of the themed issue 'Evolutionary causes and consequences of recombination rate variation in sexual organisms'.

Subtle Structural Differences Trigger Inhibitory Activity of Propafenone Analogues at the Two Polyspecific ABC Transporters: P-Glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP).

The transmembrane ABC transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are widely recognized for their role in cancer multidrug resistance and absorption and distribution of compounds. Furthermore, they are linked to drug-drug interactions and toxicity. Nevertheless, due to their polyspecificity, a molecular understanding of the ligand-transporter interaction, which allows designing of both selective and dual inhibitors, is still in its infancy. This study comprises a combined approach of synthesis, in silico prediction, and in vitro testing to identify molecular features triggering transporter selectivity. Synthesis and testing of a series of 15 propafenone analogues with varied rigidity and basicity of substituents provide first trends for selective and dual inhibitors. Results indicate that both the flexibility of the substituent at the nitrogen atom, as well as the basicity of the nitrogen atom, trigger transporter selectivity. Furthermore, inhibitory activity of compounds at P-gp seems to be much more influenced by logP than those at BCRP. Exploiting these differences further should thus allow designing specific inhibitors for these two polyspecific ABC-transporters.

HPV vaccination: acceptance and influencing factors among young men in Germany.

AIMS: This study aims to determine the factors that influence the acceptance of the HPV vaccination among German males. PATIENT & METHODS: In 2014, we conducted a population-based cross-sectional study in men aged 15-25 years. A questionnaire was mailed to male trainees of the Bayerische Motorenwerke AG (BMW) insured at the BMW health insurance company. RESULTS: The response rate was 10.8%. Of the 378 included men, 74.1% would agree to receive HPV vaccination. Most men primarily consult their physician for health-related topics, but 92.9% had never been informed about HPV infection, risk factors and prevention methods by their doctor. CONCLUSION: Our results demonstrate a high acceptance of male HPV vaccination. Education about HPV infection is low and should be intensified by medical professionals.

Thyroid Hormones and Vitamin D in Patients with Breast Cancer with Mutations in BRCA1 or BRCA2 Genes.

AIM: The thyroid hormones free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone (TSH) and vitamin D seem to be involved in the process of differentiation and proliferation of breast tissue. Little is known about these factors in breast cancer 1 and 2 (BRCA1/BRCA2)-mutation carriers with breast cancer (BC). The purpose of this investigation was to evaluate the association of thyroid gland function and vitamin D with BC in patients with BRCA mutations. PATIENTS AND METHODS: At the Department of Hereditary Breast and Ovarian Cancer of the Ludwig Maximilian University Hospital of Munich, 40 patients with BC (10 patients with mutations in the BRCA1 gene, 10 with mutations in the BRCA2 gene, and 20 without mutations, as control group) were selected for analysis of the following parameters: fT3, fT4, TSH and vitamin D. The primary diagnosis was made between 21 and 62 years of age. The patients were matched by age. Anamnestic data were evaluated concerning disorders of the thyroid gland and primary BC diagnosis. RESULTS: In patients with BC, BRCA mutations are not associated with thyroidal dysfunctions. A significantly increased level of vitamin D in BRCA2-mutation carriers compared to those without mutation (p=0.02) was detected. The grade of the tumors in the BRCA2 group was better than in those with mutation. BRCA1-mutation carriers had an increased incidence of primary BC diagnosis during pregnancy (30% vs. 0%) in comparison to those without mutation. CONCLUSION: No association between the thyroid hormones and BC in BRCA1/2-mutation carriers was found. Vitamin D was significantly elevated in BRCA2-mutation carriers and the observation of a better tumor grade in this group could be consistent with the ability of vitamin D to inhibit growth and induce differentiation.

Comparison of two surgical methods for the treatment of CIN: classical LLETZ (large-loop excision of the transformation zone) versus isolated resection of the colposcopic apparent lesion - study protocol for a randomized controlled trial.

BACKGROUND: In compliance with national and international guidelines, non-pregnant women with cervical intraepithelial neoplasia grade 3 should be treated by cervical conization. According to the definition of the large loop excision of the transformation zone (LLETZ) operation, the lesion needs to be resected, including the transformation zone. It is well known from the literature that the cone size directly correlates with the risk of preterm delivery in the course of a future pregnancy. Thus, it would be highly desirable to keep the cone dimension as small as possible while maintaining the same level of oncological safety. METHODS/DESIGN: The aim of this study is to analyze whether resection of the lesion only, without additional excision of the transformation zone, is equally as effective as the classical LLETZ operation regarding oncological outcome. We are performing this prospective, patient-blinded multicenter trial by randomly assigning women who need to undergo a LLETZ operation for cervical intraepithelial neoplasia grade 3 to either of the following two groups at a ratio of 1:1: (1) additional resection of the transformation zone or (2) resection of the lesion only. To evaluate equal oncological outcome, we are performing human papillomavirus (HPV) tests 6 and 12 months postoperatively. The study is designed to consider the lesion-only operation as oncologically not inferior if the rate of HPV high-risk test results is not higher than 5 % compared with the HPV high-risk rate of women undergoing the classical LLETZ operation. DISCUSSION: In case that non-inferiority of the "lesion-only" method can be demonstrated, this operation should eventually become standard treatment for all women at childbearing age due to the reduction in risk of preterm delivery. TRIAL REGISTRATION: German Clinical Trials Register (DRKS) Identifier: DRKS00006169 . Date of registration: 30 July 2014.

Adiponectin pretreatment counteracts the detrimental effect of a diabetic environment on endothelial progenitors.

OBJECTIVE: It has been shown that vascular progenitors from patients with diabetes are dysfunctional. However, therapeutic strategies to counteract their reduced functional capacity are still lacking. Because adiponectin has reported salutary effects on endothelial function, we investigated the functional effects of globular adiponectin (gAcrp), the active domain of adiponectin, on isolated endothelial colony-forming cells (ECFC). RESEARCH DESIGN AND METHODS: ECFC were isolated from peripheral blood of type 2 diabetic patients (dmECFC) and compared with ECFC of healthy young volunteers (yECFC) and nondiabetic age-matched control subjects (hECFC). Cells were treated with gAcrp for 48 h followed by assessment of cell counts, cell cycle analysis, and migration capacity. For in vivo evaluation, human ECFC were injected into normoglycemic or streptozotocin-induced hyperglycemic nu/nu mice after hind limb ischemia. RESULTS: Whereas dmECFC were functionally impaired compared with yECFC and hECFC, gAcrp significantly enhanced their in vitro proliferation and migratory activity. In vitro effects were significantly stronger in hECFC compared with dmECFC and were mediated through the cyclooxygenase-2 pathway. Most important, however, we observed a profound and sustained increase of the in vivo neovascularization in mice receiving gAcrp-pretreated dmECFC compared with untreated dmECFC under both normoglycemic and hyperglycemic conditions. CONCLUSIONS: Pretreatment of ECFC with gAcrp enhanced the functional capacity of ECFC in vitro and in vivo in normoglycemic and hyperglycemic environments. Therefore, preconditioning of dmECFC with gAcrp may be a novel approach to counteract their functional impairment in diabetes.

Resection of perihilar biliary schwannoma.

INTRODUCTION: Schwannomas are usually benign nerve sheath tumors, which typically arise in the head, neck, spinal cord and extremities. Schwannoma of the biliary tract is an extremely rare finding. Patients generally lack symptoms and seek medical attention when tumor growth causes obstructive jaundice. Preoperative diagnosis is difficult and resection is the treatment of choice. METHODS: A 54 year-old female with history of back and right labia minor melanoma for which she underwent complete excision and right inguinal lymph node dissection more than 10 years ago, was evaluated for new onset gastroesophageal reflux symptoms and found to have markedly abnormal liver enzymes. Imagining studies revealed intrahepatic ductal dilatation and a 5.2 cm mass in the porta hepatis that was not consistent with cholangiocarcinoma or hepatocellular carcinoma. Multiple percutaneous biopsies of the mass failed to provide a definitive diagnosis. With a high clinical suspicion of metastatic melanoma and no other evident sites of disease, operative intervention was undertaken for diagnosis and definitive treatment. RESULTS: Diagnostic laparoscopy was performed initially, but access to the mass was difficult, given its location. Subsequently, the patient underwent laparotomy, with tumor excision, common bile duct resection and hepato-jejunostomy. Pathologic examination and analysis were consistent with cellular schwannoma. Postoperatively, the patient recovered uneventfully, and liver function studies returned to normal. CONCLUSION: Schwannomas are uncommon tumors, which very rarely arise from the biliary tract and cause biliary obstruction. Exploration is indicated in order to establish the diagnosis and to render definitive treatment.