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The detection of molecular measurable residual disease (MRD) for acute myeloid leukemia (AML) has shown promise as a prognostic for AML. To support clinical and translational research into potential strategies for MRD monitoring applications in the future, a next-generation sequencing (NGS) assay, the Oncomine Myeloid MRD Assay (RUO), was developed. This assay utilizes Unique Molecular Tags (UMT) and Ion AmpliSeq HD™ technology to detect ultra-low frequency genetic alterations from blood or bone marrow samples. The workflow includes manual AmpliSeq HD library construction, templating on Ion Chef™, and sequencing on Ion GeneStudio™ sequencers. The assay contains both DNA and RNA targeted panels to detect SNVs, indels, fusion markers, and microhaplotype. The DNA panel covers 1256 hotspots in 33 genes, including key mutations in NPM1, FLT3, DNMT3A and full gene coverage for CEBPA and TP53. The panel achieved a limit of detection (LOD) of 0.1% when tested on 27 reference genomic DNA samples and over 40 clinical research samples. The RNA panel detects 1003 fusion isoforms for 27 fusion driver genes, including BCR-ABL1, RUNX1, CBFB-MYH11, and KTM2A (MLL) partial tandem duplications (PTDs) and fusions. The assay also includes a Microhaplotyping panel to monitor chimerism. The workflow is analytically verified and includes an optimized analysis pipeline for sensitive and accurate MRD detection. Overall, this comprehensive Myeloid MRD (RUO) assay reliably identifies DNA mutations and fusions with high sensitivity and provides a new tool for myeloid MRD research. RUO: For Research Use Only. Not for use in diagnostic procedures. Citation Format: Hua Zhang, Xinzhan Peng, Guobin Luo, Annie Kraltcheva, David Mandelman, Michelle Swimley, Eli Pazol, Jose Espinoza, Scott Mitchell, Bo Ding, Ratnesh Singh, Doug Fenger, Na Li, Andrew Hatch, Vadim Mozhayskiy, Steven Roman, Sophie Rozenzhak. Analytical performance of a novel next generation sequencing assay for myeloid MRD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 759.

Objective This trial was performed to examine the effects of ruminally degradable starch (RDS) levels in total mixed ration (TMR) with low corn-based starch on the milk production, whole-tract nutrient digestibility and nitrogen balance in dairy cows. Methods Eight multiparous Holstein cows (body weight [BW]: 717±63 kg; days in milk [DIM]: 169±29) were assigned to a crossover design with two dietary treatments: a diet containing 62.3% ruminally degradable starch (% of total starch, low RDS) or 72.1% ruminally degradable starch (% of total starch, high RDS). Changes to the ruminally degradable levels were conducted by using either finely ground corn or steam-flaked corn as the starch component. Results The results showed that dry matter intake, milk yield and composition in dairy cows were not affected by dietary treatments. The concentration of milk urea nitrogen was lower for cows fed high RDS TMR than low RDS TMR. The whole-tract apparent digestibility of neutral detergent fiber, acid detergent fiber and crude protein decreased, and that of starch increased for cows fed high RDS TMR over those fed low RDS TMR, with no dietary effect on the whole-tract apparent digestibility of dry matter and organic matter. The proportion of urinary N excretion in N intake was lower and that of fecal N excretion in N intake was higher for cows fed high RDS TMR than those fed low RDS TMR. The N secretion in milk and the retention of N were not influenced by the dietary treatments. Total purine derivative was similar in cows fed high RDS TMR and low RDS TMR. Consequently, estimated microbial N flow to the duodenum was similar in cows fed high RDS TMR and low RDS TMR. Conclusion Results of this study show that ruminally degradable starch levels can influence whole-tract nutrient digestibility and nitrogen balance in dairy cows fed low corn-based starch diets, with no influence on performance.

The objectives of the present research were to investigate the changes in duodenal bacterial nitrogen flow and volatile fatty acids (VFAs) with the different dietary ratios of forage and concentrate (F:C) and to determine the relationship of duodenal bacterial nitrogen flow and volatile fatty acids with ruminal odd- and branched-chain fatty acids (OBCFAs). The experimental design was a 3 × 3 Latin square. Three rumen- and duodenal-fistulated dry Holstein cows were fed three rations with different dietary ratios of forage and concentrate (F:C; 30:70, 50:50 and 70:30). The rumen and duodenal samples were collected every two hours over 3 consecutive days of each sampling period. The determined parameters included OBCFA profiles in the rumen, duodenal bacterial nitrogen flow, VFAs, ammonia nitrogen (NH3-N) content and pH. The results showed that ruminal OBCFA contents, duodenal bacterial nitrogen flow, VFAs, ammonia nitrogen (NH3-N) and pH were significantly influenced by the different dietary F:C ratios and sampling times (P

SUMMARY Late in an adenovirus infection, the viral proteinase (AVP) becomes activated to process virion precursor proteins used in virus assembly. AVP is activated by two cofactors- the viral DNA and pVIc, an 11-amino acid peptide originating from the C-terminus of the precursor protein pVI. There is a conundrum in the activation of AVP in that AVP and pVI are sequence independent DNA binding proteins with nM equilibrium dissociation constants such that in the virus particle they are predicted to be essentially irreversibly bound to the viral DNA. Here we resolve that conundrum by showing that activation of AVP takes place on the one-dimensional contour of DNA. In vitro, pVI, a substrate, slides on DNA via one-dimensional diffusion, D1 = 1.45 x 106 (bp)2/s, until it binds to AVP also on the same DNA molecule. AVP, partially activated by being bound to DNA, excises pVIc which binds to the AVP molecule that cut it out. pVIc then forms a disulfide bond with AVP forming the fully active AVP-pVIc complex bound to DNA. In vivo, in heat-disrupted immature virus, AVP was also activated by pVI in DNA-dependent reactions. This activation mechanism illustrates a new paradigm for virion maturation and a new way, by sliding on DNA, for bimolecular complexes to form among proteins not involved in DNA metabolism.

Recent single-molecule enzymology measurements with improved statistics have demonstrated that a single enzyme molecule exhibits large temporal fluctuations of the turnover rate constant at a broad range of time scales (from 1 ms to 100 s). The rate constant fluctuations, termed as dynamic disorder, are associated with fluctuations of the protein conformations observed on the same time scales. We discuss the unique information extractable from these experiments and the reconciliation of these observations with ensemble-averaged Michaelis−Menten equation. A theoretical model based on the generalized Langevin equation (GLE) treatment of Kramers' barrier crossing problem for chemical reactions accounts naturally for the observation of dynamic disorder and highly dispersed kinetics.

Electron transfer is used as a probe for angstrom-scale structural changes in single protein molecules. In a flavin reductase, the fluorescence of flavin is quenched by a nearby tyrosine residue by means of photo-induced electron transfer. By probing the fluorescence lifetime of the single flavin on a photon-by-photon basis, we were able to observe the variation of flavin-tyrosine distance over time. We could then determine the potential of mean force between the flavin and the tyrosine, and a correlation analysis revealed conformational fluctuation at multiple time scales spanning from hundreds of microseconds to seconds. This phenomenon suggests the existence of multiple interconverting conformers related to the fluctuating catalytic reactivity.

The preparation and the structure of polymer-cushioned phospholipid bilayers (PCPBs) in the solid phase through vesicle fusion was studied by atomic force microscopy. It was found that the anionic phospholipid α-l-dipalmitoylphosphatidic acid could form a good PCPB on the polyethylenimine surface but not on that of poly(dimethyldiallyammonium chloride), indicating that the attractive electrostatic force alone cannot guarantee the fusion of the vesicles. The zwitterionic phospholipid α-l-dipalmitoylphosphatidylcholine could not form a good PCPB on either polymer surface. It was found that the PCPB rearranged to an alternate monolayer and trilayer structure while it was exposed to air and turned back to a bilayer structure when it was dipped into water again. Various factors affecting the PCPB formation were discussed. The preparation of such bilayers through the LB transfer method was also investigated.

A covalently linked ultrathin multilayer film was fabricated from 2-nitro-N-methyl-diphenylamine-4-diazonium-formaldehyde resin (NDR) and phenol-formaldehyde resin (PR) in methanol-in-situ. The UV-vis, XRD, AFM and FTIR show that this multilayer is of high regularity, smoothness and stability.

Multilayer films from diazoresin (I) and poly(sodium acrylate) (II) or a hydrolyzed maleic anhydride–styrene copolymer (III) have been fabricated on mica. During the fabrication process the absorbance of the film at 380 nm, which is the characteristic absorption of the diphenylamine 4-diazonium group of I, increases about 0.033 for each fabrication cycle. This means that the thickness of the film increases regularly. Under irradiation with UV light or heating, following the decomposition of the diazonium group, the ionic bond in the film structure converts to a covalent bond; this conversion was preliminarily verified by FTIR spectrum analysis. The stability of the different films (irradiated or unirradiated) was determined by UV-vis spectroscopy and the results show that the resistance of the film to etching by polar solvents increases significantly after bond conversion.

Two kinds of ultrathin films were successfully fabricated by the self-assembly deposition technique from poly(diallyldimethylammonium chloride) (PDDAC) or nitro-containing diazoresin (NDR) as the polycation and 4,4′-di(sodium styryl-2-sulfonate) biphenyl (DSB) as the dianion. This stepwise fabrication process was monitored by UV-vis spectrometry. The regularity of the film and the morphology of the film surface were examined by X-ray diffraction (XRD) and atom force microscopy (AFM), respectively. The results show that these kinds of films are composed of well-arranged molecules, and have a very flat surface. Among them, the PDDAC/DSB film emits fluorescence when excited with UV light at 354 nm. The NDR/DSB film does not emit fluorescence, because the NDR quenches the fluorescence of DSB; however, the NDR/DSB film possesses more stability against solvent etching (such as DMF), compared with the PDDAC/DSB film.

The membranes of a double-chain amphiphile dioctadecyl-N-[4-[[p-(N,N-dimethylamino)phenyl]azo]-pyridiniumbutyryl]-l-aspartate bromide (DDPA) were investigated by atomic force microscopy (AFM) and UV−vis absorption spectroscopy. The line-shaped aggregates of the molecules were found in the Langmuir−Blodgett (LB) monolayers of DDPA. The widths of these lines were about twice the length of the molecule. A semicylindrical structure was proposed to account for the observation. It was found that the DDPA molecules self-organized into bilayers and vesicles when the LB film was immersed in aqueous solutions. The bilayer showed good compressibility. The mixed DDPA and α-l-dipalmitoylphosphatidylcholine LB films were also examined, and the phase separation was observed. From the in situ AFM observation, the self-organization process was found to occur in the DDPA region. The mechanism of self-organization in aqueous solutions was also discussed.

Two kinds of covalently tethered fullerene monolayers, which exhibit the photoelectric conversion property, are reported. The monolayers were fabricated on hydrophilic indium–tin oxide (ITO), quartz, and mica by an esterification reaction and characterized by contact angle, AFM, UV spectrum, and cyclic voltammetry. The thicknesses of the films calculated by the MM2 molecular mechanics method were in good agreement with the results of AFM, indicating the formation of monolayers. The photoelectric conversion properties of both monolayers were studied.

The layer-by-layer self-assembled NDR-PSS (nitro-containing diazoresin-polysodium p-styrenesulfonate) films were fabricated. The crosslinking structure formed from the conversion of ionic bond to covalent bond after UV irradiation, confirmed by small angle X-ray diffraction. The roughness and microtribological properties of NDR-PSS films were investigated by atomic force microscopy/friction force microscopy. The ordered multilayer films after photoreaction are better in microtribological performance than that of the monolayer film.

A kind of crystal precursor, (NH4)5[(VO)6(CO3)4(OH)9]·10H2O, less than 12 × 40 μm in dimensions was synthesized by reaction of vanadyl dichloride and ammonium hydrocarbonate in solution. The precursor microcrystals were fined to less than 10 μm with mean size of about 4 μm, by using organic solvents and surfactant in the process of the synthetical technique. Thermolysis of the precursor in a flow of nitrogen gas was investigated by TGA and DTA. The XRD powders patterns for the products from the thermolysis of the precursor show that the VO2 powder obtained at 350, 365, 380 and 410°C for 30 min were a noncrystalline phase, a monoclinic one named as B phase, a mixed phase of the B and the usual monoclinic A phase, and the pure A phase, respectively. SEM micrographs of VO2 powders from the fined precursor demonstrated that the particle sizes of these fine powders were less than 2 μm. DSC curve of the VO2 obtained at 450 °C for 30 min from the fined precursor showed that there was an endothermic phase transition at 71 °C.

Two kinds of self-assembled monolayer (SAM) and a Langmuir–Blodgett film of fullerene derivative C61(COOH)2 that have the photoelectric conversion property were first reported here. The SAMs were either fabricated directly on hydrophilic indium–tin-oxide (ITO), quartz, and mica by esterification reaction or on the aminopropylsilanated substrates by amide bonds. The SAMs were characterized by contact angle, AFM, UV spectrum and cyclic voltammetry. The film thickness calculated by molecular mechanic with the MM2 method in Molgen 4.0 coincided with that obtained by AFM measurement, indicating the formation of monolayer. All of three systems can generate stable cathodic photocurrent. The separated charge loss process in our systems is monomolecule recombination.

A monolayer ultrathin film of nitro-containing diazoresin (NDR) was fabricated on mica via a self-assembly technique. The adsorption of NDR on mica at different deposition times was monitored by de...

The response of a protein to variation of a specific coordinate can provide insights into the role of the overall architecture in the structural change. Given that the calculated potential of mean force governing the fluctuation of an electron transfer donor-acceptor distance in the NAD(P)H:Flavin oxidoreductase (Fre)/FAD complex was shown to agree with experiment, an analysis of the structural response of the rest of the protein to that distance change was made. Significant displacements are found throughout much of the protein, and the coupling pathway resulting in the structural changes was determined. A covariance analysis based on the quasiharmonic modes of the unperturbed protein was used to provide information concerning how the residue motions are correlated. It is found that, of the three regions identified as moving together in an NMR study, two undergo significant structural changes when the electron donor-acceptor distance is varied, and the third does not.

A new fullerene self-assembled monolayer (SAM) which has the property of photoelectric conversion is reported here. The SAM was fabricated on hydrophilic substrates by an esterification reaction. The SAM is characterized by contact angle, AFM, UV spectrum, and cyclic voltammetry. A cathodic photocurrent of 226 nA/cm(2) was obtained. Copyright 2000 Academic Press.

It is known that DNA-binding proteins can slide along the DNA helix while searching for specific binding sites, but their path of motion remains obscure. Do these proteins undergo simple one-dimensional (1D) translational diffusion, or do they rotate to maintain a specific orientation with respect to the DNA helix? We measured 1D diffusion constants as a function of protein size while maintaining the DNA-protein interface. Using bootstrap analysis of single-molecule diffusion data, we compared the results to theoretical predictions for pure translational motion and rotation-coupled sliding along the DNA. The data indicate that DNA-binding proteins undergo rotation-coupled sliding along the DNA helix and can be described by a model of diffusion along the DNA helix on a rugged free-energy landscape. A similar analysis including the 1D diffusion constants of eight proteins of varying size shows that rotation-coupled sliding is a general phenomenon. The average free-energy barrier for sliding along the DNA was 1.1 +/- 0.2 k(B)T. Such small barriers facilitate rapid search for binding sites.

Dynamic disorder in proteins, as demonstrated by variations in single-molecule electron transfer rates, is investigated by molecular dynamics simulations. The potential of mean force for the fluctuating donor-acceptor distance is calculated for the NAD(P)H:flavin oxidoreductase (Fre) complex with flavin adenine dinucleotide (FAD) and is found to be in agreement with that estimated from electron transfer experiments. The calculated autocorrelation function of the distance fluctuations has a simple exponential behavior at low temperatures and stretched exponential behavior at higher temperatures on femtosecond to nanosecond time scales. This indicates that the calculated dynamic disorder arises from a wide range of trapping times in potential wells on the protein energy landscape and suggests a corresponding origin for the stretched exponential behavior observed experimentally on longer time scales.

To explore the mechanism used by RB69 DNA polymerase (RB69 pol), several new experimental strategies were applied to determine rates of conformational changes by stopped-flow fluorescence (sfF). 2-...

Enzymes are biological catalysts vital to life processes and have attracted century-long investigation. The classic Michaelis-Menten mechanism provides a highly satisfactory description of catalytic activities for large ensembles of enzyme molecules. Here we tested the Michaelis-Menten equation at the single-molecule level. We monitored long time traces of enzymatic turnovers for individual β-galactosidase molecules by detecting one fluorescent product at a time. A molecular memory phenomenon arises at high substrate concentrations, characterized by clusters of turnover events separated by periods of low activity. Such memory lasts for decades of timescales ranging from milliseconds to seconds owing to the presence of interconverting conformers with broadly distributed lifetimes. We proved that the Michaelis-Menten equation still holds even for a fluctuating single enzyme, but bears a different microscopic interpretation.

Enzymes are biological catalysts vital to life processes and have attracted century-long investigation. The classic Michaelis-Menten mechanism provides a highly satisfactory description of catalytic activities for large ensembles of enzyme molecules. Here we tested the Michaelis-Menten equation at the single-molecule level. We monitored long time traces of enzymatic turnovers for individual beta-galactosidase molecules by detecting one fluorescent product at a time. A molecular memory phenomenon arises at high substrate concentrations, characterized by clusters of turnover events separated by periods of low activity. Such memory lasts for decades of timescales ranging from milliseconds to seconds owing to the presence of interconverting conformers with broadly distributed lifetimes. We proved that the Michaelis-Menten equation still holds even for a fluctuating single enzyme, but bears a different microscopic interpretation.

The fluctuation of the distance between a fluorescein-tyrosine pair within a single protein complex was directly monitored in real time by photo-induced electron transfer, and found to be a stationary, time-reversible and non-Markovian Gaussian process. Within the generalized Langevin equation (GLE) formalism, we experimentally determine the memory kernel, K(t), which is proportional to the autocorrelation function of the random fluctuating force. K(t) is a power-law decay, t- 0.51 ± 0.07 in a broad range of time-scales (10-3 s -10s. Such a long time memory effect, which is associated with sub-diffusion within a harmonic bound, has implications to protein functions.© (2005) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The fluctuation of the distance between a fluorescein-tyrosine pair within a single protein complex was directly monitored in real time by photoinduced electron transfer and found to be a stationary, time-reversible, and non-Markovian Gaussian process. Within the generalized Langevin equation formalism, we experimentally determine the memory kernel K(t), which is proportional to the autocorrelation function of the random fluctuating force. K(t) is a power-law decay, t(-0.51 +/- 0.07) in a broad range of time scales (10(-3)-10 s). Such a long-time memory effect could have implications for protein functions.

We use single-molecule fluorescence lifetimes to probe dynamics of photoinduced reversible electron transfer occurring between triphenylamine (donor) and perylenediimide (acceptor) in single molecules of a polyphenylenic rigid dendrimer embedded in polystyrene. Here, reversible electron transfer in individual donor-acceptor molecules results in delayed fluorescence that is emitted with a high photon count rate. By monitoring fluorescence decay times on a photon-by-photon basis, we find fluctuations in both forward and reverse electron transfer spanning a broad time range, from milliseconds to seconds. Fluctuations are induced by conformational changes in the dendrimer structure as well by polystyrene chain reorientation. The conformational changes are related to changes in the dihedral angle of adjacent phenyl rings located in the dendritic branch near the donor transferring the charge, a torsional motion that results in millisecond fluctuations in the “through-bond” donor-acceptor electronic coupling. Polymer chain reorientation leads to changes in the local polarity experienced by the donors and to changes in the solvation of the charge-separated state. As a result, switching between different donor moieties within the same single molecule becomes possible and induces fluctuations in decay time on a time scale of seconds.

Advanced fluorescence measurements on single molecules demand single-photon detectors with high-quantum detection efficiency, low noise, and high time resolution. We have developed a compact (82/spl times/60/spl times/30 mm) and versatile single-photon timing module (SPTM), based on a planar epitaxial single photon avalanche diodes (SPAD) working with a monolithic integrated active quenching and active reset circuit (i-AQC) and cooled by a Peltier element. The main operating parameters are computer controlled via RS-232 interface and the photon counting rate can be continuously monitored. The photon detection efficiency is 45% at 500 nm with cooling at -15/spl deg/C, the dark counting rate is 5 c/s with SPAD operating at 5 V excess bias voltage, 10c/s operating at 10 V. The time resolution obtained with tightly focused illumination has 60-ps full-width at half-maximum. Comparative tests with the SPTM prototype and with an advanced commercially available photon counting module confirmed that the time resolution and sensitivity of the SPTM make it possible to resolve and measure even short lifetime components of a single molecule. The SPTM thus made possible experiments leading to a deeper insight into angstrom-scale structural changes of single-protein molecules.

The self-assembly film fabricated via the layer-by-layer technique was studied by the dynamic contact angle (DCA) method (wilhelmy plate method). The used polyelectrolytes are poly(diallyl-dimethylammonium chloride) (PDDA), poly(etheleneimine) (PEI), diphenylamine-4-diazonium-formaldehyde resin (DR), 2-nitro-N-methyl-4-diazonium-formaldehyde resin (NDR), and poly(sodium-p-styrenesulfonate) (PSS). For the self-assembly systems of PDDA/PSS, PEI/PSS, DR/PSS, and NDR/PSS, their individual contact angle fluctuates regularly with the fabrication of each layer, while the magnitude of different systems' contact angle depends on the participant polycation. The re-organization of components and the adjacent layer interpenetration are presented here to explain this phenomena. We also found that DR or NDR can adsorb itself via the layer-by-layer method to form multilayer film, and the hydrophobic interaction is put forward to effect this process. Moreover, the procedure of washing and drying after adsorption was studied and considered as a prerequisite for the successful fabrication, especially of the same charge carried components. Copyright 2001 Academic Press.