What is the most effective treatment for nocturia or nocturnal incontinence in adult women?

Acknowledgments The authors express their thanks to F.C. Burkhard for invaluable logistic support during the conception of the manuscript.Peer reviewedPostprin

Reliability and validity of the German version of the Structured Interview of Personality Organization (STIPO)

Background: The assessment of personality organization and its observable behavioral manifestations, i.e. personality functioning, has a long tradition in psychodynamic psychiatry. Recently, the DSM-5 Levels of Personality Functioning Scale has moved it into the focus of psychiatric diagnostics. Based on Kernberg's concept of personality organization the Structured Interview of Personality Organization (STIPO) was developed for diagnosing personality functioning. The STIPO covers seven dimensions: (1) identity, (2) object relations, (3) primitive defenses, (4) coping/rigidity, (5) aggression, (6) moral values, and (7) reality testing and perceptual distortions. The English version of the STIPO has previously revealed satisfying psychometric properties. Methods: Validity and reliability of the German version of the 100-item instrument have been evaluated in 122 psychiatric patients. All patients were diagnosed according to the Diagnostic and Statistical Manual for Mental Disorders (DSM-IV) and were assessed by means of the STIPO. Moreover, all patients completed eight questionnaires that served as criteria for external validity of the STIPO. Results: Interrater reliability varied between intraclass correlations of .89 and 1.0, Crohnbach's a for the seven dimensions was .69 to .93. All a priori selected questionnaire scales correlated significantly with the corresponding STIPO dimensions. Patients with personality disorder (PD) revealed significantly higher STIPO scores (i.e. worse personality functioning) than patients without PD; patients cluster B PD showed significantly higher STIPO scores than patients with cluster C PD. Conclusions: Interrater reliability, Crohnbach's a, concurrent validity, and differential validity of the STIPO are satisfying. The STIPO represents an appropriate instrument for the assessment of personality functioning in clinical and research settings

Reliability and validity of the German version of the Structured Interview of Personality Organization (STIPO)

Background: The assessment of personality organization and its observable behavioral manifestations, i.e. personality functioning, has a long tradition in psychodynamic psychiatry. Recently, the DSM-5 Levels of Personality Functioning Scale has moved it into the focus of psychiatric diagnostics. Based on Kernberg's concept of personality organization the Structured Interview of Personality Organization (STIPO) was developed for diagnosing personality functioning. The STIPO covers seven dimensions: (1) identity, (2) object relations, (3) primitive defenses, (4) coping/rigidity, (5) aggression, (6) moral values, and (7) reality testing and perceptual distortions. The English version of the STIPO has previously revealed satisfying psychometric properties. Methods: Validity and reliability of the German version of the 100-item instrument have been evaluated in 122 psychiatric patients. All patients were diagnosed according to the Diagnostic and Statistical Manual for Mental Disorders (DSM-IV) and were assessed by means of the STIPO. Moreover, all patients completed eight questionnaires that served as criteria for external validity of the STIPO. Results: Interrater reliability varied between intraclass correlations of .89 and 1.0, Crohnbach's a for the seven dimensions was .69 to .93. All a priori selected questionnaire scales correlated significantly with the corresponding STIPO dimensions. Patients with personality disorder (PD) revealed significantly higher STIPO scores (i.e. worse personality functioning) than patients without PD; patients cluster B PD showed significantly higher STIPO scores than patients with cluster C PD. Conclusions: Interrater reliability, Crohnbach's a, concurrent validity, and differential validity of the STIPO are satisfying. The STIPO represents an appropriate instrument for the assessment of personality functioning in clinical and research settings

Use of an innovative system and nanotechnology-based strategy for therapeutic applications of Gla-rich protein (GRP)

Introduction: Gla-rich protein (GRP) is a vitamin K-dependent protein (VKDP) acting as a calcification inhibitor and anti-inflammatory agent in cardiovascular and articular systems, and THP1 monocyte/macrophage cells [1,2]. Calcification and inflammation processes are known to be involved in the etiology of several calcification-related chronic inflammatory diseases such as atherosclerosis, CKD and osteoarthritis, in a complex bi-directional interplay that drives disease progression. Here, we developed an innovative system to produce human c-carboxylated GRP (cGRP), and a nanotechnology strategy based on GRP loading into extracellular vesicles (EVs) as a gold standard delivery system for GRP in therapeutic applications. Materials and methods: Human GRP protein was co-expressed with c-carboxylase enzyme (GGCX), vitamin K oxidoreductase (GGCX) and furin, in the insect cell baculovirus system in the presence of vitamin K. GRP released in the cell culture media was characterized by mass spectrometry based techniques and Western blot analysis. EVs released by the insect cells overexpressing GRP were isolated by ultracentrifugation, and characterized for GRP content through TEM-immunogold staining, Western blot, ELISA, qPCR. Functional assays using isolated EVs containing GRP were performed in primary vascular smooth muscle cells (VSMCs) and THP1 monocyte/macrophage cells, for anti-mineralizing and anti-inflammatory screening.Results: GRP released in the cell culture media when co-expressed with GGCX, VKOR and furin in the presence of vitamin K, is processed at the pro-peptide and contain Gla residues. EVs released by the insect cells in this system were shown to be loaded with GRP protein and mRNA, and capable of reducing ECM calcium deposition of calcifying VSMCs and the production of TNFa in THP1 monocyte/macrophage cells stimulated with LPS. Discussion and conclusions: While the successful production of human cGRP constitutes a major achievement, this innovative methodology will open new opportunities for the production of other biological active VKDPs. Furthermore, EVs loaded with GRP were shown to have anti-mineralizing and anti-inflammatory properties, with promising therapeutic potentialities for calcification-related chronic inflammatory diseases.Portuguese Foundation for Science and Technology (EU/PID1003201)info:eu-repo/semantics/publishedVersio

-Tetrachlorodibenzo-p-dioxin-Mediated Impairment of B Cell Differentiation Involves Dysregulation of Paired Box 5 (Pax5) Isoform, Pax5a

ABSTRACT The persistent environmental contaminant and immunotoxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), markedly suppresses humoral immune responses. We recently reported impaired down-regulation of paired box 5 (Pax5), a repressor of B cell differentiation and concomitant suppression of the IgM response by TCDD in the murine CH12.LX B cell line. The objectives of the current study were to determine the impact of TCDD treatment on molecular outcomes characteristic of terminal B cell differentiation and to assess the role that Pax5 isoforms plays in the suppression of B cell differentiation by TCDD. In this study, we show that the highly abundant fulllength Pax5 isoform, Pax5a, and at least two additional modestly expressed Pax5 isoforms were expressed in CH12.LX and splenic B cells. In lipopolysaccharide (LPS)-activated B cells, all of the identified Pax5 isoforms were synchronously down-regulated, and in the presence of TCDD cotreatment they were abnormally and synchronously elevated, suggesting a common mechanism of regulation. Furthermore, B cell differentiation markers X-box protein-1 and major histocompatibility complex class II showed that the levels to which Pax5 was derepressed by TCDD were sufficient to impair B cell differentiation and immunoglobulin gene expression. Confirming the involvement of Pax5, ectopic expression of Pax5a in the LPS-activated CH12.LX cells closely mimicked the suppression of the IgM response by TCDD. In summary, our results demonstrate that Pax5a has a critical role in both the TCDD-mediated impairment of B cell differentiation and the suppression of the humoral immune response. Suppression of primary humoral immune responses is one of the most sensitive sequela associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous environmental contaminant. This suppression is characterized by a striking reduction in plasma cell formation and IgM secretion, and it is mediated through a direct effect by TCDD on B cells Activation by antigen or via polyclonal activators (e.g., 463 lipopolysaccharide; LPS) induces B cells to undergo cycles of intense proliferation (i.e., clonal expansion) followed by terminal differentiation into plasma cells. Terminally differentiated plasma cells specialize in secretion of antigen-specific Ig. A number of phenotypic changes distinguish terminally differentiated plasma cells from the resting B cells Materials and Methods Chemicals. TCDD, in 100% dimethyl sulfoxide (DMSO), was purchased from AccuStandard (New Heaven, CT). DMSO and LPS were purchased from Sigma-Aldrich (St. Louis, MO). Mice. Virus-free, female B6C3F1mice (six weeks old) were purchased from Charles River (Portage, MI). On arrival, mice were randomly grouped with five per plastic cage on sawdust bedding. Mice had free access to food (Purina Certified Laboratory Chow) and water at all times. The mouse holding rooms were maintained at 21 to 24°C with 40 to 60% relative humidity on a 12-h light/dark cycle. All of the experimental procedures and conditions were performed according to the guidelines of the All University Committee on Animal Use and Care at Michigan State University (East Lansing, MI). Cell Line. The CH12.LX B cell line was derived from the murine B cell lymphoma, CH12, which arose in B10.H-2aH-4bP/Wts mice (B10.A ϫ B10.129) and has been characterized previously Flow Cytometric Analysis. Cells were harvested from culture at the indicated times by centrifugation at 300g for 10 min at 4°C, washed twice in ice-cold 1ϫ Hanks' balanced-salt solution, and stained using the BD Cytofix/Cytoperm kit (BD Pharmingen, San Diego, CA) according to the manufacturer's instructions. In brief, cells were incubated with 1 g/10 6 cells of purified rat anti-mouse CD16/CD32 monoclonal antibody (BD Pharmingen) for 15 min at 4°C to prevent nonspecific binding and then stained with surface marker detection antibody [anti-fluorescein isothiocyanate (FITC)-conjugated mouse anti-mouse I-A P or anti-allophycocyanin-conjugated anti-mouse CD19] or a respective isotype control (BD Pharmingen). To exclude nonviable cells, 2 l of 7-aminoactinomycin D (7-AAD) solution (Sigma-Aldrich) containing 1 mg of 7-AAD, 50 l methanol, and 950 l of Hanks' balanced-salt solution were added simultaneously with detection antibodies to the cells in 50 l of staining buffer. The cells were then fixed, washed, and maintained in staining buffer containing 10 mM actinomycin D (Sigma-Aldrich) to prevent 7-AAD leakage from fixed cells. For the detection of nuclear Pax5 protein, cells were fixed and permeabilized before staining with anti-Pax5 antibody or isotype control (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Fluorescence detection was performed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) on 10,000 viable cells per sample. Data analysis was performed using BD CellQuest Pro software (BD Biosciences). In Vitro Polyclonal IgM Antibody-Forming Cell Response. Single-cell suspensions of splenocytes from naive mice were adjusted to 1 ϫ 10 6 cells/ml in RPMI 1640 medium (Invitrogen) supplemented with 10% bovine calf serum (HyClone Laboratories), 100 units/ml penicillin, 100 g/ml streptomycin, and 50 M 2-mercaptoethanol. Spleen cells were transferred to 48-well culture plates in 500-l aliquots with four wells per treatment group. TCDD (3, 10, or 30 nM) and/or vehicle (0.01% DMSO) were added directly to each well in 5-l aliquots. The splenocytes were sensitized with 10 g/ml LPS and cultured for 3 days in a pressurized chamber at 5.0 psi containing 10% O 2 , 7% CO 2 , and 83% N 2 gas mixture with continuous rocking at 37°C. Enumeration of the IgM antibody-forming cell (AFC) response was performed using trinitrophenol-haptenated sheep red blood cells as described previously the cell mixture was poured onto a 100 ϫ 15-mm Petri dish and immediately covered with a 45 ϫ 50-mm glass coverslip. Upon solidification of the agar, the Petri dishes were placed in a humidified 37°C incubator overnight to allow for plaque formation. The number of splenocytes from each spleen was determined using a Coulter Counter (Beckman Coulter, Fullerton, CA). Results are expressed as AFC/10 6 viable splenocytes Ϯ S.E. Splenocyte viability was measured using pronase (EMD Biosciences, San Diego, CA) as described previously Purification of Splenic B Cells. Spleens were removed aseptically and made into a single-cell suspension. B cells were then isolated using the B Cell Isolation Kit (Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer's protocol. In brief, 40 l of MACS buffer (phosphate-buffered saline solution, pH 7.2, supplemented with 0.5% bovine serum albumin and 2 mM EDTA, 4°C) per 10 7 cells was used to suspend the splenocytes, and 10 l of Biotin-Antibody Cocktail (Miltenyi Biotec Inc.) per 10 7 cells was added to label non-B cells. After incubation at 4 to 8°C for 10 min, 30 l of buffer and 20 l of Anti-Biotin Microbeads (Miltenyi Biotec Inc.) per 10 7 cells were added. The cell suspension was incubated for another 15 min at 4 to 8°C, washed with 10 to 20 times the labeling volume, and then centrifuged at 300g for 5 min. The cell pellet was finally resuspended in 500 l of buffer per 1 ϫ 10 8 cells, and passed through the prerinsed LS column (Miltenyi Biotec Inc.), followed by four washes of the column with 3 ml of buffer. The entire effluent containing the purified B cell fraction was collected. The purity of the isolated B cells was evaluated using flow cytometry to enumerate the percentage of CD19 ϩ cells through directly labeling with FITC-conjugated anti-CD19 antibody (BD Biosciences). Real-Time Polymerase Chain Reaction. Total RNA was isolated from naive or LPS-activated cells using a SV Total RNA Isolation kit (Promega, Madison, WI). To synthesize cDNA, 1000 ng/ sample of total RNA was incubated with 600 ng of random primer (Invitrogen) in 10 l of endonuclease-free water at 70°C for 10 min, cooled on ice for 10 min, and reverse transcribed in 20 l of 1ϫ First-Strand Synthesis buffer (Invitrogen), containing 0.2 mM deoxynucleotide triphosphates, 10 mM dithiothreitol, and 200 units of SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42°C for 60 min, and the reaction was stopped by incubation at 75°C for 15 min. Real-time polymerase chain reaction (PCR) detection was performed using TaqMan primers and probes Isolation of Pax5a cDNA. cDNA was obtained by PCR amplification of total RNA isolated from CH12.LX cells. Total RNA was extracted with the SV Total RNA Isolation kit (Promega), and firststrand cDNA was synthesized by reverse transcription (RT) using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). PCR was carried out as follows: one initial 2-min denaturing step at 94°C followed by denaturation for 30 s at 94°C, annealing for 30 s at 58°C, and extension for 90 s at 72°C during 30 cycles in the presence of Pfu polymerase (Stratagene, La Jolla, CA) under the conditions suggested by the supplier (at a final primer concentration of 0.5 M). Sequences of the primers designed for this PCR reaction were as follows: 5Ј-CTGTCCATTTCATCAAGTCCTGA-3Ј and 5Ј-ACC-GTCACTACCCTCAGAG-3Ј. The resulting PCR product of 1.2 kilobase was separated in 1% agarose gel electrophoresis in 1ϫ TBE buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 8.3) and stained with ethidium bromide at 1 g/ml. The PCR fragment was eluted from agarose using the QIAquick gel extraction kit (QIAGEN, Valencia, CA). Cloning and Ectopic Expression for Pax5a cDNA. Pax5a cDNA, generated as described above, was inserted into the pcDNA 3.1 V5-His-TOPO vector (Invitrogen) according to the manufacturer's protocol. The new Pax5a-V5-His tag vector sequence was confirmed by sequencing. Additional PCR reactions, in which Pax5a-V5-His vector was used as template, were performed to generate a new Pax5a expression plasmid, Pax5a-V5-His-GFP. The primer used for 5Ј-end cloning was as follows: 5Ј-CTC ACT ATA GGG AGA TCT AAG CTG GCT AGT-3Ј (BglII restriction site is underlined); and the primer used for the 3Јend cloning was as follows: 5Ј-TGA TCA GCG GGT TTA AAA GCT TTG GGA TGG TG-3Ј (HindIII restriction site is underlined). PCR products of 1.38 kilobase were obtained and isolated as described above. The Pax-5a-V5-His PCR fragment was then cloned into the vector phCMV-C-GFP (Genlantis, San Diego, CA) by a standard ligation reaction using T4-DNA ligase (New England Biolabs, Ipswich, MA). The phCMV vector from Genlantis contains an optimized cytomegalovirus promoter and intron sequences and a kanamycin/neomycin resistance gene for generation of stable cell lines. Transfection of Pax5a-V5-His-GFP. CH12.LX cells (2.5 ϫ 10 6 ) were transfected with 5 g of each plasmid using Amaxa Nucleofector (Amaxa Inc., Gaithersburg, MD). Cells, DNA, and 100 l of 90:20 (solution V supplement) were mixed and electroporated using program A-20 following the manufacturer's recommendations. A total of TCDD Impairment of B Cell Differentiation by Altered Pax5 465 2.5 ϫ 10 7 cells was used in each experiment. The backbone plasmid, phCMV-C-GFP, was used as a control in all of the transfection experiments. After electroporation, cells were incubated at 37°C in an atmosphere of 5% CO 2 for 8 h. Eight hours after transfection, cells were collected and sorted by flow cytometry for green fluorescent protein (GFP)-expressing cells. Cells that showed fluorescence above the level of fluorescence of naive CH12.LX cells were isolated using a BD FACSCalibur. The isolated cells were counted and LPS-activated for assessments of IgM secretion as determined by enzymelinked immunosorbent assay (ELISA). An aliquot of the sorted cells was also used to prepare protein extracts to determine endogenous/ ectopic Pax5a levels by Western blotting and by flow cytometry. IgM ELISA. IgM ELISA was performed as described previously Western Blotting. Proteins were extracted from transfected cells before and after sorting by flow cytometry. Protein extracts were resolved on 4 to 12% Nu-Page gradient gel (Invitrogen) and then transferred to a nitrocellulose membrane (GE Healthcare, Piscataway, NJ). Immunoblotting was performed using anti-␤-actin and anti-Pax5 antibodies (Santa Cruz Biotechnology, Inc.). SuperSignal West Pico reagent (Pierce, Rockford, IL) was used for protein detection. Statistical Analysis of Data. Mean Ϯ S.E. was determined for each treatment group of a given PCR or ELISA experiment. Statistical differences between groups in each experiment were determined by a two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test for PCR experiments and by a one-way ANOVA followed by Bonferroni's post hoc test for ELISA experiments. Results TCDD Decreases the Levels of XBP-1 in LPS-Activated B Cells. Previous studies in LPS-activated CH12.LX cells demonstrated robust suppression of the humoral IgM response by TCDD, which correlated with a marked reduction in XBP-1 protein levels Additional studies were performed to determine whether LPS and/or TCDD treatment affects the post-transcriptional modification of XBP-1. Transcriptional activity of XBP-1 is known to be enhanced by a splicing event, which removes a 26-nucleotide-long fragment from XBP-1 mRNA, resulting in an open reading frame shift and yielding a larger XBP-1 466 Schneider et al. protein that is more potent as a transcription factor TCDD Attenuates Cell Surface MHC Class II DownRegulation in LPS-Activated CH12.LX Cells. An additional event closely associated with terminal differentiation of B cells is the down-regulation of MHC class II on the cell membrane. Levels of surface MHC class II in LPS-activated CH12.LX cells and in splenic B cells (i.e., CD19 ϩ ), in the presence and absence of TCDD, were monitored by flow cytometry. LPS activation induced a down-regulation of MHC class II on the CH12.LX cell surface between 24 and 72 h of culture compared with the time-matched naive control TCDD Attenuates the LPS-Induced Down-Regulation of Pax5 Protein in LPS-Activated CH12.LX Cells. We also examined the impact of TCDD on Pax5, a repressor of B cell differentiation. Expression of Pax5 protein was characterized in splenic B cells and CH12.LX cells by flow cytometry facilitating the evaluation of individual cells in contrast to prior Western blot studies that we performed exclusively in CH12.LX cells Characterization of Pax5 Isoforms in CH12.LX Cells. In light of the altered Pax5 expression observed by flow cytometry and by real-time PCR, studies were undertaken to further characterize the effect of TCDD on Pax5 regulation. In particular, we examined the role of alternative splicing in the deregulation of Pax5 by TCDD Additional studies performed in CH12.LX cells showed that the levels of amplicons I, II, and III were down-regulated by LPS between 24 and 72 h, as determined by real-time quantitative PC

Dimensions of professional competences for interventions towards sustainability

This paper investigates sustainability competences through the eyes of professional practitioners in the field of sustainability and presents empirical data that have been created using an action research approach. The design of the study consists of two workshops, in which professional practitioners in interaction with each other and the facilitators are invited to explore and reflect on the specific knowledge, skills, attitudes and behaviours necessary to conduct change processes successfully towards sustainability in a variety of business and professional contexts. The research focuses on the competences associated with these change processes to devise, propose and conduct appropriate interventions that address sustainability issues. Labelled ‘intervention competence’, this ability comprises an interlocking set of knowledge, skills, attitudes and behaviours that include: appreciating the importance of (trying to) reaching decisions or interventions; being able to learn from lived experience of practice and to connect such learning to one’s own scientific knowledge; being able to engage in political-strategic thinking, deliberations and actions, related to different perspectives; the ability for showing goal-oriented, adequate action; adopting and communicating ethical practices during the intervention process; being able to cope with the degree of complexity, and finally being able to translate stakeholder diversity into collectively produced interventions (actions) towards sustainability. Moreover, this competence has to be practised in contexts of competing values, non-technical interests and power relations. The article concludes with recommendations for future research and practice

The differential impact of a 6-versus 12-month pharmacist-led interprofessional medication adherence program on medication adherence in patients with diabetic kidney disease: the randomized PANDIA-IRIS study

Background: For every 100 patients with diabetes, 40 will develop diabetic kidney disease (DKD) over time. This diabetes complication may be partly due to poor adherence to their prescribed medications. In this study, we aimed to evaluate the differential impact of a 6- versus 12-month pharmacist-led interprofessional medication adherence program (IMAP) on the components of adherence (i.e., implementation and discontinuation) in patients with DKD, during and after the intervention.Methods: All included patients benefited from the IMAP, which consists in face-to-face regular motivational interviews between the patient and the pharmacist based on the adherence feedback from electronic monitors (EMs), in which the prescribed treatments were delivered. Adherence reports were available to prescribers during the intervention period. Patients were randomized 1:1 into two parallel arms: a 12-month IMAP intervention in group A versus a 6-month intervention in group B. Adherence was monitored continuously for 24 months post-inclusion during the consecutive intervention and follow-up phases. In the follow-up phase post-intervention, EM data were blinded. Blood pressure was measured by the pharmacist at each visit. The repeated measures of daily patient medication intake outcomes (1/0) to antidiabetics, antihypertensive drugs, and statins were modeled longitudinally using the generalized estimated equation in both groups and in both the intervention and the follow-up phases.Results: EM data of 72 patients were analyzed (34 in group A and 38 in group B). Patient implementation to antidiabetics and antihypertensive drugs increased during the IMAP intervention phase and decreased progressively during the follow-up period. At 12 months, implementation to antidiabetics was statistically higher in group A versus group B (93.8% versus 86.8%; Δ 7.0%, 95% CI: 5.7%; 8.3%); implementation to antihypertensive drugs was also higher in group A versus B (97.9% versus 92.1%; Δ 5.8%, 95% CI: 4.8%; 6.7%). At 24 months, implementation to antidiabetics and antihypertensive drugs remained higher in group A versus B (for antidiabetics: 88.6% versus 85.6%; Δ 3.0%, 95% CI: 1.7%; 4.4% and for antihypertensive drugs: 94.4% versus 85.9%; Δ 8.5%, 95% CI: 6.6%; 10.7%). No difference in pharmacy-based blood pressure was observed between groups. Implementation to statins was comparable at each time point between groups. Three patients discontinued at least one treatment; they were all in group B. In total, 46% (16/35) of patients in the 12-month intervention versus 37% (14/38) of patients in the 6-month intervention left the study during the intervention phase, mainly due to personal reasons.Conclusion: The IMAP improves adherence to chronic medications in patients with DKD. The longer the patients benefit from the intervention, the more the implementation increases over time, and the more the effect lasts after the end of the intervention. These data suggest that a 12-month rather than a 6-month program should be provided as a standard of care to support medication adherence in this population. The impact on clinical outcomes needs to be demonstrated.Clinical Trial Registration:Clinicaltrials.gov, identifier NCT04190251_PANDIA IRIS

Specific binding of a hexanucleotide to HIV-1 reverse transcriptase: a novel class of bioactive molecules

Short oligonucleotides below 8–10 nt in length adopt relatively simple structures. Accordingly, they represent interesting and so far unexplored lead compounds as molecular tools and, potentially, for drug development as a rational improvement of efficacy seem to be less complex than for other classes of longer oligomeric nucleic acid. As a ‘proof of concept’, we describe the highly specific binding of the hexanucleotide UCGUGU (Hex-S3) to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) as a model target. Ultraviolet (UV) cross-linking studies and competition experiments with primer/template substrates and a RT-directed aptamer suggest site-specific binding of Hex-S3 to the large subunit (p66) of the viral enzyme. The affinity of 5.3 μM is related to hexanucleotide-specific suppression of HIV-1 replication in human cells by up to three orders of magnitude indicating that Hex-S3 exerts specific and biologically relevant activity. Experimental evidence described here further suggests a systematic hexamer array-based search for new tools for molecular biology and novel lead compounds in nucleic acid-based drug development

Contrast-Enhanced Ultrasound with VEGFR2-Targeted Microbubbles for Monitoring Regorafenib Therapy Effects in Experimental Colorectal Adenocarcinomas in Rats with DCE-MRI and Immunohistochemical Validation

Objectives To investigate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring therapy effects of regorafenib on experimental colon carcinomas in rats with correlation to dynamic contrast-enhanced MRI (DCE-MRI) and immunohistochemistry. Materials and Methods: Human colorectal adenocarcinoma xenografts (HT-29) were implanted subcutaneously in n =21 (n = 11 therapy group;n = 10 control group) female athymic nude rats (Hsd: RH-Foxn1 (mu)). Animals were imaged at baseline and after a one-week daily treatment with regorafenib or a placebo (10 mg/kg bodyweight), using CEUS with VEGFR2-targeted microbubbles and DCE-MRI. In CEUS tumor perfusion was assessed during an early vascular phase (wash-in area under the curve = WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity after 8 (SI8min) and 10 minutes (SI10min)), using a conventional 15L8 linear transducer (transmit frequency 7 MHz, dynamic range 80 dB, depth 25 mm). In DCE-MRI functional parameters plasma flow (PF) and plasma volume (PV) were quantified. For validation purposes, CEUS parameters were correlated with DCE-MRI parameters and immunohistochemical VEGFR2, CD31, Ki-67 and TUNEL stainings. Results: CEUS perfusion parameter WiAUC decreased significantly (116,989 +/- 77,048 a.u. to 30,076 +/- 27,095a.u.;p = 0.005) under therapy with no significant changes (133,932 +/- 65,960 a.u. to 84,316 +/- 74,144 a.u.;p = 0.093) in the control group. In the therapy group, the amount of bound microbubbles in the late phase was significantly lower in the therapy than in the control group on day 7 (SI8min: 283 +/- 191 vs. 802 +/- 460 a.u.;p = 0.006);SI10min: 226 +/- 149 vs. 645 +/- 461 a.u.;p = 0.009). PF and PV decreased significantly (PF: 147 +/- 58 mL/100 mL/min to 71 +/- 15 mL/100 mL/min;p = 0.003;PV: 13 +/- 3% to 9 +/- 4%;p = 0.040) in the therapy group. Immunohistochemistry revealed significantly fewer VEGFR2 (7.2 +/- 1.8 vs. 17.8 +/- 4.6;p < 0.001), CD31 (8.1 +/- 3.0 vs. 20.8 +/- 5.7;p < 0.001) and Ki-67 (318.7 +/- 94.0 vs. 468.0 +/- 133.8;p = 0.004) and significantly more TUNEL (672.7 +/- 194.0 vs. 357.6 +/- 192.0;p = 0.003) positive cells in the therapy group. CEUS parameters showed significant (p < 0.05) correlations to DCE-MRI parameters and immunohistochemistry. Conclusions CEUS with VEGFR2-targeted microbubbles allowed for monitoring regorafenib functional and molecular therapy effects on experimental colorectal adenocarcinomas with a significant decline of CEUS and DCE-MRI perfusion parameters as well as a significant reduction of specifically bound microbubbles under therapy, consistent with a reduced expression of VEGFR2