Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding

Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl-L-leucine for growth [Forney, Wong and Ferber (1989) Appl. Environ. Microbiol. 55, 2550-2555], are changed within one codon, replacing the B-chain residue Phe(B71) with either Cys or Leu. Increases of up to a factor of ten in k(cat)/K-m values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in K-s. Values of k(cat/)K(m) for glutaryl-L-leucine are increased at least 100-fold. A decrease in k(cat)/K-m for the CySB71 mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure of the Leu(B71) mutant protein, solved to 2 X resolution, shows a flip of the side chain of Phe(B256) into the periphery of the catalytic centre, associated with loss of the pi-stacking interactions between Phe(B256) and Phe(B71). Molecular modelling demonstrates that glutaryl-L-leucine may bind with the uncharged glutaryl group in the S-1 subsite of either the wild-type or the Leu(B71) mutant but with greater potential freedom of rotation of the substrate leucine moiety in the complex with the mutant protein. This implies a smaller decrease in the conformational entropy of the substrate on binding to the mutant proteins and consequently greater catalytic activity

Preliminary experimental results for a cryogenic brush seal configuration

Preliminary fluid nitrogen flow data are reported for a five-brush, ceramic-coated-rub-runner brush seal system, where the brushes and the rub runner were placed at each end of a centrally pressurized multifunction tester ('back-to-back' set of brushes) and tested at rotor speeds of 0, 10, 18, and 22.5 krpm. After testing, both the brushes and the ceramic-coated rub runner appeared pristine. The coating withstood both the thermomechanical and dynamic loadings with minor wear track scarring. The bristle tips showed some indication of material shearing (smearing) wear. The Ergun porous flow equation was applied to the brush seal data. The Ergun relation, which required heuristic information to characterize the coefficients, fit the gaseous data but was in poor agreement with the fluid results. The brush seal exit conditions were two phase. Two-phase, choked-flow design charts were applied but required one data point at each rotor speed to define the (C(sub f)A x Constant) flow and area coefficients. Reasonable agreement between prediction and data was found, as expected, but such methods are not to be construed as two-phase-flow brush seal analyses

Stability of Nonlinear Control Systems by the Second Method of Liapunov

This report investigates the stability of autonomous closed-loop control systems containing nonlinear elements. An n-th order nonlinear autonomous system is described by a set of n first order differential equations of the type dxi/dt=xi(x1, x2, …xn) i=1,2,…n. Liapunov\u27s second (direct) method is used in the stability analysis of such systems. This method enables one to prove that a system is stable (or unstable) if a function V=V (x1, x2, … xn) can be found which, together with its time derivative, satisfies the requirements of Liapunov\u27s stability (or instability) theorems. At the present time, there are no generally applicable straight forward procedures available for constructing these Liapunov\u27s functions. Several Liapunov\u27s functions, applicable to systems described in the canonic form of differential equations, have been reported in the literature. In this report, it is shown that any autonomous closed-loop system containing a single nonlinear element can be described by canonic differential equations. The stability criteria derived from the Liapunov\u27s functions for canonic systems give sufficient and not necessary conditions for stability. It is known that these criteria reject many systems which are actually stable. The reasons why stable systems are sometimes rejected by these simplified stability criteria are investigated in the report. It is found that a closed-loop system will always be rejected by these simplified stabi1ity criteria if the root locus of the transfer function G(s), representing the linear portion of the system, is not confined to the left-half of the s-plane for all positive values of the loop gain. A pole-shifting technique and a zero-shifting technique, extending -the applicability of the simplified stability criteria to systems that are stable for sufficiently high and/or sufficiently low values of the loop gain, are proposed in this report. New simplified stability criteria have been developed which incorporate the changes in the canonic form of differential equations caused by the application of the zero-shifting technique. Other methods of constructing Liapunov\u27s functions for nonlinear control systems are presented in Chapter III, These include the work of Pliss, Aizerman and Krasovski. Numerous other procedures, which have been reported in literature, apply to only very special cases of automatic control systems. No attempt has been made to account for all of these special cases and the presentation of methods of constructing Liapunov’s functions is limited to only those which are more generally applicable. A pseudo-canonic transformation has been developed which enables one to find stability criteria of canonic systems without the use of complex variables. The results of this research indicate that the second method of Liapunov is a very powerfuI tool of exact stability analysis of nonlinear systems. Additional research, especially in the direction of the methods of construction of Liapunov’s functions, will not only yield new analysis and synthesis procedures, but also will aid in arriving at a set of meaningful performance specifications for nonlinear control systems

Specification and Data Presentation in Linear Control Systems-Part Two

This is the second part of a 2 volume report on the specification and data presentation in linear control systems. This volume deals with Sample Data Systems, Linear Time Variable Parameter Systems, and Performance Indices, which are respectively Chapter II, III, and IV of the volume. Since these subjects are somewhat unrelated, a separate abstract is given at the beginning of each chapter, with the exception of the introductory Chapter I. The separate chapter abstracts are repeated here for the convenience of the reader. Abstract - Linear Sampled Data Control Systems The specifications recommended, for use with sampled data control systems are those recommended for linear, continuous systems [1]. These specifications must be supplemented, as is dictated by the requirements of a particular system, by compatibility considerations that are detailed in the following sections. Abstract - The Specification of Linear Time Variable Parameter Systems Linear time variable parameter (LTVP) systems are defined and subdivided into those systems with fast or slow variations and/or large or small variations. The methods of analysis of such systems are reviewed, and the following recommendations are made. Specifications 1) Time Domain Specifications (a) LTVP systems with fast variation of parameters. Simulated unfrozen system step function responses should all lie within a prescribed envelope. Whenever possible, the actual system response should be obtained. (b) LTVP systems with slow variation of parameters. Simulated or actual frozen or unfrozen system step function responses should all lie within a prescribed envelope. 2) Frequency Domain Specifications (a) LTVP system with fast variation of parameters. Frequency domain specifications are not recommended. (b) LTFP system with slow variation of parameters. The family of frequency response curves of the system frozen at different instants should all lie within a predetermined envelope. Data Presentation It is recommended that the region of variation of closed loop poles of the frozen system be exhibited on the complex plane. Thus, for example, if the only varying parameter is an open loop gain, then the region of variation of the closed loop poles will correspond to the root loci over the total range of variation of gain. It is also recommended that a family of Nyquist diagrams corresponding to the system frozen at different instants be displayed in the case of system with slow variations of parameters. Abstract - Performance Index This study was undertaken to determine whether or not Performance Indices should be used to evaluate and specify control systems* It is recommended that they not be used at this time by the Air Force for the stated purpose. A performance index is defined and detailed discussions are presented for the various performance indices. Analytical methods for evaluating performance indices are presented

Social Difficulties in Youth with Autism With and Without Anxiety and ADHD Symptoms

Social difficulties inherent to autism spectrum disorder are often linked with co‐occurring symptoms of anxiety and attention deficit hyperactivity disorder (ADHD). The present study sought to examine the relation between such co‐occurring symptoms and social challenges. Parents of adolescents with autism (N = 113) reported upon social challenges via the social responsiveness scale (SRS) and anxiety and ADHD symptomatology via the Child Behavior Checklist. Results revealed differences in SRS scores across co‐occurring symptom subgroups (Anxiety, ADHD, Both, Neither)—namely, adolescents with autism and anxiety as well as those with autism, anxiety, and ADHD showed greater scores on the SRS than the other groups. Implications for research and clinical practice are discussed and recommendations are offered. Lay Summary Anxiety and attention deficit hyperactivity disorder (ADHD) symptoms are related to greater social challenges for adolescents with autism spectrum disorder. The present study found that autism with anxiety and autism with anxiety and ADHD, was related to greater social difficulties than autism alone. Findings provide further support for the intertwined nature of anxiety and ADHD symptoms in autism. What this may mean for research and clinical practice is considered and recommendations are suggested

Social Difficulties in Youth with Autism With and Without Anxiety and ADHD Symptoms

Social difficulties inherent to autism spectrum disorder are often linked with co‐occurring symptoms of anxiety and attention deficit hyperactivity disorder (ADHD). The present study sought to examine the relation between such co‐occurring symptoms and social challenges. Parents of adolescents with autism (N = 113) reported upon social challenges via the social responsiveness scale (SRS) and anxiety and ADHD symptomatology via the Child Behavior Checklist. Results revealed differences in SRS scores across co‐occurring symptom subgroups (Anxiety, ADHD, Both, Neither)—namely, adolescents with autism and anxiety as well as those with autism, anxiety, and ADHD showed greater scores on the SRS than the other groups. Implications for research and clinical practice are discussed and recommendations are offered. Lay Summary Anxiety and attention deficit hyperactivity disorder (ADHD) symptoms are related to greater social challenges for adolescents with autism spectrum disorder. The present study found that autism with anxiety and autism with anxiety and ADHD, was related to greater social difficulties than autism alone. Findings provide further support for the intertwined nature of anxiety and ADHD symptoms in autism. What this may mean for research and clinical practice is considered and recommendations are suggested

Going places

Journeys. We all make them. Often they take us to exotic places. Sometimes they take us even further. They might take us through time. Or they might take us into a new way of life. There are times too, when we go all over the world and back again only to find that home is, after all, where it’s all happening. This book contains stories about many different types of journey. We hope you will enjoy travelling into it and finding a world that suits you

EHDV-2 Infection Prevalence Varies in Culicoides sonorensis after Feeding on Infected White-Tailed Deer over the Course of Viremia

Epizootic hemorrhagic disease viruses (EHDVs) are arboviral pathogens of white-tailed deer and other wild and domestic ruminants in North America. Transmitted by various species of Culicoides, EHDVs circulate wherever competent vectors and susceptible ruminant host populations co-exist. The impact of variation in the level and duration of EHDV viremia in white-tailed deer (Odocoileus virginianus) on Culicoides infection prevalence is not well characterized. Here we examined how infection prevalence in a confirmed North American vector of EHDV-2 (Culicoides sonorensis) varies in response to fluctuations in deer viremia. To accomplish this, five white-tailed deer were experimentally infected with EHDV-2 and colonized C. sonorensis were allowed to feed on deer at 3, 5, 7, 10, 12, 14, 18, and 24 days post infection (dpi). Viremia profiles in deer were determined by virus isolation and titration at the same time points. Blood-fed Culicoides were assayed for virus after a 10-day incubation (27 ◦C) period. We found that increases in deer EHDV blood titers significantly increased both the likelihood that midges would successfully acquire EHDV and the proportion of midges that reached the titer threshold for transmission competence. Unexpectedly, we identified four infected midge samples (three individuals and one pool) after feeding on one deer 18 and 24 dpi, when viremia was no longer detectable by virus isolation. The ability of ruminants with low-titer viremia to serve as a source of EHDV for blood-feeding Culicoides should be explored further to better understand its potential epidemiological significance