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BackgroundIn the current phase of COVID-19 pandemic, we are facing two serious public health challenges that include deficits in SARS-CoV-2 variant monitoring, and neglect of other co-circulating respiratory viruses. Additionally, accurate assessment of the evolution, extent and dynamics of the outbreak are required to understand the transmission of the virus amongst seemingly unrelated cases and provide critical epidemiological information. To address these challenges, we evaluated a new high-throughput next-generation sequencing (NGS), respiratory viral panel (RVP) that includes 40 viral pathogens with the aim of analyzing viral subtypes, mutational variants of SARS-CoV-2, model to understand the spread of the virus in the state of Georgia, USA, and to assess other circulating viruses in the same population.MethodsThis study evaluated a total of 522 samples that included 483 patient samples and 42 synthetic positive control material. The performance metrics were calculated for both clinical and reference control samples by comparing detection results with the RT-PCR assay. The limit of detection (LoD) studies were conducted as per the FDA guidelines. Inference and visualization of the phylogeny of the SARS-CoV-2 sequences were performed through the Nextstrain Command-Line Interface (CLI) tool, utilizing the associated augur and auspice toolkits.ResultsThe performance metrics calculated using both the clinical samples and the reference controls revealed a PPA, NPA and accuracy of 95.98%, 85.96% and 94.4%, respectively. The LoD was determined to be 10 copies/ml with all 25 replicates detected across two different runs. The clade for pangolin lineage B that contains certain distant variants, including P4715L in ORF1ab, Q57H in ORF 3a and, S84L in ORF8 covarying with the D614G spike protein mutation were the most prevalent, early in the pandemic, in Georgia, USA. In our analysis, isolates from the same county formed paraphyletic groups, which indicated virus transmission between counties.ConclusionThe study demonstrates the clinical and public health utility of the NGS-RVP to identify novel variants that can provide actionable information to prevent or mitigate emerging viral threats, models that provide insights into viral transmission patterns and predict transmission/ resurgence of regional outbreaks and provide critical information on co-circulating respiratory viruses that might be independent factors contributing to the global disease burden.

Objectives Fetal mutations and fetal chromosomal abnormalities can be detected by molecular analysis of circulating cell free fetal DNA (ccffDNA) from maternal plasma. This comprehensive study was aimed to investigate and verify blood collection and blood shipping conditions that enable Noninvasive Prenatal Testing. Specifically, the impact of shipping and storage on the stability and concentration of circulating cell-free DNA (ccfDNA) in Streck® Cell-Free DNA™ Blood Collection Tubes (Streck BCTs, Streck, Omaha NE). These BCTs were designed to minimize cellular degradation, and thus effectively prevent dilution of fetal ccf DNA by maternal genomic DNA, was evaluated. Design and methods Peripheral venous maternal blood was collected into Streck BCTs to investigate four aspects of handling and processing conditions: (1) time from blood draw to plasma processing; (2) storage temperature; (3) mechanical stress; and (4) lot-to-lot tube variations. Results Maternal blood stored in Streck BCTs for up to 7 days at ambient temperature provides stable concentrations of ccffDNA. The amount of fetal DNA did not change over a broad range of storage temperatures (4 °C, 23 °C, 37 °C, 40 °C), but the amount of total (largely maternal) DNA increased in samples stored at 23 °C and above, indicating maternal cell degradation and genomic DNA release at elevated temperatures. Shipping maternal blood in Streck BCTs, did not affect sample quality. Conclusions Maternal plasma DNA stabilized for 0 to 7 days in Streck BCTs can be used for non-invasive prenatal molecular applications, when temperatures are maintained within the broad parameters assessed in this study.

Understanding the structural basis for protein thermostability is of considerable biological and biotechnological importance as exemplified by the industrial use of xylanases at elevated temperatures in the paper pulp and animal feed sectors. Here we have used directed protein evolution to generate hyperthermostable variants of a thermophilic GH11 xylanase, EvXyn11. The Gene Site Saturation Mutagenesis (GSSM) methodology employed assesses the influence on thermostability of all possible amino acid substitutions at each position in the primary structure of the target protein. The 15 most thermostable mutants, which generally clustered in the N-terminal region of the enzyme, had melting temperatures (Tm) 1-8 degrees C higher than the parent protein. Screening of a combinatorial library of the single mutants identified a hyperthermostable variant, EvXyn11TS, containing seven mutations. EvXyn11TS had a Tm approximately 25 degrees C higher than the parent enzyme while displaying catalytic properties that were similar to EvXyn11. The crystal structures of EvXyn11 and EvXyn11TS revealed an absence of substantial changes to identifiable intramolecular interactions. The only explicable mutations are T13F, which increases hydrophobic interactions, and S9P that apparently locks the conformation of a surface loop. This report shows that the molecular basis for the increased thermostability is extraordinarily subtle and points to the requirement for new tools to interrogate protein folding at non-ambient temperatures.

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, 600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10 6 to 10 10 members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.

Point-of-care, home international normalized ratio (INR) monitoring (patient self-testing, or PST) provides an opportunity to optimize warfarin therapy as demonstrated in randomized trials. This study sought to determine the quality of warfarin therapy as determined by time in therapeutic INR range (TTR) in patients who perform home monitoring outside of a clinical trial setting.Retrospective analysis.The data base of an independent diagnostic testing facility was retrospectively queried over a 2.5-year period (January 2008-June 2011) and patient TTR was analyzed based on frequency of testing, age, gender, indication for therapy, duration of therapy, and critical value occurrence.A total of 29,457 patients with multiple indications for warfarin therapy comprised the database. The mean TTR for the entire group was 69.7%, with weekly testers achieving a TTR of 74% versus 68.9% for variable testers (testing every 2-4 weeks)(P.0001). In all categories analyzed (age, indication for anticoagulation, and referral site volume), weekly testers performed significantly better than variable testers. Older individuals had a higher TTR than younger patients. Weekly testers experienced significantly fewer critical values (INR1.5 or5.0) than did variable testers.Point-of-care patient self-testing at home achieves high-quality warfarin therapy outside of clinical trials and compares favorably with the results achieved in randomized trials or in anticoagulation clinic settings.

The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface environments. As a consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and specificity, we have adopted this approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease, subtilisin Bacillus lentus (SBL). A series of mono-, di- and triacidic acid methanethiosulfonates were synthesized and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site. Kinetic parameters for these chemically modified mutant (CMM) enzymes were determined at pH 8.6 under conditions which ensured complete ionization of the unnatural amino acid side-chains introduced. The presence of up to three negative charges in the S1, S1' and S2 subsites of SBL resulted in up to 11-fold lowered activity, possibly due to interference with oxyanion stabilization of the transition state of the hydrolytic reactions catalyzed. Each unit increase in negative charge resulted in a raising of K(M) and a reduction of k(cat). However, no upper limit was observed for increases in K(M), whereas decreases in k(cat) reached a limiting value. Comparison with sterically similar but uncharged CMMs revealed that electrostatic effects of negative charges at positions 62, 156 and 217 are detrimental, but are beneficial at position 166. These results indicate that the ground-state binding of SBL to the standard substrate, Suc-AAPF-pNA, to SBL is reduced, but without drastic attenuation of catalytic efficiency, and show that SBL tolerates high levels of charge at single sites.

Employing the strategy of combined site directed mutagenesis and chemical modification, we previously generated chemically modified mutant enzymes (CMMs) of subtilisin Bacillus lentus (SBL). We now report the use of these SBL-CMMs for peptide coupling reactions. The SBL-CMMs exhibit dramatically altered substrate specificity, including the acceptance of d -amino acid acyl donors, generating dipeptides containing d -Phe, d -Ala and d -Glu in up to 66% yield, which was not possible using wild-type SBL (WT-SBL). In addition, SBL-CMMs accommodate α-branched amino acids such as l -Ala-NH 2 as acyl acceptors in their S 1 ′ pockets, which WT-SBL will not.

A series of chemically modified mutants (CMMs) of subtilisin B. lentus (SBL) were generated employing the combination of site-directed mutagenesis and chemical modification. This strategy entails the mutation of a selected active site residue to cysteine and its subsequent modification with a methanethiosulfonate reagent CH3SO2S-R, where R may be infinitely variable. The present study was undertaken to evaluate the changes in specificity and pH-activity profiles that could be induced by modification of S156C and S166C in the S1 pocket of SBL with a representative range of side chain modifications, namely R −CH3, −CH2C6H5, −CH2CH2NH3+ and CH2CH2SO3−. The side chain of S156C is surface exposed and well solvated while that of S166C points into the pocket. Kinetic evaluation of the CMMs with suc-AAPF-pNA as substrate showed that the kcat/KMs changed very little for the S156C CMMs, but varied by up to 11-fold for the S166C CMMs. pH-Activity profiles were also determined, and showed that a negatively or positively charged side chain modification increased or decreased respectively, the pKa of the catalytic triad histidine for both modification sites but with more dramatic changes for the interior pointing S166C than for the solvent exposed S156C site. As an additional probe of altered specificity, inhibition of the CMMs by a representative series of 5 boronic acid transition state analogue inhibitors was determined. The KIs observed ranged from a 3.5-fold improvement over the WT value, to a 12-fold decrease in binding. Overall, greater variability in all the parameters measured, activity, pKa, and boronic acid binding resulted from modification at the inward pointing 166 site than at the solvent-exposed 156 site.

For synthetic applications of proteases, such as for peptide coupling, a combination of high esterase and low amidase activities is required. While achieving such specificity has been a long-standing goal, the decreases in amidase activity achieved to date have often also been accompanied by decreases in esterase activity. In the current study, a strategy of combined site-directed mutagenesis and chemical modification was applied to the serine protease subtilisin Bacillus lentus (SBL) to improve its esterase-over-amidase specificity. Using the crystal structure of SBL as a guide, the N62, L217, S166, and M222 active site residues were chosen for mutagenesis to cysteine and subsequent modification by alkyl methanethiosulfonate reagents. An initial rapid, combinatorial screen of the chemically modified mutant enzymes (CMMs) generated and, of their parent cysteine mutants, identified 25 promising candidates which were then subjected to detailed kinetic evaluations. Of these CMM and mutant enzymes, 20 exhibit...

Developing the ability to vary the pH−activity profile of an enzyme in a controlled manner has been a long sought-after goal. Such tailoring provides important insights into mechanism and permits optimization of enzyme performance in organic synthesis applications. The most successful approaches to date toward altering pH−activity profiles of enzymes have employed either site-directed mutagenesis or chemical modification to alter enzyme surface charge. We now report that, by combining these two methodologies, dramatic pKa changes can be induced in the serine protease subtilisin B. lentus. In particular, site specific incorporation of unnatural amino acid side chains by the following strategy, WT → Asn62Cysmutant + H3C−SO2−S−R → Asn62Cys-S−R, where R may be infinitely variable, has demonstrated that pKa shifts of up to 0.72 unit are achievable and are accompanied by significant activity enhancements. The most dramatic pKa shifts are caused by chemical modification with hydrophobic aliphatic moieties. A lin...

Specificity differences between the S1-pockets of subtilisin B. lentus (SBL), and its M222C/S mutants have been explored with boronic acid inhibitors. Similar binding trends were noted, with 2,4-dichlorophenylboronic acid being the best overall inhibitor for each enzyme. In addition, a correlation between inhibitor binding and the electrophilicity of boron was found for both the M222C and M222S enzymes. Specificity differences between the S 1-pockets of subtilisin from B. lentus (SBL), and its M222C/S mutants, have been explored with boronic acid inhibitors. Similar binding trends were noted, with 2,4-dichlorophenyl boronic acid being the best overall inhibitor for each enzyme. In addition, a correlation between inhibitor binding and the electrophilicity of boron was found for both M222C and M222S enzymes.

Chromosomal rearrangements are common markers of cancer progression across a wide range of cancer types, and therefore, identification of fusion transcripts in cancer biopsies may have potential to provide tumor-specific insight toward diagnosis, prognosis and precision treatment. Currently, routine methods for fusion detection using fluorescent in-situ hybridization (FISH) provide a low-resolution view of the aberrant fusion transcript. We describe an RNA-Seq approach designed to survey cancer fusions in a single assay by selectively enriching the cancer transcriptome using probes that target the coding regions of over 1385 cancer-associated genes. We tested the performance of the 1385 gene, RNA-Seq Pan-Cancer panel on RNA extracted from 47 patient-derived samples from brain, sarcoma and leukemia, including blood, bone marrow, and formalin-fixed paraffin-embedded (FFPE) samples. Each sample harbored at least one orthogonally verified gene fusion transcript, previously confirmed by FISH or Reverse Transcriptase PCR (RT-PCR). RNA-Seq libraries were prepared from 10-100 ng of total RNA from blood or bone marrow and 20-200 ng total RNA from FFPE tissue and subsequently enriched by hybridization to the Pan-Cancer panel. All samples yielded sufficient library and were sequenced with 76 base-pair paired-end reads on an Illumina MiSeq at 8 samples per flow cell (∼3 million reads per sample). Sequencing data was analyzed using RNA-Seq with STAR aligner and Manta fusion caller. Using this capture-based single-assay approach, we successfully detected fusions commonly associated with leukemia (BCR-ABL1, MLL-MLLT3, MLL-AFF1, RUNX1-ETV6, EBF1-PDGFRB, TCF3-PBX1, IKZF1-PAX5), sarcoma (EWSR1-ATF1, EWSR1-FLI1, JAZF1-SUZ12, SS18-SSX, FUS-DDIT3, FUS-KLF17, YWHAE-FAM22B) and brain cancer (KIAA1459-BRAF) consistent with previously confirmed RT-PCR or FISH results. Several examples of previously unknown fusion partners or additional structural information that were not identified from the FISH or RT-PCR testing were also uncovered in this study. These cases are described in detail. In summary, we show that selective enrichment of RNA-Seq libraries with cancer-specific probes enables detection of known and novel fusions across a broad range of cancer pathologies in a single reaction, creating new opportunities for discovery and translational cancer studies. Citation Format: Lisa C. Watson, Stephen M. Gross, Felix Schlesinger, Anthony Mai, Mariko Kellogg, Steve Lee, Claire Attwooll, Monica Brenca, David Swanson, Andrew Wong, Angelo P. Dei Tos, Claudia Haferlach, Torsten Haferlach, Wolfgang Kern, Roberta Maestro, Manja Meggendorfer, Niroshan Nadarajah, Maurizio Polano, Sabrina Rossi, Marta Sbaraglia, George S. Charames, Gary P. Schroth, Grace DeSantis. Enhancing the resolution and accelerating the pace of translational fusion characterization in oncology by RNA sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-329.

This paper describes a structure-based approach to elucidate the stereospecificity, including inversion of enantioselectivity, of the 2-deoxyribose-5-phosphate aldolase-catalyzed asymmetric aldol addition reaction using unnatural substrates designed for the total synthesis of epothilones. In addition, an aldolase variant with Ser-238 being altered for Asp was found to be 2.5 times more effective than the wild type in accepting the unphosphorylated substrate D-glyceraldehyde. A new H-bonding interaction between the Asp-238 carboxylate and the 3-hydroxyl of the substrate was identified and was used to rationalize the rate enhancements.Key words: aldol reaction, 2-deoxyribose-5-phosphate aldolase, mutagenesis, inversion of enantioselectivity.

In classical enzymology, intermediates and transition states in a catalytic mechanism are usually inferred from a series of biochemical experiments. Here, we derive an enzyme mechanism from true atomic-resolution x-ray structures of reaction intermediates. Two ultra–high resolution structures of wild-type and mutant d -2-deoxyribose-5-phosphate (DRP) aldolase complexes with DRP at 1.05 and 1.10 angstroms unambiguously identify the postulated covalent carbinolamine and Schiff base intermediates in the aldolase mechanism. In combination with site-directed mutagenesis and 1 H nuclear magnetic resonance, we can now propose how the heretofore elusive C-2 proton abstraction step and the overall stereochemical course are accomplished. A proton relay system appears to activate a conserved active-site water that functions as the critical mediator for proton transfer.

A transition state analogue inhibitor, boronic acid benzophenone (BBP) photoprobe, was used to study the differences in the topology of the S1 pocket of chemically modified mutant enzymes (CMMs). The BBP proved to be an effective competitive inhibitor and a revealing active site directed photoprobe of the CMMs of the serine protease subtilisin Bacillus lentus (SBL) which were chemically modified with the hydrophobic, negatively charged and positively charged moieties at the S1 pocket S166C residue. As expected, in all cases BBP bound best to WT-SBL. BBP binding to S166C-SCH2C6H5 and S166C-CH2-c-C6H11, with their large hydrophobic side chains, was reduced by 86-fold and 9-fold, respectively, compared to WT. Relative to WT, BBP binding to the charged CMMs, S166C-S-CH2CH2SO3− or S166C-S-CH2CH2NH3+, was reduced 170-fold and 4-fold respectively. Photolysis of the WT-SBL–BBP enzyme–inhibitor (EI) complex, inactivated the enzyme and effected the formation of a covalent crosslink between WT and BBP. The crosslink was identified at Gly127 by peptide mapping analysis and Edman sequencing. Gly127 is located in the S1 hydrophobic pocket of SBL and its modification thus established binding of the benzophenone moiety in S1. Photolysis of the EI complex of S166C-SCH2C6H5, S166C-S-CH2CH2SO3−, or S166C-S-CH2CH2NH3+ and BBP under the same conditions did not inactivate these enzymes, nor effect the formation of a crosslink. These results corroborated the kinetic evidence that the active site topology of these CMMs is dramatically altered from that of WT. In contrast, while photolysis of the S166C-CH2-c-C6H11–BBP EI complex only inactivated 50% of the enzyme after 12 h, it still effected the formation of a covalent crosslink between the CMM and BBP, again at Gly127. However, this photolytic reaction was less efficient than with WT, demonstrating that the S1 pocket of S166C-CH2-c-C6H11 is significantly restricted compared to WT, but not as completely as for the other CMMs.

In both protein chemistry studies and organic synthesis applications, it is desirable to have available a toolbox of inexpensive proteases with high selectivity and diverse substrate preferences. Toward this goal, we have generated a series of chemically modified mutant enzymes (CMMs) of subtilisin B. lentus (SBL) possessing expanded S(1) pocket specificity. Wild-type SBL shows a marked preference for substrates with large hydrophobic P(1) residues, such as the large Phe P(1) residue of the standard suc-AAPF-pNA substrate. To confer more universal P(1) specificity on S(1), a strategy of chemical modification in combination with site-directed mutagenesis was applied. For example, WT-SBL does not readily accept small uncharged P(1) residues such as the -CH(3) side chain of alanine. Accordingly, with a view to creating a S(1) pocket that would be of reduced volume providing a better fit for small P(1) side chains, a large cyclohexyl group was introduced by the CMM approach at position S166C with the aim of partially filling up the S(1) pocket. The S166C-S-CH(2)-c-C(6)H(11) CMM thus created showed a 2-fold improvement in k(cat)/K(M) with the suc-AAPA-pNA substrate and a 51-fold improvement in suc-AAPA-pNA/suc-AAPF-pNA selectivity relative to WT-SBL. Furthermore, WT-SBL does not readily accept positively or negatively charged P(1) residues. Therefore, to improve SBL's specificity toward positively and negatively charged P(1) residues, we applied the CMM methodology to introduce complementary negatively and positively charged groups, respectively, at position S166C in S(1). A series of mono-, di-, and trinegatively charged CMMs were generated and all showed improved k(cat)/K(M)s with the positively charged P(1) residue containing substrate, suc-AAPR-pNA. Furthermore, virtually arithmetic improvements in k(cat)/K(M) were exhibited with increasing number of negative charges on the S166C-R side chain. These increases culminated in a 9-fold improvement in k(cat)/K(M) for the suc-AAPR-pNA substrate and a 61-fold improvement in suc-AAPR-pNA/suc-AAPF-pNA selectivity compared to WT-SBL for the trinegatively charged S166C-S-CH(2)CH(2)C(COO(-))(3) CMM. Conversely, the positively charged S166C-S-CH(2)CH(2)NH(3)(+) CMM generated showed a 19-fold improvement in k(cat)/K(M) for the suc-AAPE-pNA substrate and a 54-fold improvement in suc-AAPE-pNA/suc-AAPF-pNA selectivity relative to WT-SBL.

By combining site-directed mutagenesis with chemical modification, we have altered the S1 and S1' pocket specificity of subtilisin Bacillus lentus (SBL) through the incorporation of unnatural amino acid moieties, in the following manner: WT --Cysmutant + H3CSO2SR --Cys-SR, where R may be infinitely variable. A paradigm between extent of activity changes and surface exposure of the modified residue has emerged. Modification of M222C, a buried residue in the S1' pocket of SBL, caused dramatic changes in kcat/KM, of an up to 122-fold decrease, while modification of S166C, which is located at the bottom of the S1 pocket and is partially surface exposed, effected more modest activity changes. Introduction of a positive charge at S166C does not alter kcat/KM, whereas the introduction of a negative charge results in lowered activity, possibly due to electrostatic interference with oxyanion stabilization. Activity is virtually unaltered upon modification of S156C, which is located toward the bottom of the S1 pocket and surface exposed and whose side chain is solvated. An unexpected structure-activity relationship was revealed for S166C-SR enzymes in that the pattern of activity changes observed with increasing steric size of R was not monotonic. Molecular modeling analysis was used to analyze this unprecedented structure-activity relationship and revealed that the position of the beta-carbon of Cys166 modulates binding of the P1 residue of the AAPF product inhibitor.

Gene site saturation mutagenesis (GSSM) technology is applied for the directed evolution of a nitrilase. The nitrilase effectively catalyzes the desymmetrization of the prochiral substrate 3-hydroxyglutaronitrile to afford (R)-4-cyano-3-hydroxybutyric acid, a precursor to the valuable cholesterol-lowering drug Lipitor. The discovered wild-type enzyme effectively performs the reaction at the industrially relevant 3 M substrate concentration but affords a product enantiomeric excess of only 87.6% ee. Through GSSM, a mutagenesis technique that effects the combinatorial saturation of each amino acid in the protein to each of the other 19 amino acids, combined with a novel high-throughput mass spectroscopy assay, a number of improved variants were identified, the best of which is the Ala190His mutant that yields product enantiomeric excess of 98.5% at 3 M substrate loading and a volumetric productivity of 619 g L-1 d-1.

Chemical Modification of Cysteine Mutants of Subtilisin Bacillus lentus Can Create Better Catalysts Than the Wild-Type Enzyme

By focusing on East European Jewish and Lithuanian ethnic groups in the United States, this paper offers a three stage model for understanding the process by which assimilating groups create their ethnicity: Stage One, ethnic community and participatory organizations; Stage Two, individual perpetuation and representational organizations; Stage Three, informal behavior and residual symbols. The model considers the impact of both cultural and structural variables, instead of treating them as rival claimants to primacy. In applying the model to data gathered in the groups' Chicago communities, we found that despite particular divergencies, both groups presented a similar response to the United States, enabling their members to maintain a dual ethnic identity beyond the tightly-knit ghetto and urban village.