Magnetic hydrogel particles improve nanopore sequencing of SARS-CoV-2 and other respiratory viruses.

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with Nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40 × improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher automation platform.

Hydrogel particles improve detection of SARS-CoV-2 RNA from multiple sample types.

Here we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from contrived transport medium and saliva samples using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus from 1 mL samples prior to RNA extraction, substantially improving detection of virus using real-time RT-PCR across a range of viral titers (100-1,000,000 viral copies/mL) and enabling detection of virus using the 2019 nCoV CDC EUA Kit down to 100 viral copies/mL. This method is compatible with commercially available nucleic acid extraction kits (i.e., from Qiagen) and a simple heat and detergent method that extracts viral RNA directly off the particle, allowing a sample processing time of 10 min. We furthermore tested our method in transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2, showing that our method not only correctly identified all positive samples but also substantially improved detection of the virus in low viral load samples. The average improvement in cycle threshold value across all viral titers tested was 3.1. Finally, we illustrate that our method could potentially be used to enable pooled testing, as we observed considerable improvement in the detection of SARS-CoV-2 RNA from sample volumes of up to 10 mL.

Synthesis and Electrochemistry of O3-type NaFeO2-NaCo0.5Ni0.5O2 Solid Solutions for Na-Ion Positive Electrodes.

The synthesis, structure, and electrochemistry in Na cells of NaFe xM1- xO2 positive electrode materials with M = Ni, Co0.5Ni0.5, and Co are reported. In particular, the properties of O3-NaFeO2-NaCo0.5Ni0.5O2 solid solutions having compositions NaFe x(Co0.5Ni0.5)1- xO2 with 0 ≤ x ≤ 0.5 are explored. It is found that the substitution of Fe in NaNi0.5Co0.5O2 causes an increase in first cycle energy density from 320 to 440 mWh/g in a 1.5-4.0 V test. However, capacity retention is generally reduced when x is increased for all M = Ni, Co0.5Ni0.5, and Co. In general, NaFe xM1- xO2 samples with M = Co had the highest capacity retention for all values of x. Ex situ X-ray diffraction and Mössbauer results of as-prepared and charged materials are directly compared for NaFe x(Co0.5Ni0.5)1- xO2 and NaFe xCo1- xO2 ( x = 0.4, 0.5). Iron was found to be in the +3 oxidation state in the as-prepared materials. A significant fraction of Fe3+ is oxidized to Fe4+ in these samples when they are charged to 4.0 V.

Role of CuO in improving NH3 and SO2 capture on nanoporous Fe2O3 sorbents.

In this work, mixed Fe/Cu oxides as sorbents for SO2 and NH3 removal were investigated. Nanoporous iron oxide mixed with 10, 20 and 30 at.% CuO were prepared by thermal decomposition of the corresponding oxalates at 250 °C for 5 h in air. The mixed Fe/Cu oxalates were obtained from the co-precipitation of iron/copper sulfate and ammonium oxalate during ultrasonication. The physical properties of the oxalate precursors and the resulting mixed Fe/Cu oxides were characterized with SEM, TGA-DSC, FTIR, powder XRD and Mössbauer spectroscopy. The porosity was studied by N2 adsorption-desorption isotherms and small angle X-ray scattering. Evenly dispersed CuO hindered the crystallization of Fe2O3, which significantly increased the specific BET surface area from 211 m2/g for Fe2O3 to 354 m2/g for Fe0.8Cu0.2Ox. As a result, SO2 and NH3 adsorption on Fe0.8Cu0.2Ox were enhanced by about 70% compared to Fe2O3. Compared to Fe2O3-impregnated activated carbons, nanoporous Fe0.8Cu0.2Ox could capture five times more SO2 per unit weight, which will be attractive for applications in respirators with lower weight and smaller size.

THE EARTH SYSTEM PREDICTION SUITE: Toward a Coordinated U.S. Modeling Capability.

The Earth System Prediction Suite (ESPS) is a collection of flagship U.S. weather and climate models and model components that are being instrumented to conform to interoperability conventions, documented to follow metadata standards, and made available either under open source terms or to credentialed users. The ESPS represents a culmination of efforts to create a common Earth system model architecture, and the advent of increasingly coordinated model development activities in the U.S. ESPS component interfaces are based on the Earth System Modeling Framework (ESMF), community-developed software for building and coupling models, and the National Unified Operational Prediction Capability (NUOPC) Layer, a set of ESMF-based component templates and interoperability conventions. This shared infrastructure simplifies the process of model coupling by guaranteeing that components conform to a set of technical and semantic behaviors. The ESPS encourages distributed, multi-agency development of coupled modeling systems, controlled experimentation and testing, and exploration of novel model configurations, such as those motivated by research involving managed and interactive ensembles. ESPS codes include the Navy Global Environmental Model (NavGEM), HYbrid Coordinate Ocean Model (HYCOM), and Coupled Ocean Atmosphere Mesoscale Prediction System (COAMPS®); the NOAA Environmental Modeling System (NEMS) and the Modular Ocean Model (MOM); the Community Earth System Model (CESM); and the NASA ModelE climate model and GEOS-5 atmospheric general circulation model.

Characterization of the covalent chromatography of thymidylate synthase on thiopropyl-Sepharose 6B.

We have examined the covalent chromatography of Lactobacillus casei thymidylate synthase on thiopropyl-Sepharose 6B resin. This enzyme is a dimer of identical subunits, each of which contains one active site that features a catalytic sulfhydryl group (Cys-198). Reversible coupling was achieved via the attack of one of the enzyme's two catalytic cysteine residues, causing displacement of 2-thiopyridone, the reactive moiety of the resin. To establish the usefulness of this matrix for immobilization and the conditions required for chromatography, model studies were conducted with 2,2'-dithiodipyridine. The chemical modification of thymidylate synthase with 2,2'-dithiodipyridine was shown to be specific for the catalytic sulfhydryl groups of the native dimer, titrating 1.51 sulfhydryl groups, while 2.93 cysteines were modified in the GnHCl-denatured protein. The former reaction, which resulted in total loss of enzyme activity, was reversible with complete recovery of control activity within 30 min after addition of 100 mM 2-mercaptoethanol. Characterization of the protein pools generated in the covalent chromatography procedure indicated that the enzyme fraction washing through the column without attachment had substantially lower catalytic and ligand binding activities than the original protein stock; conversely, the enzyme fraction eluted from the column by 2-mercaptoethanol exhibited higher levels of these activities. Gel electrophoresis studies further illustrated that the unique application of the covalent chromatography technique described herein fractionated homogeneous thymidylate synthase protein into enzyme pools exhibiting distinct biochemical properties. As immobilization reaction times were increased beyond 6 h, the coupling of thymidylate synthase was demonstrated to occur through more than one enzymic sulfhydryl group. Interestingly, no covalent coupling was detected in attempts using activated thiol-Sepharose 4B, a result underlining the importance of the structure of the resin linker arm in enzyme immobilization.

Modification of tyrosine residues in dihydrofolate reductase from Lactobacillus casei.

Dihydrofolate reductase from Lactobacillus casei was inactivated by reaction with tetranitromethane and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Loss of activity occurred with modification of four of the five tyrosine residues present in the enzyme. The presence of either substrate, NADPH or 7,8-dihydrofolate, as well as NADP and folate, provided extensive protection against inactivation, while NADH and tetrahydrofolate exhibited none. This protection from inactivation occurred on protection of two of the four susceptible tyrosines from modification. Nitration of the enzyme adversely affected its ability to bind substrates. Restoration of the pKa of the nitrated tyrosines by reduction of the nitro group to an amino group did not result in a regeneration of enzymatic activity. However, fluorotyrosine-containing enzyme, prepared by growing the bacterium in the presence of fluorotyrosine, exhibited specific activity identical to that of native enzyme over the pH range of 4.5-8. These results suggest that inactivation of dihydrofolate reductase by tyrosine modification occurs primarily due to a steric effect and that the active site tyrosines may participate in substrate binding.

Characterization of the parameters affecting covalent binding stoichiometry in binary and ternary complexes of thymidylate synthase.

Covalent binding stoichiometries for both the enzyme:5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) binary complex and the enzyme:FdUMP:5,10-methylenetetrahydrofolate (inhibitory ternary) complex at equilibrium were measured by the trichloroacetic acid precipitation assay and shown to be a function of temperature, time, pH, salt concentration, buffer composition and thiol concentration. Incubation at 37 degrees C yielded the maximum covalent binding ratio (mol FdUMP/mol enzyme) for the latter binary (0.7) and ternary (1.7) complexes. In most buffers studied, the maximum covalent binding ratio (1.5-1.7) for the inhibitory ternary complex occurred over a broad pH range (4.5-8.0), while the optimum covalent binding ratio for binary complex was observed at a much narrower region centered between pH 5.5-6.5. In the presence of increasing concentrations of phosphate buffer, the maximum binding ratio for the covalent binary complex decreased from 0.63 in the absence of phosphate to 0.1 in the presence of 225 mM phosphate, while that for the inhibitory ternary complex was unchanged. When a ternary complex was formed with enzyme, FdUMP and (+/-)-tetrahydrofolate in the absence of phosphate, the FdUMP:enzyme covalent binding ratio was 1.8, while in the presence of 75 mM phosphate, the binding ratio was only 1.0. When exogenous thiol was removed by centrifugal column chromatography, the maximum binding stoichiometry of the resulting inhibitory ternary complex was 1.7 and was independent of added thiol over a 2 h incubation period at 37 degrees C. When extensive dialysis at 5 degrees C was used to remove the thiol, the maximum binding stoichiometry of the resulting inhibitory ternary complex was found to be dependent on both the concentration of added thiol and the time of incubation at 37 degrees C and did not exceed a value of 1.0.

High expression and steady-state kinetic characterization of methionine site-directed mutants of Escherichia coli methionyl- and selenomethionyl-dihydrofolate reductase.

A high expression system that produces Escherichia coli dihydrofolate reductase (DHFR) at 30% total cellular protein was constructed. This expression vector, named pCOCK, allowed for the purification of nearly 100 mg of homogeneous DHFR from a 11 bacterial culture. A simple, single Q-Sepharose anion exchange column purification was developed on an FPLC instrument. Methionine site-directed mutants were constructed in DHFR to assess the role of Met within the enzymes. These mutants consisted of a Met16leucine (Leu), Met20Leu, Met42Leu, Met92Leu, Met16,20Leu and Met16,20,42Leu. Steady-state kinetic studies showed that the Met16Leu, Met42Leu and Met92Leu mutants possessed essentially the same kcat, Km(DHF) and Km(NADPH) as that of wild-type (wt) DHFR (13.7 s-1, 0.97 microM and 2.52 microM, respectively). Mutants which contained a Leu at position 20 possessed substantially elevated specific activity and kcat values. The specific activity and kcat of wt, Met20Leu, Met16,20Leu and Met16,20,42Leu were 45.9, 92.7, 90.2 and 172 mumol/min/mg and 13.7, 24.6, 25.2 and 52.7 s-1, respectively. Upon substitution of Met by selenomethionine (SeMet) in the aforementioned mutants, further information as to the effect of SeMet incorporation into proteins was ascertained. Steady-state kinetic parameters of the SeMet substituted Met16Leu, Met20Leu, Met42Leu and Met92Leu mutants were nearly identical to those of their Met containing counterparts. These data indicate that Met apparently has a limited role in the protein structure and function of DHFR and that SeMet incorporation has no effect on the steady-state kinetic constants of DHFR.

Phosphorus-31 nuclear magnetic resonance studies of complexes of thymidylate synthase.

The interactions of thymidylate synthase (TS) with deoxyuridylate (dUMP), deoxythymidylate (dTMP) and 5-fluorodeoxyuridylate (FdUMP) were examined by 31P-NMR. Single 31P resonances appeared at 3.3 ppm, 3.2 ppm and 3.0 ppm from the standard, 85% phosphoric acid, for unbound dUMP, dTMP, and FdUMP, respectively. Incubation of the enzyme with either dUMP or dTMP, alone, resulted in new resonances at 3.9 and 3.6 ppm, respectively, which were assigned to noncovalent complexes with the enzyme. The same experiment employing FdUMP as the ligand gave two new resonances appearing at 3.6 and 4.6 ppm, which were attributed to noncovalent and covalent binary complexes, respectively. When the cofactor, CH2H4 folate, was present in the solution with enzyme and FdUMP, a new resonance appeared at 5.1 ppm, corresponding to the covalent inhibitory ternary complex. The ternary complex comprised of the enzyme, dUMP and the quinazoline folate CB 3731 produced a resonance at 5.0 ppm at the expense of the resonance due to the enzyme-dUMP binary complex at 3.9 ppm. Similarly, the ternary complex consisting of TS with dTMP and CB 3731 showed a deshielding of the resonance at 3.6 ppm by 0.8 ppm. A maximum binding of 1.5 nucleotides per enzyme dimer was found for dUMP and dTMP in both the presence and the absence of the quinazoline folate. The deshielding observed was attributed to changes in the interaction of the phosphate group with the nearby residues of the active site of the enzyme.

Differences in natural ligand and fluoropyrimidine binding to human thymidylate synthase identified by transient-state spectroscopic and continuous variation methods.

Thymidylate synthase (TS) is a central target for the design of chemotherapeutic agents due to its vital role in DNA synthesis. Structural studies of binary complexes between Escherichia coli TS and various nucleotides suggest the chemotherapeutic agent FdUMP and the natural ligand dUMP bind similarly. We show, however, that FdUMP binding to human TS yields a substantially greater decrease in fluorescence than does dUMP. Because the difference in quenching due to ligand binding was approximately two-fold and this difference was not seen when using ecTS, the intriguing result indicated a significant difference in the mode of FdUMP binding to the human enzyme. We compared the binding affinities of dUMP, FdUMP, and TMP to TS from both species and found no significant differences for the individual ligands. Because binding affinities were not different among the ligands, the method of continuous variation was employed to determine binding stoichiometry. Similar to that found for dUMP binding to human and ecTS, FdUMP displayed single site occupancy with both enzymes. These results show that nucleotide binding differences exist for FdUMP and dUMP binding to the human enzyme. The observed differences are not due to differences in stoichiometry or ligand affinity. Therefore, although the crystal structure of human TS with various nucleotide ligands has not been solved, these results show that the differences observed using fluorescence methods result from as yet unidentified differential interactions between the human enzyme and nucleotide ligands.

Modification of lysyl residues of dihydrofolate reductase with 2,4-pentanedione.

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) from an amethopterin-resistant strain of Lactobacillus casei was inactivated by 2,4-pentanedione. The inactivation appears to be due to the specific interaction of 2,4-pentanedione with lysyl residues. Inactivation is concomitant with with the modification of three lysyl residues. Both NADPH and dihydrofolate protect the enzyme against inactivation, suggesting that the critical residue(s) lies at or near their binding sites. Unlike native dihydrofolate reductase, 2,4-pentanedione-modified enzyme does not form binary complexes with either NADPH, dihydrofolate or amethopterin which are stable to gel filtration. Treatment of the modified enzyme with nucleophilic reagents such as hydroxylamine, failed to promote reactivation of the enzyme. Reactivation was achieved following gel filtration at pH 6.0 and was found to be dependent on the degree to which the enzyme was inactivated.

Purification and characterization of recombinant mouse thymidylate synthase.

Recombinant mouse thymidylate synthase (TS) expressed at high levels in Escherichia coli was purified to homogeneity in greater than 70% yield by a rapid three-step procedure. Both 0.1% Triton X-100 and 10% glycerol were required to stabilize the enzyme whose activity remained unchanged after 1 month when stored at -20 degrees C. Thermal inactivation of the enzyme was a first-order process at 37 degrees C, with t1/2 values of 6.9, 15.6 and 3.0 min at pH 5.5, 7.0 and 8.5, respectively. The presence of saturating levels of dUMP at pH 8.5 increased the t1/2 of inactivation of 38 min. The pH profile for enzyme activity showed a narrow optimum region centered at pH 7.0, which was mirrored by the shape of the Km, dUMP/Vmax plot. The pH dependence of Kd for the covalent inhibitory ternary complex of enzyme, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate exhibited a broad minimum between pH 5.5 and 8.5, and ranged between 3.1, 0.8 and 1.1 nM at pH 5.5, 7.0 and 8.5, respectively. The UV/VIS spectrum of the native enzyme exhibited a maximum at 280 nm (epsilon = 98,200 M-1 cm-1), while that of the inhibitory ternary complex showed an additional maximum at 320 nm. The 19F-NMR spectrum of the mouse enzyme:FdUMP binary complex revealed two new resonances at -2.8 and -34.8 ppm. The most deshielded resonance represented the noncovalent binary complex while the other resonance was assigned to the nucleotide covalently bound to the enzyme. The alteration of nucleotide binding equilibria produced by addition of H4 folate was exemplified by both an increase in intensity and a 5 ppm deshielding of the resonance attributed to the covalent FdUMP-enzyme complex. Addition of formaldehyde to the latter mixture produced the covalent ternary complex which resulted in the collapse of the resonances at -2.8 and -39.5 ppm and the appearance of a new resonance at -12.4 ppm.

Digital supine bicycle stress echocardiography: a new technique for evaluating coronary artery disease.

OBJECTIVES: The objective of this study was to determine the accuracy of digital supine bicycle stress echocardiography, a new technique for evaluating coronary artery disease during peak exercise. BACKGROUND: Prior stress echocardiographic techniques have not utilized peak exercise imaging to determine the extent and location of coronary artery disease. METHODS: Two-hundred twenty-two patients were studied: 180 underwent both supine bicycle stress echocardiography and coronary arteriography; 42 had a < 5% likelihood of disease. Forty-three patients had normal coronary arteries, 55 had single-vessel, 42 had double-vessel and 40 had triple-vessel coronary artery disease. RESULTS: Supine bicycle stress echocardiography was 93% sensitive, 86% specific and 92% accurate for identifying patients with coronary artery disease irrespective of prior myocardial infarction or achievement of > or = 85% maximal predicted heart rate. The "normalcy" rate in the low probability group was 100%. Supine bicycle stress echocardiography was 87% sensitive, 89% specific and 88% accurate for specific vessel identification. The sensitivity was greatest for the left anterior descending compared with the right coronary artery and the left circumflex coronary artery (95% vs. 81% vs. 78%, p < 0.01) and for vessels in patients with double- and triple-vessel compared with single-vessel disease (90% vs. 89% vs. 78%, p < 0.05). The procedure was significantly more sensitive for detection of vessels with 90% to 100% compared with 50% to 70% diameter stenosis (91% vs. 81%, p < 0.05) and was 88% correct in the prediction of multivessel disease. CONCLUSIONS: Supine bicycle stress echocardiography is a highly accurate tool for evaluating coronary artery disease, identifying both the patient with coronary artery disease and the location and extent of disease.

Crystallization and crystallographic data for new forms of thymidylate synthase from Lactobacillus casei.

Several new crystal forms of thymidylate synthase (5,10-methlenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) were obtained by controlled pH change. In the crystals the dimeric molecule has a 2-fold symmetry axis coinciding with crystallographic symmetry. The crystals scatter to at least 2.7 A resolution in the synchrotron X-ray beam and appear to be suitable for high-resolution X-ray diffraction analysis. The crystals were successfully derivatized and preliminary results are reported for the covalent inhibitory ternary complex of thymidylate synthase, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate.

Propagation and properties of Kaposi's sarcoma-derived cell lines obtained from patients with AIDS: similarity of cultured cells to smooth muscle cells.

Cells derived from Kaposi's sarcoma (KS) were propagated in vitro using conditions which resulted in elimination of contaminating fibroblasts and the emergence of homogeneous cell populations which morphologically resembled smooth muscle cells and had neoplastic characteristics. In long-term culture, they differentiated into large ribbon-like cells with longitudinal fibrillarity of their cytoplasm. These fibrils stained red by Masson trichrome staining, and were reactive with antibodies to desmin. Dense bodies typical of myoblasts were observed in some cells by electron microscopy. The cells did not form capillary structures like endothelial cells, they lacked Weible-Palade bodies, and did not express the blood-clotting Factor VIII-related antigen or receptors for the lectin Ulex europaeus agglutinin I. They did express four other antigens, however, in common with endothelial cells. The cells did not form tumors in athymic nude mice; however, they formed colonies in soft agar, manifested tumor-like growth on muscle organ cultures, and were invasive in an artificial basement membrane invasion assay. The results indicate that a component of KS is closely related to leiomyoblasts and and has neoplastic properties.

Efficient synthesis of mouse thymidylate synthase in Escherichia coli.

The coding region of the mouse thymidylate synthase (TS)-encoding cDNA (ts) was inserted downstream from the phage T7 promoter and translation initiation signals of the expression vector, pET-3a, and transformed into Escherichia coli BL21(DE3)[pLysS]. When the wild-type (wt) cDNA sequence was used, mouse TS was synthesized in the bacterial cells in response to induction, but the level of expression was low. When the second codon (Leu) was changed from CUG, found in the normal mRNA, to CUU, the level of expression increased 17-fold and TS represented 5-10% of total cell protein. The recombinant enzyme was purified to homogeneity by affinity chromatography. The recombinant TS had the same Mr as the enzyme from cultured mouse fibroblasts. Kinetic studies with the recombinant enzyme showed that the apparent Km values for deoxyuridylate and 5,10-methylenetetrahydrofolate were 10.5 and 22 microM, respectively, which were similar to the values for TS from mouse cell extracts. The mouse ts expression vector will be useful for the large-scale production of the wt enzyme and for the creation and analysis of mutant enzymes by protein engineering techniques.

Phosphonates and phosphinates: novel leaving groups for benzisothiazolone inhibitors of human leukocyte elastase.

A novel class of alkyl and aryl phosphonate and phosphinate acid-based leaving groups has been developed for utilization in the synthesis of benzoisothiazolone (BIT) inhibitors of human leukocyte elastase (HLE). A number of BITs were synthesized with phosphonate and phosphinate acid-based leaving groups and were found to be potent inhibitors of HLE. Compound 3c with a diethyl phosphonate leaving group is the most potent inhibitor synthesized in this series with Ki* = 0.035 nM and ED50 = 2.0 mg/kg.

A novel class of cyclic beta-dicarbonyl leaving groups and their use in the design of benzisothiazolone human leukocyte elastase inhibitors.

Human leukocyte elastase (HLE) has been proposed to be a primary mediator of pulmonary emphysema, and inhibitors of this enzyme should be effective in the treatment of emphysema and other pulmonary diseases. We have discovered a novel class of alicyclic and heterocyclic leaving groups which share one common structural feature, a cyclic beta-dicarbonyl. This design concept for leaving groups has not been previously reported. A structure-activity relationship has been developed and the concept extended to several types of alicyclic and heterocyclic beta-dicarbonyl systems. This work led to the identification of a potent (K*i of 0.066 nM) and tissue stable (in vitro: blood t1/2 = 160 min, liver t1/2 > 240 min) benzisothiazolone HLE inhibitor, WIN 65936 (13b).