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Current practices for structure analysis of extremely large molecular weight polysaccharides via solution-state NMR spectroscopy incorporate partial depolymerization protocols that enable polysaccharide solubilization in suitable solvents. Non-specific depolymerization techniques utilized for glycosidic bond cleavage, such as chemical degradation or ultrasonication, potentially generate structure fragments that can complicate the complete characterization of polysaccharide structures. Utilization of appropriate enzymes for polysaccharide degradation, on the other hand, requires prior structure information and optimal enzyme activity conditions that are not available to the analyst working with novel or unknown compounds. Herein, we describe the application of a permethylation strategy that allows the complete dissolution of the intact polysaccharides for NMR structure characterization. This approach is utilized for NMR analysis ofXylella fastidiosaEPS, which is essential for the virulence the plant pathogen that affects multiple commercial crops and is responsible for multibillion dollar losses each year.

Upon host infection, Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) into the cytosol of infected macrophages, leading to host cell death by necroptosis. TNT hydrolyzes NAD+ in the absence of any exogenous cofactor, thus classifying it as a β-NAD+ glycohydrolase. However, TNT lacks sequence similarity with other NAD+ hydrolyzing enzymes and lacks the essential motifs involved in NAD+ binding and hydrolysis by these enzymes. In this study, we used NMR to examine the enzymatic activity of TNT and found that TNT hydrolyzes NADP+ as fast as NAD+ but does not cleave the corresponding reduced dinucleotides. This activity of TNT was not inhibited by ADP-ribose or nicotinamide, indicating low affinity of TNT for these reaction products. A selection assay for nontoxic TNT variants in Escherichia coli identified four of six residues in the predicted NAD+-binding pocket and four glycine residues that form a cradle directly below the NAD+-binding site, a conserved feature in the TNT protein family. Site-directed mutagenesis of residues near the predicted NAD+-binding site revealed that Phe727, Arg757, and Arg780 are essential for NAD+ hydrolysis by TNT. These results identify the NAD+-binding site of TNT. Our findings also show that TNT is an NAD+ glycohydrolase with properties distinct from those of other bacterial glycohydrolases. Because many of these residues are conserved within the TNT family, our findings provide insights into understanding the function of the >300 TNT homologs.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93-based mechanism-of-action studies and drug discovery efforts.

The Gag protein of avian sarcoma virus (ASV) lacks an N-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. Similarly to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; however, it is unclear whether the phospholipid PI(4,5)P2 binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. Moreover, the role of PI(4,5)P2 in ASV Gag localization and budding has been controversial. Here, we report that substitution of residues that define the PI(4,5)P2-binding site in the ASV MA domain (reported in an accompanying paper) interfere with Gag localization to the cell periphery and inhibit the production of virus-like particles (VLPs). We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipase Cδ (PH-PLC), two proteins that bind PI(4,5)P2, affects ASV Gag trafficking to the PM and budding. Replacement of the N-terminal 32 residues of HIV-1 MA, which encode its N-terminal myr signal and its PI(4,5)P2-binding site, with the structurally equivalent N-terminal 24 residues of ASV MA created a chimera that localized at the PM and produced VLPs. In contrast, the homologous PI(4,5)P2-binding signal in ASV MA could target HIV-1 Gag to the PM when substituted, but did not support budding. Collectively, these findings reveal a basic patch in both ASV and HIV-1 Gag capable of mediating PM binding and budding for ASV but not for HIV-1 Gag. We conclude that PI(4,5)P2 is a strong determinant of ASV Gag targeting to the PM and budding.

BackgroundStreptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis are leading causes of invasive diseases including bacteraemic pneumonia and meningitis, and of secondary infections post-viral respiratory disease. They are typically transmitted via respiratory droplets. We investigated rates of invasive disease due to these pathogens during the early phase of the COVID-19 pandemic.MethodsLaboratories in 26 countries across six continents submitted data on cases of invasive disease due to S pneumoniae, H influenzae and N meningitidis from 1 January 2018 to 31 May 2020. Weekly cases in 2020 vs 2018-2019 were compared. Streptococcus agalactiae data were collected from nine laboratories for comparison to a non-respiratory pathogen. The stringency of COVID-19 containment measures was quantified by the Oxford COVID-19 Government Response Tracker. Changes in population movements were assessed by Google COVID-19 Community Mobility Reports. Interrupted time series modelling quantified changes in rates of invasive disease in 2020 relative to when containment measures were imposed.FindingsAll countries experienced a significant, sustained reduction in invasive diseases due to S pneumoniae, H influenzae and N meningitidis, but not S agalactiae, in early 2020, which coincided with the introduction of COVID-19 containment measures in each country. Similar impacts were observed across most countries despite differing stringency in COVID-19 control policies. There was no evidence of a specific effect due to enforced school closures.InterpretationThe introduction of COVID-19 containment policies and public information campaigns likely reduced transmission of these bacterial respiratory pathogens, leading to a significant reduction in life-threatening invasive diseases in many countries worldwide.

Clostridium perfringens is a leading cause of food-poisoning and causes avian necrotic enteritis, posing a significant problem to both the poultry industry and human health. No effective vaccine against C. perfringens is currently available. Using an antiserum screen of mutants generated from a C. perfringens transposon-mutant library, here we identified an immunoreactive antigen that was lost in a putative glycosyltransferase mutant, suggesting that this antigen is likely a glycoconjugate. Following injection of formalin-fixed whole cells of C. perfringens HN13 (a laboratory strain) and JGS4143 (chicken isolate) intramuscularly into chickens, the HN13-derived antiserum was cross-reactive in immunoblots with all tested 32 field isolates, whereas only 5 of 32 isolates were recognized by JGS4143-derived antiserum. The immunoreactive antigens from both HN13 and JGS4143 were isolated, and structural analysis by MALDI-TOF-MS, GC-MS, and 2D NMR revealed that both were atypical lipoteichoic acids (LTAs) with poly-(β1→4)-ManNAc backbones substituted with phosphoethanolamine. However, although the ManNAc residues in JGS4143 LTA were phosphoethanolamine-modified, a few of these residues were instead modified with phosphoglycerol in the HN13 LTA. The JGS4143 LTA also had a terminal ribose and ManNAc instead of ManN in the core region, suggesting that these differences may contribute to the broadly cross-reactive response elicited by HN13. In a passive-protection chicken experiment, oral challenge with C. perfringens JGS4143 lead to 22% survival, whereas co-gavage with JGS4143 and α-HN13 antiserum resulted in 89% survival. This serum also induced bacterial killing in opsonophagocytosis assays, suggesting that HN13 LTA is an attractive target for future vaccine-development studies.

The impact of food consumption on vaccine responses is unknown. Streptococcus pneumoniae (the pneumococcus) is an important human pathogen, and its polysaccharide capsule is used as a vaccine. We show that capsule type 10A in a pneumococcal vaccine shares an antigenic epitope, βGal(1-6), with pectin, which is in many plant foods and is widely consumed. Immune response to 10A is comparable to that seen with other capsule types, and pectin ingestion may have little impact on vaccine responses. However, antibody to pectin can kill serotype 10A pneumococci and this shared epitope may be considered in pneumococcal vaccine designs., We are exposed daily to many glycans from bacteria and food plants. Bacterial glycans are generally antigenic and elicit antibody responses. It is unclear if food glycans’ sharing of antigens with bacterial glycans influences our immune responses to bacteria. We studied 14 different plant foods for cross-reactivity with monoclonal antibodies (MAbs) against 24 pneumococcal serotypes which commonly cause infections and are included in pneumococcal vaccines. Serotype 15B-specific MAb cross-reacts with fruit peels, and serotype 10A MAb cross-reacts with many natural and processed plant foods. The serotype 10A cross-reactive epitope is 1,6-β-galactosidase [βGal(1-6)], present in the rhamno-galacturonan I (RG-I) domain of pectin. Despite wide consumption of pectin, the immune response to 10A is comparable to the responses to other serotypes. An antipectin antibody can opsonize serotype 10A pneumococci, and the shared βGal(1-6) may be useful as a simple vaccine against 10A. Impact of food glycans should be considered in host-pathogen interactions and future vaccine designs. IMPORTANCE The impact of food consumption on vaccine responses is unknown. Streptococcus pneumoniae (the pneumococcus) is an important human pathogen, and its polysaccharide capsule is used as a vaccine. We show that capsule type 10A in a pneumococcal vaccine shares an antigenic epitope, βGal(1-6), with pectin, which is in many plant foods and is widely consumed. Immune response to 10A is comparable to that seen with other capsule types, and pectin ingestion may have little impact on vaccine responses. However, antibody to pectin can kill serotype 10A pneumococci and this shared epitope may be considered in pneumococcal vaccine designs.

During the late phase of the HIV-1 replication cycle, the viral Gag polyproteins are targeted to the plasma membrane for assembly. The Gag-membrane interaction is mediated by binding of Gag's N-terminal myristoylated matrix (MA) domain to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The viral envelope (Env) glycoprotein is then recruited to the assembly sites and incorporated into budding particles. Evidence suggests that Env incorporation is mediated by interactions between Gag's MA domain and the cytoplasmic tail of the gp41 subunit of Env (gp41CT). MA trimerization appears to be an obligatory step for this interaction. Insufficient production of a recombinant MA trimer and unavailability of a biologically relevant membrane system have been barriers to detailed structural and biophysical characterization of the putative MA-gp41CT-membrane interactions. Here, we engineered a stable recombinant HIV-1 MA trimer construct by fusing a foldon domain (FD) of phage T4 fibritin to the MA C terminus. Results from NMR experiments confirmed that the FD attachment does not adversely alter the MA structure. Employing hydrogen-deuterium exchange MS, we identified an MA-MA interface in the MA trimer that is implicated in Gag assembly and Env incorporation. Utilizing lipid nanodiscs as a membrane mimetic, we show that the MA trimer binds to membranes 30-fold tighter than does the MA monomer and that incorporation of PI(4,5)P2 and phosphatidylserine enhances the binding of MA to nanodiscs. These findings advance our understanding of a fundamental mechanism in HIV-1 assembly and provide a template for investigating the interaction of MA with gp41CT. This work was supported by National Institutes of Health Grants 5R01GM117837 and 9R01AI150901 (to J.S.S.) Sí

Pneumococcal conjugate vaccines have been successful, but their use has increased infections by nonvaccine serotypes. Oral streptococci often harbor capsular polysaccharide (PS) synthesis loci (cps). Although this has not been observed in nature, if pneumococcus can replace its cps with oral streptococcal cps, it may increase its serotype repertoire. In the current study, we showed that oral Streptococcus strain SK95 and pneumococcal strain D39 both produce structurally identical capsular PS, and their genetic backgrounds influence the amount of capsule production and shielding from nonspecific killing. SK95 is avirulent in a well-established in vivo mouse model. When acapsular pneumococcus was transformed with SK95 cps, the transformant became virulent and killed all mice. Thus, cps from oral Streptococcus strains can make acapsular pneumococcus virulent, and interspecies cps transfer should be considered a potential mechanism of serotype replacement. Our findings, along with publications from the US Centers for Disease Control and Prevention, highlight potential limitations of the 2013 World Health Organization criterion for studying pneumococcal serotypes carried without isolating bacteria. We show that an oral streptococcal strain, SK95, and a pneumococcal strain, D39, both produce chemically identical capsular PS. We also show that transferring SK95 cps into noncapsulated, avirulent pneumococcus gave it the capacity for virulence in a mouse model.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca

For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA(87)) to lipids and liposomes. We report that MA(87) binds to the polar head of phosphoinositides such as PI(4,5)P(2). We found that MA(87) binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA(87)–IP binding is governed by charge–charge interactions. Using a sensitive NMR-based liposome-binding assay, we show that binding of MA(87) to liposomes is enhanced by incorporation of PI(4,5)P(2) and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding of MA(87) to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly.

The Gag protein of avian sarcoma virus (ASV) lacks an N-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. Similarly to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; however, it is unclear whether the phospholipid PI(4,5)P(2) binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. Moreover, the role of PI(4,5)P(2) in ASV Gag localization and budding has been controversial. Here, we report that substitution of residues that define the PI(4,5)P(2)-binding site in the ASV MA domain (reported in an accompanying paper) interfere with Gag localization to the cell periphery and inhibit the production of virus-like particles (VLPs). We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipase Cδ (PH-PLC), two proteins that bind PI(4,5)P(2), affects ASV Gag trafficking to the PM and budding. Replacement of the N-terminal 32 residues of HIV-1 MA, which encode its N-terminal myr signal and its PI(4,5)P(2)-binding site, with the structurally equivalent N-terminal 24 residues of ASV MA created a chimera that localized at the PM and produced VLPs. In contrast, the homologous PI(4,5)P(2)-binding signal in ASV MA could target HIV-1 Gag to the PM when substituted, but did not support budding. Collectively, these findings reveal a basic patch in both ASV and HIV-1 Gag capable of mediating PM binding and budding for ASV but not for HIV-1 Gag. We conclude that PI(4,5)P(2) is a strong determinant of ASV Gag targeting to the PM and budding.

Subcellular distribution of calmodulin (CaM) in human immunodeficiency virus type-1 (HIV-1)-infected cells is distinct from that observed in uninfected cells. CaM co-localizes and interacts with the HIV-1 Gag protein in the cytosol of infected cells. Although it has been shown that binding of Gag to CaM is mediated by the matrix (MA) domain, the structural details of this interaction are not known. We have recently shown that binding of CaM to MA induces a conformational change that triggers myristate exposure, and that the CaM-binding domain of MA is confined to a region spanning residues 8-43 (MA-(8-43)). Here, we present the NMR structure of CaM bound to MA-(8-43). Our data revealed that MA-(8-43), which contains a novel CaM-binding motif, binds to CaM in an antiparallel mode with the N-terminal helix (α1) anchored to the CaM C-terminal lobe, and the C-terminal helix (α2) of MA-(8-43) bound to the N-terminal lobe of CaM. The CaM protein preserves a semiextended conformation. Binding of MA-(8-43) to CaM is mediated by numerous hydrophobic interactions and stabilized by favorable electrostatic contacts. Our structural data are consistent with the findings that CaM induces unfolding of the MA protein to have access to helices α1 and α2. It is noteworthy that several MA residues involved in CaM binding have been previously implicated in membrane binding, envelope incorporation, and particle production. The present findings may ultimately help in identification of the functional role of CaM in HIV-1 replication.

Staphylococcus aureus is an opportunistic human pathogen able to transfer virulence genes to other cells through the mobilization of S. aureus pathogenicity islands (SaPIs). SaPIs are derepressed and packaged into phage-like transducing particles by helper phages like 80α or φNM1. Phages 80α and φNM1 encode structurally distinct dUTPases, Dut80α (type 1) and DutNM1 (type 2). Both dUTPases can interact with the SaPIbov1 Stl master repressor, leading to derepression and mobilization. That two structurally distinct dUTPases bind the same repressor led us to speculate that dUTPase activity may be important to the derepression process. In type 1 dUTPases, Stl binding is inhibited by dUTP. The purpose of this study was to assess the involvement of dUTP binding and dUTPase activity in derepression by DutNM1. DutNM1 activity mutants were created and tested for dUTPase activity using a novel NMR-based assay. We found that all DutNM1 null activity mutants interacted with the SaPIbov1 Stl C-terminal domain, formed DutNM1–Stl heterodimers, and caused the release of the Pstr promoter. However, promoter release was inhibited in the presence of dUTP or dUMP. We tested two φNM1 mutant phages that had null enzyme activity and found that they could still mobilize SaPIbov1. These results show that only the apo form of DutNM1 is active in Stl derepression and that dUTPase activity is not necessary for the mobilization of SaPIbov1 by DutNM1.

This paper presents a new statistical design method for maximizing the manufacturing yield of engineering systems for which the realizations of design parameters are assumed to be dependent random variables. Like in many practical situations, the method assumes that the joint distribution of design parameters is unknown and their marginal distributions can be estimated but are not necessarily symmetrical. This is a difficult problem to which little research has been devoted, other than using some brute force search methods. We use a Frank copula to construct the joint distribution of correlated random variables. Kumaraswamy density function is used to approximate their marginal distributions because of its flexibility and simplicity. The proposed method is based on the approximation of the yield integral over the largest rectangular hypercube (n-box) that is contained in the feasible region. It tries to maximize the yield by relocating and rescaling the box so that higher portion of the manufacturing yield is captured by this box. No integration is necessary since the yield of any given design is approximated by evaluating the cumulative distribution of the copula at the endpoints of the associated n-box. Finally, the actual yield of the optimal design is tested using Monte-Carlo simulation. This requires generating correlated random samples from the chosen copula distribution. One tutorial example and two practical design problems demonstrate the applications of the proposed method. Computational results show that the optimal designs significantly increase the manufacturing yield and this observation is verified using Monte-Carlo simulation.

Localization of the HIV type-1 (HIV-1) Gag protein on the plasma membrane (PM) for virus assembly is mediated by specific interactions between the N-terminal myristoylated matrix (MA) domain and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ]. The PM bilayer is highly asymmetric, and this asymmetry is considered crucial in cell function. In a typical mammalian cell, the inner leaflet of the PM is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) and contains minor populations of phosphatidylcholine (PC) and PI(4,5)P 2 . There is strong evidence that efficient binding of HIV-1 Gag to membranes is sensitive not only to lipid composition and net negative charge, but also to the hydrophobic character of the acyl chains. Here, we show that PS, PE, and PC interact directly with MA via a region that is distinct from the PI(4,5)P 2 binding site. Our NMR data also show that the myristoyl group is readily exposed when MA is bound to micelles or bicelles. Strikingly, our structural data reveal a unique binding mode by which the 2′-acyl chain of PS, PE, and PC lipids is buried in a hydrophobic pocket whereas the 1′-acyl chain is exposed. Sphingomyelin, a major lipid localized exclusively on the outer layer of the PM, does not bind to MA. Our findings led us to propose a trio engagement model by which HIV-1 Gag is anchored to the PM via the 1′-acyl chains of PI(4,5)P 2 and PS/PE/PC and the myristoyl group, which collectively bracket a basic patch projecting toward the polar leaflet of the membrane.

The cytoplasmic tail of gp41 (gp41CT) remains the last HIV-1 domain with an unknown structure. It plays important roles in HIV-1 replication such as mediating envelope (Env) intracellular trafficking and incorporation into assembling virions, mechanisms of which are poorly understood. Herein, we present the solution structure of gp41CT in a micellar environment and characterize its interaction with the membrane. We show that the N-terminal 45 residues are unstructured and not associated with the membrane. However, the C-terminal 105 residues form three membrane-bound amphipathic a-helices with distinctive structural features such as variable degree of membrane penetration, hydrophobic and basic surfaces, clusters of aromatic residues, and a network of cation-p interactions. This work fills a major gap by providing the structure of the last segment of HIV-1 Env, which will provide insights into the mechanisms of Gag-mediated Env incorporation as well as the overall Env mobility and conformation on the virion surface.

Targeting of the Gag polyprotein to the plasma membrane (PM) for assembly is a critical event in the late phase of immunodeficiency virus type-1 (HIV-1) infection. Gag binding to the PM is mediated by interactions between the myristoylated matrix (MA) domain and PM lipids. Despite the extensive biochemical and in vitro studies of Gag and MA binding to membranes over the last two decades, the discovery of the role of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] in Gag binding to the PM has sparked a string of studies aimed at elucidating the molecular mechanism of retroviral Gag–PM binding. Electrostatic interactions between a highly conserved basic region of MA and acidic phospholipids have long been thought to be the main driving force for Gag–membrane interactions. However, recent studies suggest that the mechanism is rather complex since other factors such as the hydrophobicity of the membrane interior represented by the acyl chains and cholesterol also play important roles. Here we summarize the current understanding of HIV-1 Gag–membrane interactions at the molecular and structural levels and briefly discuss the underlying forces governing interactions of other retroviral MA proteins with the PM.

Engineering systems are usually designed deterministically, but if there are uncertainties in parameters, an appropriate approach is to use probabilistic methods. For reliability estimation it is necessary to have at least the first two moments including means and covariances of the output variables. Most of the existing methods applied to problems involving linear systems need inverses of matrices. There are two problems with these approaches. First, for large sparse linear systems (for example, a tri-diagonal system) the inverse is fully dense. Second, the mean of the random matrix has to be non-singular. In this paper, we present a new method to automatically formulate the moment equations that aims to overcome the drawbacks of these methods and apply it on electrical networks. This method does not require an inverse and able to solve problems when the mean matrix of a system is singular. In addition, it takes advantage of both sparsity (zero elements) and deterministic coefficients. This method can be used to solve both uncorrelated and correlated cases. To demonstrate the feasibility of this method, a quantitative comparison with another existing method requiring the inverse and with Monte Carlo results is done. Copyright © 2005 John Wiley & Sons, Ltd.

TIM proteins are known to promote viral entry into host cells. Unexpectedly, a recent study has shown that TIM proteins also inhibit HIV-1 release from the host cell by directly binding to phosphatidylserine exposed on the virus surface, providing details on a new role of TIM proteins in HIV replication.

This paper presents a new method for finding optimal solutions of systems with design parameters which are random variables distributed with various general and possibly non-symmetrical distributions. A double-bounded density function is used to approximate the distributions. Specifications may require tracking constraints in time domain and stability conditions in frequency domain. Using sensitivity information, the proposed method first finds a linearized feasible region. Afterwards it attempts to place a tolerance box of the design parameters such that the region with higher yield lies in the feasible region. The yield is estimated by the joint cumulative density function over a portion of the tolerance box contained in the feasible region. Optimal designs are found for a fourth-order servomechanism and actual yields are evaluated by Monte-Carlo simulation. Copyright © 2001 John Wiley & Sons, Ltd.

This paper presents a general method for maximizing manufacturing yield when the realizations of system components are independent random variables with arbitrary distributions. Design specifications define a feasible region which, in the nonlinear case, is linearized using a first-order approximation. The method attempts to place the given tolerance hypercube of the uncertain parameters such that the area with higher yield lies in the feasible region. The yield is estimated by using the joint cumulative density function over the portion of the tolerance hypercube that is contained in the feasible region. A double-bounded density function is used to approximate various bounded distributions for which optimal designs are demonstrated on a tutorial example. Monte Carlo simulation is used to evaluate the actual yields of optimal designs.

Switched periodic networks are widely used in power engineering and sensitivities of their steady-state to element changes are of interest to the designers. The paper develops a method to obtain steady-state sensitivities for networks in which semiconductor and other switching devices are modelled as ideal short circuits without additional elements. Such modelling has the advantage that switching transients need not be calculated and correct initial conditions after switching are obtained with only four integration steps. It is also shown that sensitivities need not be calculated during the process of reaching the steady-state. Once the steady-state is available, integration over two additional periods is sufficient to provide time domain steady state sensitivity to the element changes.

This paper presents an application of advanced first-order second-moment (AFOSM) reliability method to probabilistic design of integrated circuits with correlated parameters. The method avoids the transformation to the space of uncorrelated parameters and provides a conservative estimate of the yield. Optimal nominal values are found such that the cost of tolerances is minimized while the desired yield is achieved. Numerical results are presented for a switched capacitor filter and verified by Monte Carlo simulation.

This paper presents a general method for statistical design centering of integrated circuits with correlated parameters. It unifies worst-case design, nominal design and tolerance design in a single framework by selecting appropriate norms to measure the distances from the nominal values. The method uses an advanced first-order second moment technique as an alternative to the simplicial algorithm. Yield estimation is calculated in the original space and no transformation to uncorrelated variables is required. The solution algorithms are based on the recently developed interior-point methods for semi-definite programming. One tutorial and one practical example explain the application.

To improve the simulation efficiency of switched circuits, it is advantageous to model their switching devices as short and open circuits. The disadvantage is that such models may cause inconsistent initial conditions (IIC), and Dirac impulses may appear. Conventional simulators do not handle such situations correctly. This paper introduces a new algorithm for analyzing circuits with IIC by a mixed numerical-symbolic method. The method detects the order of the Dirac impulse and also finds the correct initial conditions after switching. It is applicable to nonlinear circuits.

This paper presents an accelerated method to calculate the quasi-periodic state of switched networks. Switching may be performed either by external clock or by internally controlled switches and the frequencies of switching and signal need not be harmonically related. The algorithm uses time domain integration and special signal sampling which repeatedly projects the initial condition to the time t=0. Acceleration based on extrapolation makes the algorithm efficient.

The timing of sample collections for the biological monitoring of occupational exposure profoundly affects the resulting data. Sampling time with respect to the day in the working week and the end of exposure is crucial for measurements of rapidly excreted indicators of exposure. Owing to the cumulation of slowly excreted exposure indicators, timing of sample collection with respect to the duration of employment is essential. The steady state is established within a week, if the exposure indicator is excreted rapidly (with a half-life shorter than 45 h), or within months or years, if it is excreted slowly. In this study, exposure indicators are characterized by the elimination half-life. A monocompartmental model is used to calculate the biological levels at steady state and the duration of occupational exposure needed to reach the apparent steady state.

The authors have worked in computer-aided design (CAD) and in simulation of switched networks over many years. In this brief, they consider all CAD aspects related to simulation of these networks and present practical suggestions and methods useful for analysis in time and frequency domain.

This paper describes SWANN, a programme for fast simulation of non-linear switched analogue networks. the switches act as open and short circuits and are used to model switching diodes, transistors or thyristors. Non-linearities of other elements such as capacitors, inductors, resistors, conductors and dependent sources, are modelled by polynomials. Possible inconsistent conditions which can appear in networks with ideal switches are correctly handled. At switching instants the values from left and right are obtained and are available for plotting. Examples show applications in communications and power electronics.

Inconsistent initial conditions and/or Dirac impulses can appear in networks with ideal switches, and in such cases simulators may give wrong results. To avoid errors, many simulators do not allow ideal switching. This paper presents simple CAD methods which can handle correctly, and automatically, inconsistent initial conditions of linear as well as nonlinear networks. For easy understanding, all explanations are done on simple examples, with the minimum of mathematics. >

The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD) and that of the Fas-associated protein (FADD) interact to form the core of the death-inducing signaling complex (DISC), a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD). However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR), biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas-mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209-239 (Fas-Pep1) and 251-288 (Fas-Pep2) constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD-CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling pathway.

SUMMARY The paper describes models of a constant-phase element consisting of passive R and C components. The models offer any input impedance argument (phase) between −90° and 0° over a selectable frequency band covering several decades. The design procedure makes it possible to choose values of average phase, phase ripple, frequency bandwidth, and total number of R and C elements. The model can cover three frequency decades with as few as five resistors and five capacitors. The models can be used for practical realization of fractional analog differentiators and integrators, fractional oscillators, chaotic networks or for analog simulation of fractional control systems. Copyright © 2011 John Wiley & Sons, Ltd.

Ideal switches are rarely modelled in network analysis because inconsistent conditions can occur at the instant of switching. This causes standard integration routines to fail. If the initial conditions can be determined, the use of ideal switches can considerably speed up analysis and can make the results easier to understand. This paper, partly a review, presents a method which can handle ideal switching, inconsistent initial conditions, Dirac impulses and signals with discontinuities. It is applicable to networks containing linear or non-linear elements.

In the analysis of switched networks discontinuity of network variables and the presence of impulsive voltages and currents at the switching instants are often encountered. A theory for obtaining consistent initial conditions of electrical networks containing ideal switches and impulsive sources is proposed. The method is based on charge and flux conservation, and requires the solution of three one-element-kind networks. It is applicable immediately to networks with linear resistors, linear controlled sources, and linear or nonlinear capacitors and inductors. >

This paper describes an experimental mixed-mode simulation system which couples an incremental time analogue simulator to an event-driven switch level simulator. the analogue timing programme uses a variable-step-size, multi-order (VSMO) numerical integration algorithm which detects and uses latency while maintaining wave-form synchronization. the switch level programme uses piecewise linear wave-forms rather than discrete states to model the operation of logical networks. A unified format in which time responses are events is used for communication between the two programmes. the mixed-mode simulator operates without backtracking or translation between logical states and analogue wave-forms.

This chapter provides simplified steps for the synthesis of inductor capacitor (LC) filters. Synthesis of electrical networks is an area of electrical engineering in which one attempts to find the network from given specifications. In most cases, this is applied to filters constructed with various elements. Filters are linear devices that allow the use of linear network theories. Specifications of filters are almost always given as frequency domain responses, which leads to the use of Laplace transform. Filters can be built with passive elements that do not need any power supply to retain their properties and with active elements that work only when electrical power is supplied from a battery or from a power supply. The chapter also discusses frequency domain responses, normalization, and scaling.