Three-step Protocols Research Articles

Memory in non-monotonic stress response of an athermal disordered solid

Athermal systems across a large range of length scales, ranging from foams and granular bead packings to crumpled metallic sheets, exhibit slow stress relaxation when compressed. Experimentally they show a non-monotonic stress response when decompressed somewhat after an initial compression, i.e., under a two-step, Kovacs-like protocol. It turns out that from this response one can tell for how long the system was in a compressed state, suggesting an interpretation as a memory effect. In this paper we use a model of an athermal jammed solid, specifically a binary mixture of soft harmonic particles, to explore this phenomenon through in silico experiments. Using extensive simulations under conditions analogous to those in experiment, we observe identical phenomenology in the stress response under a two-step protocol. Our model system also recovers the behavior under a more recently studied three-step protocol, which consists of a compression followed by a decompression and then a final compression. We show that the observed response in both two-step and three-step protocols can be understood using linear response theory. In particular, a linear scaling with age for the two-step protocol arises generically for slow linear responses with power law or logarithmic decay and does not in itself point to any underlying aging dynamics.

Photocatalytic Stoichiometric Oxidant-Free Synthesis of Linear Unsaturated Ketones from 1,2-Disubstituted Cyclopropanols

AbstractA one-step catalytic oxidant-free synthesis of unsaturated ketones from 1,2-disubstituted cyclopropanols is reported. Previously for this transformation, only two- and three-step protocols have been developed. The reaction proceeds under irradiation with visible light in the presence of catalytic amounts of both an acridinium photocatalyst and a cobaloxime complex. 2-Aryl-substituted cyclopropanols react giving α,β-unsaturated ketones, while dehydrogenative ring opening of 2-alkyl-substituted substrates affords mixtures of α,β- and β,γ-enones. The reaction starts with one-electron oxidation of a cyclopropanol to cyclopropyloxy radical, presumably, by the photoexcited acridinium catalyst. We also found that Co(dmgBF2)2(MeCN)2 complex under an air atmosphere and irradiation with blue LEDs or upon heating can serve as a hydroxycyclopropane oxidant.

Comparison of three multi-cryoprotectant loading protocols for vitrification of porcine articular cartilage

Vitrification is a cryopreservation technique for the long-term storage of viable tissue, but the success of this technique relies on multiple factors. In 2012, our group published a working vitrification protocol for intact human articular cartilage and reported promising chondrocyte recovery after using a four-step multi-cryoprotectant (CPA) loading method that required 570 min. However, this protocol requires further optimization for clinical practice. Herein, we compared three multi-step CPA loading protocols to investigate their impact on chondrocyte recovery after vitrification of porcine articular cartilage on a bone base, including our previous four-step protocol (original: 570 min), and two shorter three-step protocols (optimized: 420 min, and minimally vitrifiable: 310 min). Four different CPAs were used including glycerol, dimethyl sulfoxide, ethylene glycol and propylene glycol. As vitrification containers, two conical tubes (50 ml and 15 ml) were evaluated for their heat transfer impact on chondrocyte recovery after vitrification. Osteochondral dowels were cored into two diameters of 10.0 mm and 6.9 mm with an approximately 10-mm thick bone base, and then allocated into the twelve experimental groups based on CPA loading protocol, osteochondral dowel size, and vitrification container size. After vitrification at −196 °C and tissue warming and CPA removal, samples in all groups were assessed for both chondrocyte viability and metabolic activity. The optimized protocol proposed based on mathematical modelling resulted in similar chondrocyte recovery to our original protocol and it was 150 min shorter. Furthermore, this study illustrated the role of CPA permeation (dowel size) and heat transfer (container size) on vitrification protocol outcome.

How the Complex Interplay between Different Blocks Determines the Isothermal Crystallization Kinetics of Triple-Crystalline PEO-b-PCL-b-PLLA Triblock Terpolymers

PEO-b-PCL-b-PLLA triblock terpolymers are fascinating triple-crystalline materials. In this work, the isothermal crystallization kinetics of these terpolymers evaluated by differential scanning calorimetry (DSC) is presented for the first time and compared to analogous PCL-b-PLLA diblock copolymers and to PLLA, PCL, and PEO homopolymers. The results are complemented by in situ SAXS/WAXS synchrotron experiments. One-, two-, and three-step crystallization protocols were employed to study the crystallization kinetics of the blocks. At PLLA block crystallization temperatures, both PCL and PEO molten chains caused a strong plasticizing effect on the PLLA block crystallization, and the overall crystallization rate of the PLLA block in the terpolymers was higher than that in the PLLA-b-PCL diblock copolymers. In the case of the PCL block, the crystallization was followed after PLLA was fully crystallized (two-step crystallization). A nucleating effect induced by the previously formed PLLA crystals was observed. ...

Local Hypothesis Testing Between a Pure Bipartite State and the White Noise State

In this paper, we treat a local discrimination problem in the framework of asymmetric hypothesis testing. We choose a known bipartite pure state $\ket{\Psi}$ as an alternative hypothesis, and the completely mixed state as a null hypothesis. As a result, we analytically derive an optimal type 2 error and an optimal POVM for one-way LOCC POVM and Separable POVM. For two-way LOCC POVM, we study a family of simple three-step LOCC protocols, and show that the best protocol in this family has strictly better performance than any one-way LOCC protocol in all the cases where there may exist difference between two-way LOCC POVM and one-way LOCC POVM.

Propensity of Self-Assembled Leucine-Lysine Diblock Copolymeric α-Helical Peptides To Remain in Parallel and Antiparallel Alignments in Water.

Molecular dynamics simulation study of α-helical diblock copolypeptides preassembled in parallel and antiparallel alignments in water are presented. The assembled peptide lamellar structures were not disrupted even after performing three-step simulation protocols. Primarily hydrogen bonds between peptide are responsible for the stability. The analysis of the trajectory also suggests that water plays a significant role in favoring self-assembly. We have detected continuous hydrogen bonded network structure, which is further responsible for the stability of the lamellar structures. We have performed a detailed analysis of the hydrogen bonded network structure and its length. Further, free energy calculations revealed that the degree of stability for both lamellae are similar. The present study provides structural insight into the stability of self-assembled structures of block copolypeptides.

A Library of Well-Defined and Water-Soluble Poly(alkyl phosphonate)s with Adjustable Hydrolysis

Poly(alkyl ethylene phosphonate)s with different alkyl side chains exhibit significant differences in their degradation behavior. Three novel 2-alkyl-2-oxo-1,3,2-dioxaphospholanes, cyclic monomers for the ring-opening polymerization (ROP) toward poly(alkyl alkylene phosphonate)s, were synthesized by robust two- or three-step protocols in reasonable yields and high purity. The polymerization was promoted by the organocatalysts 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) and proceeded with high control over molecular weight and narrow molecular weight distributions (Đ < 1.2) up to full conversion. These polymers with methyl, ethyl, and isopropyl side chains are perfectly soluble in water (up to 25 mg mL–1) without a temperature-dependent phase separation. They showed no toxicity against HeLa cells after 24 h of incubation at any tested concentration. Polymers with butyl side chains exhibit decreased solubility and concentration-dependent cloud point temperatures and show toxicity against HeLa cells at concentrations above 25 μg mL–1. The polymers showed no acetylcholinesterase inhibition. All polymers exhibited significantly different degradation times under both neutral as well as basic conditions (variation of the alkyl side chain allowed stabilities from 8 h up to 6 days).

Asymptotic local hypothesis testing between a pure bipartite state and the completely mixed state

In this paper, we treat an asymptotic hypothesis testing (or state discrimination with asymmetric treatment of errors) between an arbitrary fixed bipartite pure state $|\ensuremath{\Psi}\ensuremath{\rangle}$ and the completely mixed state by one-way local operation and classical communication (LOCC), two-way LOCC, and separable positive operator-valued measures (POVMs). As a result, we derive single-letterized formulas for the Stein's lemma type of optimal error exponents under one-way LOCC, two-way LOCC, and separable POVMs, the Chernoff bounds under one-way LOCC POVMs and separable POVMs, and the Hoeffding bounds under one-way LOCC POVMs in the whole region of a parameter and under separable POVMs on a restricted region of a parameter. We also numerically calculate the Chernoff and the Hoeffding bounds under a class of three-step LOCC protocols in low-dimensional systems and show that these bounds not only outperform the bounds for one-way LOCC POVMs but also almost approximate the bounds for separable POVMs in the parameter region where analytical bounds for separable POVMs are derived.

Rapid PCR protocols for forensic DNA typing on six thermal cycling platforms.

Rapid PCR protocols for the amplification of typing STR multiplexes were evaluated on six different thermal cyclers. Through the use of a faster DNA polymerase coupled with the use of rapid thermal cyclers the amplification cycling times were reduced down to as little as 14 min using PCR primers from the commercially available multiplex STR typing kit Identifiler. Previously described two-step and three-step thermal cycling protocols were evaluated for the six thermal cyclers on 95 unique single-source DNA extracts. CE characterization of the PCR products indicates good peak balance between loci (median values greater than 0.84), and N minus four stutter ratios on averages were 30 to 40% higher than for standard Identifiler PCR conditions. Nonspecific amplification artifacts were observed, but were not observed to migrate within the allele calling bins. With the exception of one locus (D18S51) in a single sample, genotyping results were concordant with manufacturer's recommended amplification conditions utilizing standard thermal cycling procedures. Assay conditions were robust enough to routinely amplify 250 to 500 pg of template DNA. This work describes the protocols for the rapid PCR amplification of STR multiplexes on various PCR thermal cyclers with the future intent to support validation for typing single-source samples in a database laboratory.

N-Acetylgalactosamino Dendrons as Clearing Agents to Enhance Liver Targeting of Model Antibody-Fusion Protein

Dendrimer clearing agents represent a unique class of compounds for use in multistep targeting (MST) in radioimmunotherapy and imaging. These compounds were developed to facilitate the removal of excess tumor-targeting monoclonal antibody (mAb) prior to administration of the radionuclide to minimize exposure of normal tissue to radiation. Clearing agents are designed to capture the circulating mAb, and target it to the liver for metabolism. Glycodendrons are ideally suited for MST applications as these highly branched compounds are chemically well-defined, thus advantageous over heterogeneous macromolecules. Previous studies have described glycodendron 3 as a clearing agent for use in three-step MST protocols, and early in vivo assessment of 3 showed promise. However, synthetic challenges have hampered its availability for further development. In this report we describe a new sequence of chemical steps which enables the straightforward synthesis and analytical characterization of this class of dendrons. With accessibility and analytical identification solved, we sought to evaluate both lower and higher generation dendrons for hepatocyte targeting as well as clearance of a model protein. We prepared a series of clearing agents where a single biotin is connected to glycodendrons displaying four, eight, sixteen or thirty-two α-thio-N-acetylgalactosamine (α-SGalNAc) units, resulting in compounds with molecular weights ranging from 2 to 17 kDa, respectively. These compounds were fully characterized by LCMS and NMR. We then evaluated the capacity of these agents to clear a model (131)I-labeled single chain variable fragment antibody-streptavidin ((131)I-scFv-SAv) fusion protein from blood and tissue in mice, and compared their clearing efficiencies to that of a 500 kDa dextran-biotin conjugate. Glycodendrons and dextran-biotin exhibited enhanced blood clearance of the scFv-SAv construct. Biodistribution analysis showed liver targeting/uptake of the scFv-SAv construct to be 2-fold higher for compounds 1 to 4, as well as for the 500 kDa dextran, over saline. Additionally, the data suggest the glycodendrons clear through the liver, whereas the dextran through reticuloendothelial system (RES) metabolism.

Differences of PCR efficiency between two-step PCR and standard three-step PCR protocols in short tandem repeat amplification

The effects on STR typing results, using a two-step PCR protocol that combines the annealing and extension steps, were determined using samples at various DNA concentrations. DNA samples ranging from 62.5 to 500 pg were amplified using the reagents contained within the AmpFlSTR Identifiler PCR Amplification Kit. At 250–500 pg of DNA template, the rates of detecting alleles were close to those found when using three-step PCR (the standard Identifiler protocol) and two-step PCR protocols. The two-step PCR increased by 8.1% and 64.2% the average number of amplified alleles compared with the three-step PCR at 125 pg and 62.5 pg of template DNA, respectively. At 62.5–500 pg of DNA, the average peak heights were 23.3–65.0% higher than heights using the three-step PCR protocol. When two different PCR protocols were applied to old bones, the average number of amplified loci was similar. These results suggested that the two-step PCR protocol can generate more STR alleles in low template DNA samples; however, the method may be limited with samples of very low-quality, i.e., degraded DNA, compared with the three-step protocol. A better understanding of the effects of the two-step PCR protocol on amplification conditions might help researchers improve STR typing.

Reproducibility of Plantar Pressure Measurements in Patients with Chronic Arthritis: A Comparison of One-Step, Two-Step, and Three-Step Protocols and an Estimate of the Number of Measurements Required

Plantar pressure measurement may be a helpful evaluation tool in patients with foot complaints. Determination of dynamic pressure distribution under the foot may give information regarding gait, progress of disorders, and the effect of treatment. However, for these measurements to have clinical application, reproducibility, consistency, and accuracy must be ascertained. We compared the reproducibility of measurements among one-step, two-step, and three-step protocols for data collection in patients with arthritis. In addition, the number of measurements needed for a consistent average was determined for the protocol that was found to be the most reproducible. Twenty patients with foot complaints secondary to arthritis participated in the study. Each patient was tested with a pressure platform system using two of the three testing protocols. Reproducibility of contact time and maximal peak pressure were assessed. Intraclass Correlation Coefficients (ICC) were calculated for measurement results among protocols. In stage two of the study, the number of measurements needed for a consistent average was determined by calculating the first three measurements, the first five measurements, and then all seven measurements for both feet. ICC of three, five, and seven measurements were compared. The two-step protocol (13 patients), which was found in stage one of the study to be the most reproducible, was used for this determination. Reproducibility was found to be reasonable or good for all three measurement protocols. The mean values of contact time for the one-step protocol were found to be higher than the mean values of contact time for the two-step or three-step protocols in both feet. The differences between the one-step and three-step protocols were statistically significant for the left foot only. The mean peak pressure did not show statistically-significant differences among the three protocols. The one-step and three-step protocols were not used for stage two of the study. Using the two-step protocol, three measurements were found to be sufficient for obtaining a consistent average. The results of our study indicated that the one-step, two-step, and three-step protocols of collecting plantar pressure measurements in patients with foot complaints secondary to chronic arthritis were all similar. However, the use of the two-step protocol is recommended over the one-step and three-step protocols; the one-step protocol produced a longer stance phase that did not resemble normal walking and when comparing the two-step and three-step protocols, the two-step protocol was less time consuming and less strenuous for patients with painful feet.

Influence of Cooling Rates and Plunging Temperatures in an Interrupted Slow-Freezing Procedure for Semen of the African Catfish, Clarias gariepinus

The objective of this study was to optimize interrupted slow-freezing protocols for African catfish semen. Semen diluted with methanol and extender was frozen in 1-ml vials in a programmable freezer. The temperatures of the freezer (Tchamber) and of the semen (Tsemen) were measured simultaneously. We first tested two-step freezing protocols with different cooling rates (−2, −5, and −10°C/min) and different temperatures at plunging into liquid N2. The difference between Tsemen and Tchamber increased with faster cooling rates. In all programs, survival of spermatozoa, expressed as hatching rates, increased from near zero when Tsemen at plunging was higher than −30°C to values equal to those of control when Tsemen at plunging was equal to or lower than −38°C. The inclusion of an isothermal holding period before plunging into liquid N2 (three-step freezing protocols) resulted in an equilibration between Tsemen and Tchamber and improved semen survival. Semen could be plunged at temperatures as high as −36°C when cooled at −5 or −10°C/min, without compromising postthaw semen survival. Cooling at −2°C/min in combination with a 5-min holding period reduced postthaw survival. We conclude that with slow cooling rates of −2 to −5°C/min, hatching rates can be maximized by plunging as soon as Tsemen reaches −38°C. The isothermal holding period is beneficial when faster rates are used. A simple and efficient protocol for freezing African catfish semen can be obtained by cooling at a rate of −5 to −10°C/min combined with a 5-min holding period in the freezer, at −40°C

Fluorescent staining of κ opioid receptors using naltrexamine derivatives and phycoerythrin

An immunofluorescent technique that is more sensitive than radioligand binding was developed in order to detect opioid receptors expressed on leukocytes. The current study was designed to optimize the method for fluorescently labeling κ opioid receptors. For these experiments, the opioid antagonist naltrexamine was conjugated to either fluorescein (FITC-NTXamine) or biotin (biotin-NTXamine). One-step, two-step, and three-step protocols were compared to determine which procedure resulted in optimal staining of the κ opioid receptor expressed on intact, unfixed R1E/TL8x.1.OUA r.1 (R1EGO) cells, a thymoma known to express κ opioid receptors. The one-step method involved incubating cells with FITC-NTXamine, and the fluorescein intensity was measured by flow cytometry. In the two-step method, cells were incubated with biotin-NTXamine, followed by extravidin-conjugated phycoerythrin, and the phycoerythrin fluorescence was measured. Finally, in the three-step protocol, cells were incubated with FITC-NTXamine, followed by biotin-conjugated anti-fluorescein IgG, then extravidin-phycoerythrin. The one-step protocol stained the cells, but the signal was not diminished in the presence of opioid competitors. The two-step approach did not stain cells significantly above background levels. Only the three-step approach yielded staining that was displaced by the κ-selective antagonist nor-binaltorphimine. Thus, the addition of a secondary biotinylated antibody, resulting in the amplification of binding, which was detected using phycoerythrin as a fluorophore, was required to detect low levels of opioid receptor expression on leukocytes.

Intraepithelial and postinvasive neoplasia as a stochastic continuum of clonal evolution, and its relationship to mechanisms of chemopreventive drug action

The progression of intraepithelial and postinvasive neoplasia depends on the occurrence of clonal evolution, defined as the continuous development of mutations and selective clonal expansions in the neoplastic cell population. The two continuously repeating events of clonal evolution, mutation and clonal expansion, occur at unpredictable times and locations. Therefore the neoplastic process is best characterized as a stochastic, i.e., probabilistic, continuum. The rate of intraepithelial neoplastic progression is continuously driven by the dosage level of exposure to mutagens and mitogens. For example, in chronic smokers the length of time before development of lung cancer depends on the number of cigarettes smoked per day. A commonly held misconception is that human carcinogenesis develops after an initial short period of mutation followed by a long period of stimulated proliferation (the multistage model). This incorrect idea derives from the sequential nature of the consecutive two- or three-step operational protocols imposed on experimental animal models by the experimenter. In reality, human carcinogenesis develops as the result of simultaneous and continuous exposure to mutagens and mitogens over the entire period of tumor development. A recent example is the finding that the intraepithelial neoplasia of colorectal adenomas continuously progresses through serial waves of mutation and clonal expansion. The rational design of chemopreventive agents should be based on blocking the two parameters which continuously drive neoplasia: mutagenesis and mitogenesis. In addition to blocking exposure, chemopreventive agents may act at many points during activation and DNA adduction of mutagens, or during stimulation of the proliferation signal pathway by mitogens. Based on the chemopreventive strategy of blocking mutagenesis and mitogenesis, chemopreventive agents are classed as either antimutagenic or antimitogenic. A third class, the antioxidants, are both antimutagenic and antimitogenic, and operate by the common mechanism of breaking free radical chain reactions initiated by reactive oxygen species. In the program of the Chemoprevention Investigational Studies Branch, Division of Cancer Prevention and Control, National Cancer Institute, preclinical development of antimutagens, antimitogens, and antioxidants is well under way, and some of these agents are highlighted here.

Investigations of the chemical structure of sulfonated amine-formaldehyde resins. 4. Variations in structure with preparation procedure

The chemical structures of resins prepared according to ASC (addition-sulfonation-condensation) and ACS (addition-condensation-sulfonation) protocols were compared, and it was shown that the ACS protocol leads to resins with a higher molecular weight, whereas the ASC protocol results in resins with a higher degree of sulfonation. Two-step and three-step ASC protocols were also compared. It was found that combining the addition and sulfonation steps is one step resulted in a resin that was very similar to that obtained from a three-step ASC protocol. Therefore, it was concluded that the urea-formaldehyde reaction is much faster than the formaldehyde-sulfite reaction