Intercalation of proflavine and a platinum derivative of proflavine into double-helical Poly(A)

The equilibria and kinetics of the interactions of proflavine (PR) and its platinum-containing derivative [PtCl(tmen)(2)HNC(13)H(7)(NHCH(2)CH(2))(2)](+) (PRPt) with double-stranded poly(A) have been investigated by spectrophotometry and Joule temperature-jump relaxation at ionic strength 0.1 M, 25 degrees C, and pH 5.2. Spectrophotometric measurements indicate that base-dye interactions are prevailing. T-jump experiments with polarized light showed that effects due to field-induced alignment could be neglected. Both of the investigated systems display two relaxation effects. The kinetic features of the reaction are discussed in terms of a two-step series mechanism in which a precursor complex DS(I) is formed in the fast step, which is then converted to a final complex in the slow step. The rate constants of the fast step are k(1) = (2.5 +/- 0.4) x 10(6) M(-1) s(-1), k(-1) = (2.4 +/- 0.1) x 10(3) s(-1) for poly(A)-PR and k(1) = (2.3 +/- 0.1) x 10(6) M(-1) s(-1), k(-1) = (1.6 +/- 0.2) x 10(3) s(-1) for poly(A)-PRPt. The rate constants for the slow step are k(2) = (4.5 +/- 0.5) x 10(2) s(-1), k(-2) = (1.7 +/- 0.1) x 10(2) s(-1) for poly(A)-PR and k(2) = 9.7 +/- 1.2 s(-1), k(-2) = 10.6 +/- 0.2 s(-1) for poly(A)-PRPt. Spectrophotometric measurements yield for the equilibrium constants and site size the values K = (4.5 +/- 0.1) x 10(3) M(-1), n = 1.3 +/- 0.5 for poly(A)-PR and K = (2.9 +/- 0.1) x 10(3) M(-1), n = 2.3 +/- 0.6 for poly(A)-PRPt. The values of k(1) are similar and lower than expected for diffusion-limited reactions. The values of k(-1) are similar as well. It is suggested that the formation of DS(I) involves only the proflavine residues in both systems. In contrast, the values of k(2) and k(-2) in poly(A)-PRPt are much lower than in poly(A)-PR. The results suggest that in the complex DS(II) of poly(A)-PRPt both proflavine and platinum residues are intercalated. In addition, a very slow process was detected and ascribed to the covalent binding of Pt(II) to the adenine.     

DNA variability and recombination rates at X-linked loci in humans.

We sequenced 11,365 bp from introns of seven X-linked genes in 10 humans, one chimpanzee, and one orangutan to (i) provide an average estimate of nucleotide diversity (pi) in humans, (ii) investigate whether there is variation in pi among loci, (iii) compare ratios of polymorphism to divergence among loci, and (iv) provide a preliminary test of the hypothesis that heterozygosity is positively correlated with the local rate of recombination. The average value for pi was low 0.063%, SE = 0.036%, about one order of magnitude smaller than for Drosophila melanogaster, the species for which the best data are available. Among loci, pi varied by over one order of magnitude. Statistical tests of neutrality based on ratios of polymorphism to divergence or based on the frequency spectrum of variation within humans failed to reject a neutral, equilibrium model. However, there was a positive correlation between heterozygosity and rate of recombination, suggesting that the joint effects of selection and linkage are important in shaping patterns of nucleotide variation in humans.     

CHO cell production and sequence improvement in the 13C6FR1 anti-Ebola antibody

From March 2014 through February 2015, the Ebola virus spread rapidly in West Africa, resulting in almost 30,000 infections and approximately 10,000 deaths. With no approved therapeutic options available, an experimental antibody cocktail known as ZMapp? was administered to patients on a limited compassionate-use basis. The supply of ZMapp? was highly constrained at the time because it was in preclinical development and a novel production system (tobacco plants) was being used for manufacturing. To increase the production of ZMapp? for an uncertain future demand, a consortium was formed in the fall of 2014 to quickly manufacture these anti-Ebola antibodies in Chinese hamster ovary (CHO) cells using bioreactors for production at a scale appropriate for thousands of doses. As a result of the efforts of this consortium, valuable lessons were learned about the processing of the antibodies in a CHO-based system. One of the ZMapp? cocktail antibodies, known as c13C6FR1, had been sequence-optimized in the framework region for production in tobacco and engineered as a chimeric antibody. When transfected into CHO cells with the unaltered sequence, 13C6FR1 was difficult to process. This report describes efforts to produce 13C6FR1 and the parental murine hybridoma sequence, 13C6mu, in CHO cells, and provides evidence for the insertion of a highly conserved framework amino acid that improved the physical properties necessary for high-level expression and purification. Furthermore, it describes the technical and logistical lessons learned that may be beneficial in the event of a future Ebola virus or other pandemic viral outbreaks where mAbs are considered potential therapeutics.     

Effects of Filtering the Center of Pressure Feedback Provided in Visually Guided Mediolateral Weight Shifting

Thirty healthy adults completed a mediolateral weight-shifting balance task in which they were instructed to shift their weight to visually displayed target regions. A model-based filter and three different moving average filters employing 10, 34, and 58 samples were applied to the center of pressure visual feedback that guided the activity. The effects of filter selection on both the displayed feedback and the shift performance were examined in terms of shift time and non-minimum phase behavior. Shift time relates to feedback delay and shift speed, whereas non-minimum phase behavior relates to the force applied in shift initiation. Results indicated that increasing the number of samples in moving average filters (indicative of stronger filtering) significantly increases shift speed and shift initiation force. These effects indicate that careful selection and documentation of data filtering is warranted in future work and suggest opportunities for strategic filtering of visual feedback in clinical weight-shifting balance activities in order to improve outcomes based on such feedback.     

Testing the Effectiveness of 3D Film for Laboratory-Based Studies of Emotion

Research in psychology and affective neuroscience often relies on film as a standardized and reliable method for evoking emotion. However, clip validation is not undertaken regularly. This presents a challenge for research with adolescent and young adult samples who are exposed routinely to high-definition (HD) three-dimensional (3D) stimuli and may not respond to older, validated film clips. Studies with young people inform understanding of emotional development, dysregulated affect, and psychopathology, making it critical to assess whether technological advances improve the study of emotion. In the present study, we examine whether 3D film is more evocative than 2D using a tightly controlled within-subjects design. Participants (n  =  408) viewed clips during a concurrent psychophysiological assessment. Results indicate that both 2D and 3D technology are highly effective tools for emotion elicitation. However, 3D does not add incremental benefit over 2D, even when individual differences in anxiety, emotion dysregulation, and novelty seeking are considered.     

Variations in the mode of stolon growth among different genera of colonial hydroids,and their evolutionary implications

Stolonic growth has been compared among several genera of calyptoblastic (Campanularia flexuosa, C. calceolifera, Gonothyraea and Sertularia) and gymnoblastic (Pennaria, Bougainvillia, Eudendrium and Cordylophora) hydroids with respect to such features as the (1) complexity and uniformity of the tip movements, (2) constancy of growth cycle duration, (3) variability in growth among cycles, (4) growth rate, and (5) variability of retractions among cycles. The “growth cycle,” previously described in C. flexuosa, is the basis for elongation in all species observed. Its pattern is indistinguishable between the Campanularia species; however, at the generic level the tip movements show peculiarities which delineate each genus from all others. In addition, the movements in all calyptoblasts are uniform from cycle to cycle and comparatively simple, whereas those of the gymnoblasts are complicated by the variable appearance of one or more secondary forward thrusts between crests. A dichotomy is seen between the calyptoblasts and most gymnoblasts in other respects as well. Cycle time is very predictable and cycle-to-cycle variability in growth is relatively narrow in all calyptoblasts. However, in most gymnoblasts both features are much more loosely regulated: only Pennaria shows some degree of control to its timing mechanism, and only in Bougainvillia and Eudendrium does the uniformity of growth per cycle reach the calyptoblast level. On the premise that calyptoblasts are evolutionarily the more advanced group, simplicity and regularity of growth movements are concluded to be the evolved, and complexity and variability the primitive, conditions. On this basis the evolutionary relations among the four gymnoblasts, as deduced from their stolonic growth behavior alone, are consistent in many but not all respects with those interpreted from morphological considerations.     

Regional cerebral blood flow thresholds during cerebral ischemia.

The development of methods of determining regional cerebral blood flow (rCBF) has made possible the determination of thresholds for the appearance of cerebral ischemia. These thresholds vary depending on the method used for assessing cerebral ischemia. The following thresholds have been determined in man and nonhuman primates: 20 cc/100 g per min, electroencephalogram (EEG) and evoked cortical potential abnormalities appear, paralysis seen in waking monkeys; 15 cc/100 g per min. EEG and evoked cortical potential are lost; 12 cc/100 g per min, flows at this level in excess of 120 min produce infarction in waking animals; and 6 cc/100 g per min, massive loss of intracellular [K+]. The residual rCBF and the duration of ischemia determine the appearance of infarction in waking Macaca irus monkeys.