Minimizing Structural Heterogeneity in DNA Self-Assembled Dye Templating via DNA Origami-Tuned Conformations.

With recent advances in DNA-templated dye aggregation for leveraging and engineering molecular excitons, a need exists for minimizing structural heterogeneity. Holliday Junction complexes (HJ) are commonly used to covalently template dye aggregates on their core; however, the global conformation of HJ is detrimentally dynamic. Here, the global conformation of the HJ is selectively tuned by restricting its position and orientation by using a sheet-like DNA origami construct (DOC) physisorbed on glass. The HJ arms are fixed with four different designed interduplex angles (IDAs). Atomic force microscopy confirmed that the HJs are bound to the surface of DOC with tuned IDAs. Dye orientation distributions were determined by combining dipole imaging and super-resolution microscopy. All IDAs led to dye orientations having dispersed distributions along planes perpendicular to the HJ plane, suggesting that stacking occurred between the dye and the neighboring DNA bases. The dye-base stacking interpretation was supported by increasing the size of the core cavity. The narrowest IDA minimizes structural heterogeneity and suggests dye intercalation. A strong correlation is found between the IDA and the orientation of the dye along the HJ plane. These results show that the HJ imposes restrictions on the dye and that the dye-DNA interactions are always present regardless of global conformation. The implications of our results are discussed for the scalability of dye aggregates using DNA self-assembly. Our methodology provides an avenue for the solid-supported single-molecule characterization of molecular assemblies templated on biomolecules─such as DNA and protein templates involved in light-harvesting and catalysis─with tuned conformations and restricted in position and orientation.

Q methodology: An underutilised tool in pharmacy practice research.

Pharmacy practice research is often concerned with opinions, perspectives, values, or a variety of other subjective domains, whether that be in regard to the experiences of patients, views of stakeholders about innovative pharmacy services, or culture in pharmacy practice. This article offers a brief introduction to Q methodology, which is a philosophical, conceptual, and technical framework well-suited to shed light on such subjective views. Q methodology combines qualitative and quantitative processes to uncover distinct viewpoints present about any given topic. While other textual analyses focus on identifying the constituent themes about a topic, Q methodology instead detects and interprets holistic and shared perspectives. The introduction covers key theoretical principles, as well as the logistics and procedures involved in completing a Q-methodological study. Example data from a study investigating views on pharmacist integration into general practice in New Zealand are presented to highlight the potential of Q methodology for pharmacy practice research.

Sialochemical analysis in polytraumatized patients in intensive care units.

The profiles of polytraumatized patients in intensive care units were characterized. Serum and salivary markers were compared with normality between Classes I and II of APACHE II and between periods of hospitalization; these results were correlated. This was a prospective study on saliva charts and collection (n = 70). Profile: male, 27 years old, blunt traumas and collisions. Serum parameters with normality: decrease in pH, creatinine at admission to Class I, and at 48 and 72 hours in both classes; K+ at 48 h in Class II; Ca+ on admission in both classes and at 72 h in Class I. Increase in urea at 72 h in Class II, glucose at all times and in all classes, and Ca+ at 48 h in both classes. Class II had high Na+ at 48 and 72 h compared to Class I. In Class I, creatinine reduction occurred in 48 h and 72 h compared to admission and an increase of Ca+ at 48 h with admission. In Class II, pH and Na+ increased at 48 h and 72 h compared to admission. K+ decreased from admission to 48 h and increased from 48 h to 72 h. Urea increased from 48 to 72 hours. Creatinine decreased from admission to 48 and 72 hours. Ca+ increased from admission to 48 hours and decreased from 48 to 72 hours. There was an increase in the saliva levels in both classes and times in relation to normality. There was an increase in urea at admission, glucose at 72 h, and Ca+ at 48 h in Class II compared with Class I. Class I urea increased from admission to 48 h and Ca+ decreased from admission to 48 h. Class II urea decreased from 48 h to 72 h. Strong or very strong positive correlation was identified between blood and creatinine saliva at all times and regular and negative Ca+ at 72 h. This study provides evidence that salivary and serum biomarkers can be used together to monitor the evolution of the clinical symptoms of ICU patients.

Specificity and false positive rates of the Test of Memory Malingering, Rey 15-item Test, and Rey Word Recognition Test among forensic inpatients with intellectual disabilities.

This study evaluated the specificity and false positive (FP) rates of the Rey 15-Item Test (FIT), Word Recognition Test (WRT), and Test of Memory Malingering (TOMM) in a sample of 21 forensic inpatients with mild intellectual disability (ID). The FIT demonstrated an FP rate of 23.8% with the standard quantitative cutoff score. Certain qualitative error types on the FIT showed promise and had low FP rates. The WRT obtained an FP rate of 0.0% with previously reported cutoff scores. Finally, the TOMM demonstrated low FP rates of 4.8% and 0.0% on Trial 2 and the Retention Trial, respectively, when applying the standard cutoff score. FP rates are reported for a range of cutoff scores and compared with published research on individuals diagnosed with ID. Results indicated that although the quantitative variables on the FIT had unacceptably high FP rates, the TOMM and WRT had low FP rates, increasing the confidence clinicians can place in scores reflecting poor effort on these measures during ID evaluations.

Multiple effects of Escherichia coli Nissle 1917 on growth, biofilm formation, and inflammation cytokines profile of Clostridium perfringens type A strain CP4.

Clostridium perfringens is an important Gram-positive pathogen responsible for food poisoning, necrotic enteritis, gas gangrene, and even death. Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic strain with demonstrated benefits. In this study, we evaluated the effects of EcN on growth, toxin production, biofilm formation, and inflammatory cytokine responses of C. perfringens. In vitro co-culture experiments demonstrated that EcN inhibited growth, gas production, and toxin production (α-toxin and NetB) of C. perfringens in a dose-dependent manner. The growth inhibition effect was not observed when C. perfringens was incubated with EcN cell-free supernatants (CFSE), suggesting that growth inhibition was caused by nutrition competition during co-incubation. In vitro studies demonstrated that pre-incubation with EcN did not inhibit C. perfringens attachment to Caco-2 cells, but did reduce C. perfringens total number, toxin production, and cytotoxicity after 24 h. The similar growth inhibition results were also observed during the formation of C. perfringens biofilm. Finally, pre-incubation of EcN with RAW264.7 cells significantly decreased the production of inflammatory cytokines caused by the introduction of C. perfringens. Our results indicate that EcN can inhibit many of the pathological effects of C. perfringens in vitro conditions.

Development of biotechnology products in pre-filled syringes: technical considerations and approaches.

A monoclonal antibody (mAb) product development case study is presented to address some of the issues faced during developing a pre-filled syringe (PFS) product for a biotherapeutic. In particular, issues involving incompatibility with silicone oil and a stability-based approach for selection of PFS barrel and tip cap components have been discussed. Silicone spiking studies followed by exposure to agitation stress or accelerated temperature conditions were used to check for incompatibilities of the mAb with silicone oil, a necessary product contact material in PFS. In addition, screening studies to compare various closure materials as well as syringe barrel processing methods were used to select the optimum closure materials as well as the correct syringe processing method. Results indicate that the model mAb formulation used was sensitive to high levels of silicone oil especially under accelerated temperature conditions resulting in formation of protein-silicone particles in the solution for samples that were spiked with the silicone oil. Agitation stress did not have any significant impact on the quality attributes tested. Samples stored in syringe barrels that were processed with sprayed-on silicone had higher levels of subvisible particles as compared to those that were processed with the baked-on process. The tip cap comparability study resulted in one tip cap material having superior compatibility among the three that were tested. The quality attribute that was most impacted by the tip cap materials was mAb oxidation. An approach for evaluation of primary packaging components during the development of pre-filled syringe presentations for biotechnology-based compounds has been highlighted.

The association between bullying dynamics and psychological distress.

This study examined the association between past experience of victimization (PEV), perceived risk of victimization (PRV), and nonspecific psychological distress (NSPD). Repeated measures-analysis of variance and hierarchical regression analyses were conducted on 186 seventh grade middle school students from an urban university-research-affiliated school. Results indicated that gender, PEV, and PRV significantly predicted NSPD. There were no gender differences in either the total number of past experience of victimization or depressive and/or anxious feelings reported. However, the types of victimization experienced as well as perceived risk of victimization appeared to be gender-related in that boys were significantly higher than girls on past experience of physical aggression and property aggression but significantly lower than girls on past experience of emotional aggression and perceived risk of victimization. In gender-specific analyses, PRV mediated the effects of PEV on NSPD for girls but not boys. The reasons for these findings, as well as implications for social policies and future directions, are discussed.

Food selection changes under stress.

Two studies investigate the effect of stress on food choice. Experiment 1 demonstrates experimentally that stress causes changes in food choice away from healthy low fat foods (grapes) to less healthy high fat foods (M&Ms), confirming previous survey research. Experiment 2, a survey study, finds that more females than males report increasing food consumption when stressed. A much larger percentage of those who report increasing their food consumption when stressed (71%) are restrained eaters (i.e., dieters) than are people who undereat or who do not change the amount they eat when stressed (35%). The foods that they report overeating when stressed are foods they normally avoid for weight-loss or health reasons (i.e., highly caloric high fat snack foods). They report eating these foods to feel better. Both studies show that stress not only increases consumption in certain individuals but also shifts their food choice from lower fat to higher fat foods.

Zn2+-dependent deoxyribozymes that form natural and unnatural RNA linkages.

We report Zn(2+)-dependent deoxyribozymes that ligate RNA. The DNA enzymes were identified by in vitro selection and ligate RNA with k(obs) up to 0.5 min(-)(1) at 1 mM Zn(2+) and 23 degrees C, pH 7.9, which is substantially faster than our previously reported Mg(2+)-dependent deoxyribozymes. Each new Zn(2+)-dependent deoxyribozyme mediates the reaction of a specific nucleophile on one RNA substrate with a 2',3'-cyclic phosphate on a second RNA substrate. Some of the Zn(2+)-dependent deoxyribozymes create native 3'-5' RNA linkages (with k(obs) up to 0.02 min(-)(1)), whereas all of our previous Mg(2+)-dependent deoxyribozymes that use a 2',3'-cyclic phosphate create non-native 2'-5' RNA linkages. On this basis, Zn(2+)-dependent deoxyribozymes have promise for synthesis of native 3'-5'-linked RNA using 2',3'-cyclic phosphate RNA substrates, although these particular Zn(2+)-dependent deoxyribozymes are likely not useful for this practical application. Some of the new Zn(2+)-dependent deoxyribozymes instead create non-native 2'-5' linkages, just like their Mg(2+) counterparts. Unexpectedly, other Zn(2+)-dependent deoxyribozymes synthesize one of three unnatural linkages that are formed upon the reaction of an RNA nucleophile other than a 5'-hydroxyl group. Two of these unnatural linkages are the 3'-2' and 2'-2' linear junctions created when the 2'-hydroxyl of the 5'-terminal guanosine of one RNA substrate attacks the 2',3'-cyclic phosphate of the second RNA substrate. The third unnatural linkage is a branched RNA that results from attack of a specific internal 2'-hydroxyl of one RNA substrate at the 2',3'-cyclic phosphate. When compared with the consistent creation of 2'-5' linkages by Mg(2+)-dependent ligation, formation of this variety of RNA ligation products by Zn(2+)-dependent deoxyribozymes highlights the versatility of transition metals such as Zn(2+) for mediating nucleic acid catalysis.

Small-angle X-ray scattering study of the interaction of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) triblock copolymers with lipid bilayers.

The relationship between molecular architecture and the nature of interactions with lipid bilayers has been studied for a series of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers using small-angle X-ray scattering (SAXS) and thermal analysis (differential scanning calorimetry, DSC). The number of molecular repeat units in the hydrophobic poly(propylene oxide), PPO, block has been found to be a critical determinant of the nature of triblock copolymer-lipid bilayer association. For dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-based biomembrane structures, polymers possessing a PPO chain length commensurate with the acyl chain dimensions of the lipid bilayer yield highly ordered, swollen lamellar structures consistent with well-integrated (into the lipid bilayer) PPO blocks. Triblock copolymers of lesser PPO chain length yield materials with structural characteristics similar to a simple dispersion of DMPC in water. Increasing the concentration (from 4 to 12 mol %) of well-integrated triblock copolymers enhances the structural ordering of the lamellar phase, while concentrations exceeding 16 mol % result in the formation of a hexagonal phase. Examination of temperature-induced changes in the structure of these mesophases (complex fluids) reveals that if the temperature is reduced sufficiently, all compositions exclude polymer and thus exhibit the characteristic SAXS pattern for hydrated DMPC bilayers. Increasing the temperature promotes better insertion of the polymers possessing PPO chain lengths sufficient for membrane insertion. No temperature-induced structural changes are observed in compositions prepared with PEO-PPO-PEO polymers that feature PPO length insufficient to permit full incorporation into the lipid bilayer.

Optimization and generality of a small deoxyribozyme that ligates RNA.

In vitro evolution was previously used to identify a small deoxyribozyme, 7Q10, that ligates RNA with formation of a 2'-5' phosphodiester linkage from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. Ligation occurs in a convenient "binding arms" format analogous to that of the well-known 10-23 and 8-17 RNA-cleaving deoxyribozymes. Here, we report the optimization and generality of 7Q10 as a 2'-5' RNA ligase. By comprehensive mutagenesis of its 16-nucleotide enzyme region, the parent 7Q10 sequence is shown to be optimal for RNA ligation yield, although several mutations are capable of increasing the ligation rate approximately fivefold at the expense of yield. The 7Q10 deoxyribozyme ligates any RNA substrates that form the sequence motif UA GR (arrowhead=ligation site and R=purine), providing at least 30% yield of ligated RNA in approximately 1-2 hours at 37 degrees C and pH 9.0. Comparable yields are obtained in approximately 12-24 hours at pH 7.5, which may be more suitable for larger RNAs that are more sensitive to non-specific degradation. For RNA substrates that form the related ligation junction UA GY (Y=pyrimidine), somewhat lower yields are obtained, but significant ligation activity is still observed. These data establish that 7Q10 is a generally applicable RNA ligase. A plot of log(k(obs)) versus pH from pH 6.9 to 9.0 has a slope of just under 1, suggesting that a single deprotonation occurs during the rate-determining reaction step. The compact 7Q10 deoxyribozyme has both practical utility and the potential for increasing our structural and mechanistic understanding of how nucleic acids can mediate chemical reactions.

Deoxyribozymes with 2'-5' RNA ligase activity.

In vitro selection was used to identify deoxyribozymes that ligate two RNA substrates. In the ligation reaction, a 2'-5' RNA phosphodiester linkage is created from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The new Mg(2+)-dependent deoxyribozymes provide 50-60% yield of ligated RNA in overnight incubations at pH 7.5 and 37 degrees C, and they afford 40-50% yield in 1 h at pH 9.0 and 37 degrees C. Various RNA substrate sequences may be joined by simple Watson-Crick covaration of the DNA binding arms that interact with the two RNA substrates. The current deoxyribozymes have some RNA substrate sequence requirements at the nucleotides immediately surrounding the ligation junction (either UAUA GGAA or UAUN GGAA, where the arrow denotes the ligation site and N equals any nucleotide). One of the new deoxyribozymes was used to prepare by ligation the Tetrahymena group I intron RNA P4-P6 domain, a representative structured RNA. Nondenaturing gel electrophoresis revealed that a 2'-5' linkage between nucleotides A233 and G234 of P4-P6 does not disrupt its Mg(2+)-dependent folding (DeltaDeltaG degrees ' < 0.2 kcal/mol). This demonstrates that a 2'-5' linkage does not necessarily interfere with structure in a folded RNA. Therefore, these non-native linkages may be acceptable in modified RNAs when structure/function relationships are investigated. Deoxyribozymes that ligate RNA should be particularly useful for preparing site-specifically modified RNAs for studies of RNA structure, folding, and catalysis.