Academic literature on the topic 'CcdB substitutions'

The method described here allows editing of the bacterial genome without leaving any secondary changes (scars) behind. This method uses a tripartite selectable and counterselectable cassette comprising an antibiotic-resistance gene (catorkan) and thetetRrepressor gene linked to a Ptetpromoter-ccdBtoxin gene fusion. In the absence of induction, thetetRgene product represses the Ptetpromoter, preventingccdBexpression. The cassette is first inserted at the target site by selecting for chloramphenicol or kanamycin resistance. It is subsequently replaced by the sequence of interest by selecting for growth in the presence of anhydrotetracycline (AHTc), which inactivates the TetR repressor thereby causing CcdB-induced lethality. Unlike other CcdB-based counterselection schemes, which require specifically designed λ-Red delivery plasmids, the system described here uses the popular plasmid pKD46 as the source of λ-Red functions. This protocol allows a wide variety of modifications, including the intragenic insertion of fluorescent or epitope tags, gene replacements, deletions, and single base-pair substitutions, to be made. In addition, the procedure can be used to place the inducible Ptetpromoter at a chosen position in the bacterial chromosome.

Most amino acid substitutions in a protein either lead to partial loss of function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss of function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. When coupled to inactive mutants, they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we determined the crystal structures of three such suppressors to obtain insight into the specific molecular interactions responsible for the observed effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increments, thermodynamic stabilisation is neither necessary nor sufficient for suppressor action. Instead, in diverse systems, we observe that individual global suppressors greatly enhance the foldability of buried site mutants, primarily through increase in refolding rate parameters measured in vitro. In the crowded intracellular environment, mutations that slow down folding likely facilitate off-pathway aggregation. We suggest that suppressor mutations that accelerate refolding can counteract this, enhancing the yield of properly folded, functional protein in vivo.

Abstract INTRODUCTION Mass spectrometry (MS) is a potentially useful tool for the study of the hemostasic system and its imbalances that lead to bleeding and thrombotic disorders. By harnessing the high throughput and broad scope of MS, vast data may be available to investigators and clinicians to help predict or manage hemostatic events. Although utilizing MS to evaluate coagulation proteins appears promising, amino acid (AA) substitutions resulting from genetic variation may yield a spectrum of mass-to-charge ratios (m/z) that can impede accurate protein identification. The goals of this study were to describe 1) the proteins present in a blood sample that might be involved in or otherwise affect coagulation, 2) the realm of variations that might occur with a single nucleotide substitution (SNS) in the reference coding sequences of these proteins, and 3) the variation of peptide fragments of these proteins when only one of the nucleotides is a variant. METHODS We obtained protein lists from the NCBI BioSystems database for the terms: Blood Clotting Cascade, Complement Cascade, Formation of Fibrin Clot, Hemostasis, Platelet Activation, Platelet Aggregation Plug Formation, Platelet Degranulation, Platelet Homeostasis, and Thrombin Signaling (e.g., http://www.ncbi.nlm.nih.gov/biosystems/198840). We linked the Symbol (gene) to the CCDS ID (consensus coding sequence, http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi ). For each nucleotide, we enumerated the effect of a SNS relative to the other three nucleotides. We then generated every peptide of length 5-20, determined the change in mass based on the average, as opposed to the monoisotopic, mass of the substituted AA, and assessed whether the peptide was unique among those in the system. Finally, we determined whether possible N-linked glycosylation sites were preserved, destroyed, or created by SNS. We considered a putative site N[^P][S|T], that is N in position 1, not P in position 2, and either a S or T in position 3. RESULTS The proteins in the biosystems that were also in the CCDS database comprised 517 distinct Symbols and 951 distinct CCDS ID's, comprising 2,180,352 codons. The duplicate Symbols include transcript variants, for instance, the Symbol F8 linked to CCDS ID's 35457.1 and 44026.1, thereby diminishing the uniqueness of the peptides (transcript variants share some, if not most, of the reading frames). All of the codons were susceptible to an AA substitution; at least one variant nucleotide substation in position 1 or 2 of the codon always resulted in an AA substitution. SNS caused premature termination signals (stop codons) in 240,100 of these codons. Table 1 details the variations. A map of the N-glycosylation sites is available for each protein, although this may not affect MS directly. Of the 83,510 potential N-Linked Glycosylation sites, a SNS disrupted the putative AA sequence in 53,067 (64%). A SNS created a novel potential N-Linked Glycosylation site at 52,787 loci. Table 1. Wild-type Only Wild-type and Variants Peptide Length Peptides Distinct Peptides Peptides Distinct Peptides Relative Change in Mass 5 722,174 292,485 24,470,567 2,464,873 0.052250 10 717,564 350,570 47,911,535 20,982,641 0.025968 15 712,954 355,446 71,052,562 31,777,153 0.017273 20 708,344 357,177 93,893,423 42,465,377 0.012939 CONCLUSIONS Variant peptides due to a single SNS per peptide greatly outnumber wild-type peptides. The ability to identify a protein based on uniqueness of one of its peptides increases as the peptide size increases, but AA variations in those peptides that arise from one SNS will require 1) increased mass resolution and 2) both a search algorithm and database that accounts for the possible variations. Patients with hemostatic or thrombotic disorders may be more likely to have a variant, and these results highlight the need to know the genetic sequence associated with proteins being analyzes by MS if this technology is to be adopted for research and clinical purposes. The inclusion of currently identified SNPs and the effect of INDELs that preserve the reading frame is ongoing. Disclosures No relevant conflicts of interest to declare.

To reduce the amount of cement used in cemented coal gangue backfill (CCGB, a mixture of coal gangue, cement, fly ash, and water), mechanical and deformation properties of CCGB in which CSFA partially replaces the cement (0, 10, 20, 30, and 40 wt%) were studied. Compressive strength, acoustic emission during uniaxial loading, shear strength, and drying shrinkage were analysed. The compressive strength, shear strength, and drying shrinkage tests were performed at different curing times. The results showed that cemented coal gangue and corn stalk fly ash backfill (CGCAB) presented better performance, and the CGCAB with a 20% substitution rate had the best performance at day 28. Despite having the largest drying shrinkage value, 20% is the best choice for the substitution rate of CSFA. A 20% CSFA addition can enhance the bearing capacity of CGCAB and improve its failure mode, which is of great significance to support the upper overburden load and maintain the surface stability of the goaf.

To functionally characterize transport properties of the apical anion exchanger of rabbit β-intercalated cells, the mean change in anion exchange activity, dpHi/d t(where pHi is intracellular pH), was measured in response to lumen Cl− replacement with gluconate in perfused cortical collecting ducts (CCDs). β-Cell apical anion exchange was not affected by 15-min exposure to 0.2 mM lumen DIDS in the presence of 115 mM Cl−. In contrast, apical anion exchange was significantly inhibited by 0.1 mM lumen DIDS in the absence of Cl−. β-Cell apical anion exchange was unchanged by 15 mM maleic anhydride, 10 mM phenylglyoxal, 0.2 mM niflumic acid, 1 mM edecrin, 1 mM furosemide, 1 mM probenecid, or 0.1 mM diphenylamine-2-carboxylate. However, β-cell apical anion exchange was inhibited by α-cyano-4-hydroxycinnamic acid, with an IC50 of 2.4 mM. Substitution of either sulfate or gluconate for lumen Cl− resulted in a similar rate of alkalinization. Conversely, pHi was unchanged by substitution of sulfate for lumen gluconate, confirming the lack of transport of sulfate on the β-cell apical anion exchanger. Taken together, the results demonstrate a distinct “fingerprint” of the rabbit CCD β-cell apical anion exchanger that is unlike that of other known anion exchangers.

State-of-the-art detector calibration in the UV/VUV and soft X-ray spectral ranges at the Physikalisch-Technische Bundesanstalt (PTB) is based on the primary detector standard SYRES, a cryogenic electrical substitution radiometer capable of measuring radiant power of a few µW. At the PTB radiometry laboratory at the synchrotron radiation facility BESSY, two dedicated beamlines are operated, providing monochromatic radiation of high spectral purity, high radiant power and tunable photon energy in the 3–1500 eV range. The spectral responsivity of detectors, e.g. photodiodes, can be measured with a relative uncertainty of about 1% by direct comparison with SYRES, as will be demonstrated for PtSi/Si and GaAsP/Au Schottky and silicon n-on-p photodiodes. The calibration of photon-counting detectors traceable to SYRES can by accomplished by exploiting the unique capability to scale the spectral photon flux over several orders of magnitude by changing the stored electron current. Calibrations of CCDs and photomultipliers are presented as examples.

In Europe, the toxicological safety of genetically modified (GM) crops is routinely evaluated using rodent feeding trials, originally designed for testing oral toxicity of chemical compounds. We aimed to develop and optimize methods for advancing the use of zebrafish feeding trials for the safety evaluation of GM crops, using maize as a case study. In a first step, we evaluated the effect of different maize substitution levels. Our results demonstrate the need for preliminary testing to assess potential feed component-related effects on the overall nutritional balance. Next, since a potential effect of a GM crop should ideally be interpreted relative to the natural response variation (i.e., the range of biological values that is considered normal for a particular endpoint) in order to assess the toxicological relevance, we established natural response variation datasets for various zebrafish endpoints. We applied equivalence testing to calculate threshold equivalence limits (ELs) based on the natural response variation as a method for quantifying the range within which a GM crop and its control are considered equivalent. Finally, our results illustrate that the use of commercial control diets (CCDs) and null segregant (NS) controls (helpful for assessing potential effects of the transformation process) would be valuable additions to GM safety assessment strategies.

We used liquid ion exchanger and conventional microelectrodes to evaluate the effects of mineralocorticoids on the intracellular K activity (aiK) and K transport properties of principal cells (PC) of isolated cortical collecting ducts (CCDs). Hoffman modulation optics and electrophysiological methods were used to identify PC. K activity was measured with two single-barreled electrodes. We found that aiK of PC from deoxycorticosterone acetate (DOCA)-treated rabbits (97.6 mM) was not different from controls (94.8 mM). The driving forces for K transport across the basolateral membrane favored cell to bath (reabsorption) in PCs from controls and bath to cell (secretion) in PCs from DOCA-treated rabbits. However, the driving force for K secretion across the apical membrane was not significantly different between the two groups. We used the intracellular aiKs and bath ion substitutions (gluconate for Cl and K for Na) to evaluate the effects of DOCA on the ion-selective properties of the basolateral membrane of PC. DOCA increased PK/PCl from 0.33 to 0.89. Our conclusion was as follows: in PC of control rabbits K is above electrochemical equilibrium across the basolateral membrane. However, the basolateral K conductance is probably too small for significant K recycling. In PC of DOCA-treated rabbits the aiK is below electrochemical equilibrium across the basolateral membrane and the K conductance is increased. These effects enhance K secretion across this border while maintaining cell K constant.

Objective: The existence of keto-enol tautomerism in β-diketones can typically study by a choice of analytical technique. The position of the keto-enol equilibrium depends on a number of factors like solvent, temperature, and substituents. Here an attempt was made to examine the effect of indole, a heterocyclic moiety with the moderately high polar surface area to examine its effect on ketonisation of β-diketone.Methods: The β-diketone studied and synthesized is a structural analog of magical drug curcumin. The structural influence of indole on ketonisation of β-diketone is studied to give a hypothesis on factors contributing towards ketonisation. This work involves the synthesis of 6-(1H-Indol-3-yl)-hex-5ene-2, 4-dione and the study on the single crystal structure of indole-3-carboxaldehyde, major functional component to result in the reaction. The tautomer was studied for its ability to bind with tetrahydrofolate reductase enzyme using Discovery Studio 3.5 version to differentiate the pharmacological significance of conformations.Results: The single crystal XRD structure of this compound was deposited in Cambridge crystallographic data center bearing CCDC No.1536311. The structural characterization of synthesized ligand was carried out by using IR, Mass, 1H NMR spectroscopic techniques. The docking study reveals that keto isomer found to exhibit more inhibition of the enzyme tetrahydrofolate reductase hence more pharmacologically active.Conclusion: The experimental evidence proves that indole substitution shifted the keto-enol equilibrium towards keto form of 6-(1H-Indol-3-yl)-hex-5ene-2, 4-dione.

A wide variety of retailers (supermarkets, home electronics, Internet shops, etc.) provide scanner data containing information at the level of the barcode, e.g. the Global Trade Item Number (GTIN). As scanner data provide complete transaction information, we may use the expenditure shares of items as weightsfor calculating price indices at the lowest (elementary) level of data aggregation. The challenge here is the choice of the index formula which should be able to reduce chain drift bias and substitution bias. Multilateral index methods seem to be the best choice due to the dynamic character of scanner data. These indices work on a wholetime window and are transitive, which is key to the elimination of the chain drift effect. Following what is called an identity test, however, it may be expected that even when only prices return to their original values, the index becomes one. Unfortunately, the commonly used multilateral indices (GEKS, CCDI, GK, TPD, TDH) do not meet the identity test. The paper discusses the proposal of two multilateral indices and their weighted versions. On the one hand, the design of the proposed indices is based on the idea of the GEKS index. On the other hand, similarly to the Geary-Khamis method, it requires quality adjusting. It is shown that the proposed indices meet the identity test and most other tests. In an empirical and simulation study, these indices are compared with the SPQ index, which is relatively new and also meets the identity test. The analytical considerations as well as empirical studies confirm the high usefulness of the proposed indices.

Determining the precise effects of thousands of point mutants on protein structure, stability, and activity is a challenging task. Chapter 1 introduces how next-generation sequencing technologies exploit phenotypic screens of mutant libraries to simultaneously measure the effects of thousands of mutants in a single experiment. In the recent past, several approaches have been developed to predict fitness effects of individual members of mutational libraries constructed on a target gene by treating them under different conditions, optionally indexing them based on their barcodes and finally, generating a fitness landscape to ascertain the effect of each mutant. The model system used in our work is the ccdAB operon which is tightly autoregulated by the CcdAB complex. CcdAB is a TypeII toxin-antitoxin (TA) system composed of a labile CcdA antitoxin and a stable CcdB toxin. At low toxin: antitoxin ratio, the operator/promoter region is repressed by a multimeric chain of alternating CcdA2-CcdB2 modules spiralling around the promoter DNA. Degradation of the labile antitoxin causes a decrease in the toxin: antitoxin ratio, which results in formation of a V-shaped derepressing CcdB2-CcdA2-CcdB2 heterohexamer. xii In Chapter 2, we developed a facile method to deduce the structural and functional determinants of the globular, cytotoxic protein, CcdB from E.coli, solely from mutational data. We generated a comprehensive site-saturation mutagenesis library of CcdB in its native operon. We examined the effects of mutations on Gyrase binding activity of the CcdB toxin in an E.coli strain which was sensitive to the toxic activity of the toxin. In conjunction with this screen, we also developed a RelE reporter assay in which the ccd promoter precedes the RelE gene to examine the effects of mutations in CcdB on CcdA binding activity, in a strain resistant to the toxic action of CcdB but sensitive to RelE toxicity. In vivo studies with individual point mutants of CcdB were used to validate our deep sequencing results. In vitro studies further support the molecular mechanisms inferred from deep sequencing data. Since autoregulation and rejuvenation are mechanistically intertwined in the CcdAB TA system, residues that are inferred as being important for antitoxin binding are also important for rejuvenating CcdB from the CcdB-Gyrase complex. In this study, we delineated protein-protein interaction interface residues for two different interacting partners, namely the antitoxin CcdA and cellular target Gyrase, and discriminated buried residues from residues involved in partner binding, with the help of differential mutational patterns observed for these different classes of residues. Chapter 3 describes studies which demonstrate that the majority of the mutants that were destabilised in the absence of the antitoxin, when expressed in the operonic context, displayed a phenotype like the WT in the RelE reporter stain, and exhibited toxicity less than WT in a strain sensitive to the toxicity of the CcdB toxin. In vivo studies showed that the folding defects of several buried site mutants were relieved in the presence of CcdA. In vitro characterisation of these destabilised mutants showed that they form a complex with CcdA similar to that of the WT. In vivo solubility assays confirmed that the folding defects inherently present in these buried site CcdB mutants were alleviated in the presence of its interacting partner, CcdA, likely via cotranslational folding. xiii In Chapter 4, we measured the effects of all possible single-site synonymous substitutions for each position of CcdB in their native operonic context. It is important to establish genotype-phenotype relationships under physiological conditions. The ability to perform genetic screens for both antitoxin binding and toxin binding to its cellular target helped to probe and understand effects of synonymous mutations on gene function. The data suggest that control of translational initiation is important for determining protein abundance inside the cell. There is an interplay of several factors, namely, codon usage, t-RNA abundance, generation of internal Shine-Dalgarno-like sequences, mRNA structure and evolutionary conservation, in dictating the translational efficiency of a gene. Amongst all the amino acids, synonymous mutations of Arginine displayed the maximum phenotypic and codon-specific effects on in vivo protein abundance in the ccdB gene, in its operonic context. In Chapter 5, we used the same methodology, to estimate the effects of single-site synonymous substitutions in combination with a mutation in the N-terminal region of CcdB, that likely alters the translation rate, thereby affecting the cotranslational folding process of the protein. We observed that introduction of potential pause sites in the middle of the gene because of the synonymous mutation, results in increased CcdB toxicity. Our data suggest that double-site synonymous mutations result in translational uncoupling, along with alteration in translational kinetics. This is possibly accompanied by improved in vivo protein stability or folding kinetics, in the case of CcdB protein. In summary, extensive analysis of the deep sequencing data obtained for both non-synonymous and synonymous CcdB substitutions helps understand the molecular basis of the observed mutant phenotypes. Such studies provide novel insights into gene regulation, protein folding, stability, and activity.

Symbolic substitution is essentially a combination of recognition and substitution process. In addition, although symbolic substitution is not restricted to space invariant operations, it is indeed based on the space invariant connectivity of optics. Therefore, holographic associative memory techniques may be alternatively applied to implement a symbolic substitution logic system. In this paper, we propose to use a high efficiency joint transform correlator to implement a symbolic subsitiution operation. Consider that the search pattern fs(x,y) and an input image which consists of M × N input patterns fm,n(x−xm,y−yn), are to be displayed on an input plane in a joint transform correlator architecture. The power spectrum recorded by a CCD camera is then replicated, using dedicated video hardware, to produce a L × L spectra array on a second spatial light modulator. Upon illuminated by a quasi-monochromatic, partially coherent parallel light source, the object irradiance at the correlation plane will be increased by L2 times that of a conventional joint transform correlator.1 Therefore, if fmn matches with the search pattern fs, a bright correlation peak would be located at a specific location determined by (xm,yn). To eliminate all of the unwanted light, a set of sampling pinholes is placed at the correlation plane. As a result, a spatial distribution of point light sources is generated. The substitution process can be accomplished by inserting a multiplexed Fourier transform hologram of the substituting pattern behind the collimating system after the sampling plane. Each of the point light source produces a plane wave front to read out the hologram. Consequently, each correlated input pattern produces a substituted output pattern at the corresponding position on the output plane, and symbolic substitution operation is completed.

This paper reports the design and fabrication of a prototype holographic optical processor array—HOPE—for parallel optical logic, arithmetic and interconnect operations. The volume holographic technology gives the inherent advantages of high resolution, large dynamic range, large recording area, low cost and 3-D interconnection capabilities. A set of rules and input/output lookup tables can be recorded in a holographic associative memory, and then the HOPE performs parallel operations by input/output symbolic substitutions and thresholds. A practical N4 recording scheme has been developed to record a 2-D array of holographic elements. The automatic recording system is capable of constructing an array of up to 256 × 256 holograms on a 128 × 128 mm2 holographic plate, each hologram 0.5 × 0.5 mm2 in size. Based on large array of holographic elements, the HOPE has the potential of highly parallel computation between thousands of optical processors in a compact system. The aim of this research study is toward the concept design of a HOPE architecture with the electronic counterpart-connection machine in mind. A prototype HOPE system is designed and fabricated with moderate number of holographic elements (approximately 1000 holograms of 1 × 1 mm2 in size) which employs commercially available devices such as high contrast liquid crystal television and CCD camera as input and output interface. Evaluation of the system performance is also discussed.

Fatty acid methyl esters (biodiesel), prepared from transesterification of vegetable oils or animal fats, have gained great importance in substituting petroleum based diesel for combating environmental problems and higher diesel prices. Moringa oleifera fatty acids are among the newly investigated potentials for biodiesel production in recent years. In getting rid of soap formation and thus large waste washing water from biodiesel produced from homogenous catalysts, the use of heterogeneous catalysts is currently preferred due to easily separation and purification of the final products. In this study, biodiesel was produced from moringa oleifera oil using sulfated tin oxide enhanced with SiO2 (SO42−/SnO2−SiO2) as super acid solid catalyst. The experimental design was done using design of experiment (DoE), specifically, response surface methodology based on three-variable central composite design (CCD) with alpha (α) = 2. The reaction parameters in the optimization process were reaction temperature (60°C to 180°C), reaction period (1 to 3 hrs) and methanol to oil ratio (1:6 to 1:24 mol/mol). It was observed that the yield up to 84wt% of moringa oleifera methyl esters can be obtained with reaction conditions of 150°C temperature, 150 minutes reaction time and 1:19.5 methanol to oil ratio, while catalyst concentration and agitation speed are kept at 3wt% and 350 rpm respectively.

Abstract γ-TiAl, gamma titanium aluminide, is a unique metal matrix composite, the structure of which provides exceptional properties that include low density, high yield strength, high stiffness, high specific stiffness, etc. Consequently, γ-TiAl alloys are long being considered for substituting several superalloys for various applications in the fields of automotive, aerospace and biomedical industries. However, γ-TiAl is well-known as very difficult to machine, which has hindered the widespread application of γ-TiAl. In this paper, a central composite design (CCD) of experiments is adopted to investigate various turning process responses with hexagonal shape inserts. Results show that, among three turning factors, the depth of cut factor has the greatest impact on cutting force responses, while the feed rate factor has the most dominating effect on specific cutting energy and surface finish. Quadratic equations are developed, and the corresponding two-dimensional and three-dimensional plots are established. By means of regression analysis and response surface methodology (RSM), a series of optimal turning parameters is obtained, which renders the minimum machining energy, leading to increased tool life and improved machinability. Furthermore, it is feasible to achieve an industry-accepted surface finish. The results of this work cover a machinability investigation of a gamma titanium aluminide that can find application in automobile and aerospace industries for machining of high-temperature resistance parts.

The feasibility of using adaptive optics (AO) systems on large ground based optical telescopes for producing compensated images of both astronomical sources and artificial earth satellites has been demonstrated by a number of research groups. AO systems consist conceptually of a wavefront sensor (WFS) which measures atmospherically induced wavefront phase perturbations faster than the rate at which they occur and a wavefront phase compensation device, typically a deformable mirror, which takes the WFS data and produces a conjugate phase correction nulling the distortion. In the case of faint sources, the ability of an AO system to produce phase corrections is limited by the signal-to-noise ratio (SNR) in the WFS. In the case of the Shack Hartmann (SH) WFS this limit is a function of the SNR required to estimate with sufficient accuracy the centroid positions of unresolved images of the source formed by a lenslet array on an array of quadrant detectors composing the subapertures of the WFS. Although the use of laser guidestars may allow the substitution of faint source radiation with a bright artificial AO beacon, considerations of cost and complexity motivate a continuing interest in improving the detection sensitivity of a source referenced, low light level (LLL) WFS. This paper explores the promise of aluminum gallium arsenide/gallium arsenide (AlGaAs/GaAs) heterostructure photocathodes coupled to shot noise limited photon counting (PC) cameras and compares their performance with the read noise limited charge coupled device (CCD) cameras often used in this application. Figure (1) illustrates the quantum efficiency of the various detectors to be considered in the study, including two high performance AlGaAs/GaAs photocathodes recently obtained from Litton Electro-Optical Systems.