Direct measurement of a pK(a) near neutrality for the catalytic cytosine in the genomic HDV ribozyme using Raman crystallography

The hepatitis delta virus (HDV) ribozyme uses a cytosine to facilitate general acid-base catalysis. Biochemical studies suggest that C75 has a pKa perturbed to near neutrality. To measure this pKa directly, Raman spectra were recorded on single ribozyme crystals using a Raman microscope. A spectral feature arising from a single neutral cytosine was identified at 1528 cm(-1). At low pH, this mode was replaced with a new spectral feature. Monitoring these features as a function of pH revealed pKa values for the cytosine that couple anticooperatively with Mg2+ binding, with values of 6.15 and 6.40 in the presence of 20 and 2 mM Mg2+, respectively. These pKa values agree well with those obtained from ribozyme activity experiments in solution. To correlate the observed pKa with a specific nucleotide, crystals of C75U, which is catalytically inactive, were examined. The Raman difference spectra show that this mutation does not affect the conformation of the ribozyme. However, crystals of C75U did not produce a signal from a protonatable cytosine, providing strong evidence that protonation of C75 is being monitored in the wild-type ribozyme. These studies provide the first direct physical measurement of a pKa near neutrality for a catalytic residue in a ribozyme and show that ribozymes, like their protein enzyme counterparts, can optimize the pKa of their side chains for proton transfer.     

Tyrosyl rotamer interconversion rates and the fluorescence decays of N-acetyltyrosinamide and short tyrosyl peptides

It has long been recognized that the fluorescence lifetimes of amino acid residues such as tyrosine and tryptophan depend on the rotameric configuration of the aromatic side chain, but estimates of the rate of interchange of rotameric states have varied widely. We report measurements of the rotameric populations and interchange rates for tyrosine in N-acetyltyrosinamide (NATyrA), the tripeptide Tyr-Gly-Gly (YGG), and the pentapeptide Leu-enkephalin (YGGFL). The fluorescence lifetimes were analyzed to determine the rotameric interchange rates in the context of a model incorporating exchange among three rotameric states. Maximum entropy method analysis verified the presence of three fluorescence decay components for YGGFL and two for YGG and NATyrA. Rotameric exchange between the gauche(-) and trans states occurred on the nanosecond time scale, whereas exchange with the gauche(+) state occurred on a longer time scale. Good agreement was obtained with rotameric populations and exchange rates from molecular dynamics simulations. Quenching by iodide was used to vary the intrinsic fluorescence lifetimes, providing additional constraints on the determined interchange rates. The temperature dependence was measured to determine barriers to exchange of the two most populated rotamers of 3, 5, and 7 kcal/mol for NATyrA, YGG, and YGGFL, respectively.     

Interface Conditions for a Phase Field Model with Anisotropic and Non-Local Interactions

An alternative formulation of the phase field method is utilized from an integral equation perspective. The technique allows one to derive macroscopic conditions at the interface from the microscopic potentials. Differential geometry and asymptotic analysis yield interface conditions, in arbitrary spatial dimension, for interactions that may include anisotropy as well as non-local potentials. The interface conditions can be expressed in various formulations, for example, in terms of the principal curvature directions of the interface, or the second order directional derivatives of the (signed) distance function and the Hessian of the surface tension.     

Assessing chiral self-recognition using chiral stationary phases

Chiral stationary phases were synthesized and their ability to separate racemic precursors from which they were derived was assessed. Taken in conjunction with homochiral recognition previously observed in the solid state, the results of this study reveal that a geometrically controlling π-π interaction has a profound influence on molecular recognition.     

Preliminary investigation of Spartina alterniflora for phytoextraction of selected heavy metals in soils from Southwest Louisiana

Spartina alterniflora plants were collected at the Rockefeller Wildlife Refuge near Lake Charles, Louisiana for phytoremediation (phytoextraction) studies. In order to determine whether the plant would thrive in different soils and undergo phytoremediation, three soils were used and analyzed: experimental controlled soil (minimum heavy metal contamination), spiked soil (spiked with 100 ppm of selected heavy metals of Cr, Cu, Pb, Fe and Zn) collected from a local store and contaminated soil collected from the known contaminated (with metals) Bayou d'Inde in Lake Charles, Louisiana. The plant grew evenly in each of the three soils. Based on the inductively coupled plasma-optical emission spectrometric determination of the plant in each soil, it was shown that S. alterniflora exhibited potential for phytoremediation of metals in soils via phytoextraction into the roots.     

Rapid identification of bacteria with a disposable colorimetric sensing array

Rapid identification of both species and even specific strains of human pathogenic bacteria grown on standard agar has been achieved from the volatiles they produce using a disposable colorimetric sensor array in a Petri dish imaged with an inexpensive scanner. All 10 strains of bacteria tested, including Enterococcus faecalis and Staphylococcus aureus and their antibiotic-resistant forms, were identified with 98.8% accuracy within 10 h, a clinically important time frame. Furthermore, the colorimetric sensor arrays also proved useful as a simple research tool for the study of bacterial metabolism and as an easy method for the optimization of bacterial production of fine chemicals or other fermentation processes.     

Implications of partial tryptic digestion in organic-aqueous solvent systems for bottom-up proteome analysis

For bottom-up MS, the digestion step is critical and is typically performed with trypsin. Solvent-assisted digestion in 80% acetonitrile has previously been shown to improve protein sequence coverage at shorter digestion times. This has been attributed to enhanced enzyme digestion efficiency in this solvent. However, our results demonstrate that tryptic digestion in 80% acetonitrile is less efficient than that of conventional (aqueous) digestion. This is a consequence of decreased enzyme activity beyond ∼40% acetonitrile, increased enzyme autolysis and lower protein solubility in 80% acetonitrile. We observe multiple missed cleavages and reduced concentration of fully cleaved digestion products. Nonetheless we confirm, through room temperature solvent-assisted digestion, a consistent improvement in protein sequence coverage when analyzed by mass spectrometry. These results are explained through the increased number of unique digestion products available for detection. Thus, while solvent-assisted digestion has clear merits for proteome analysis, one should be aware of the inefficiency of protein digestion though this protocol, particularly with absolute protein quantitation experiments.     

Nonlinearity in the dynamics of financial markets

A new set of methodologies extracts key nonlinearities in the dynamics of financial markets from data that would appear to be completely random with ordinary linear time series methods. The understanding acquired from this analysis forms a basis for modeling conflicting and competing motivations in market decisions. By standardizing the daily changes using the mean and standard deviation, it then becomes possible to compare the quantitative impact of very different variables such as price trend and valuation, and the nonlinear relationship between them. The analysis of a large data set of closing stock prices provides strong statistical evidence that relative daily price change is positively influenced by valuation, recent price trend, short term volatility, volume trend and the M2 money supply. However, there is a strong nonlinearity in the influence of the price trend, so that a significantly large recent uptrend has a negative influence on the subsequent day’s relative price change. The nonlinearity is the key to understanding the conflicting role of price trend, since a single large data set exhibits both underreaction and overreaction in different regimes of the independent variables. The role of long term volatility is not a clear-cut risk/return inverse relation. But rather there is an ambiguous and complicated relationship between volatility and return. There is limited support for resistance when prices near the quarterly high. Mixed effects regressions are used after standardizing the data by subtracting the mean and dividing by one standard deviation individually for each of the 119 closed-end funds. A valuation variable is constructed in terms of the recent history of net asset value.     

A Jost–Pais-Type Reduction of Fredholm Determinants and Some Applications

We study the analog of semi-separable integral kernels in \({\mathcal {H}}\) of the type $$ K(x, x') = \left\{\begin{array}{ll} F_1(x) G_1(x'), \quad& a < x' < x < b,\\ F_2 (x)G_2(x'), \quad& a < x < x' < b,\end{array}\right.$$ where \({-\infty \leqslant a < b \leqslant \infty}\) , and for a.e. \({x \in (a, b)}\) , \({F_j (x) \in \mathcal{B}_2(\mathcal{H}_j, \mathcal{H})}\) and \({G_j(x) \in \mathcal {B}_2(\mathcal {H},\mathcal {H}_j)}\) such that F _j (·) and G _j (·) are uniformly measurable, and $$\begin{array}{ll} || F_j ( \cdot) ||_{\mathcal {B}_2(\mathcal {H}_j,\mathcal {H})} \in L^2((a, b)), ||G_j (\cdot)||_{\mathcal {B}_2(\mathcal {H},\mathcal {H}_j)} \in L^2((a, b)), \quad j=1,2, \end{array}$$ with \({\mathcal {H}}\) and \({\mathcal {H}_j}\) , j = 1, 2, complex, separable Hilbert spaces. Assuming that K(·, ·) generates a trace class operator K in \({L^2((a, b);\mathcal {H})}\) , we derive the analog of the Jost–Pais reduction theory that succeeds in proving that the Fredholm determinant \({{\rm det}_{L^2((a,b);\mathcal{H})}}\) (I ? α K), \({\alpha \in \mathbb{C}}\) , naturally reduces to appropriate Fredholm determinants in the Hilbert spaces \({\mathcal{H}}\) (and \({\mathcal{H}_1 \oplus \mathcal{H}_2}\) ). Explicit applications of this reduction theory to Schrödinger operators with suitable bounded operator-valued potentials are made. In addition, we provide an alternative approach to a fundamental trace formula first established by Pushnitski which leads to a Fredholm index computation of a certain model operator.     

On index theory for non‐Fredholm operators: A (1 + 1)‐dimensional example

Using the general formalism of 12 , a study of index theory for non‐Fredholm operators was initiated in 9 . Natural examples arise from (1 + 1)‐dimensional differential operators using the model operator in of the type , where , and the family of self‐adjoint operators in studied here is explicitly given by Here has to be integrable on and tends to zero as and to 1 as (both functions are subject to additional hypotheses). In particular, , , has asymptotes (in the norm resolvent sense) as , respectively. The interesting feature is that violates the relative trace class condition introduced in 9 , Hypothesis 2.1 ]. A new approach adapted to differential operators of this kind is given here using an approximation technique. The approximants do fit the framework of 9 enabling the following results to be obtained. Introducing , , we recall that the resolvent regularized Witten index of , denoted by , is defined by whenever this limit exists. In the concrete example at hand, we prove Here denotes the spectral shift operator for the pair of self‐adjoint operators , and we employ the normalization, , .