Correction: Self-limiting stoichiometry in SnSe thin films.

Correction for 'Self-limiting stoichiometry in SnSe thin films' by Jonathan R. Chin et al., Nanoscale, 2023, 15, 9973-9984, https://doi.org/10.1039/D3NR00645J.

Self-limiting stoichiometry in SnSe thin films.

Unique functionalities can arise when 2D materials are scaled down near the monolayer limit. However, in 2D materials with strong van der Waals bonds between layers, such as SnSe, maintaining stoichiometry while limiting vertical growth is difficult. Here, we describe how self-limiting stoichiometry can promote the growth of SnSe thin films deposited by molecular beam epitaxy. The Pnma phase of SnSe was stabilized over a broad range of Sn : Se flux ratios from 1 : 1 to 1 : 5. Changing the flux ratio does not affect the film stoichiometry, but influences the predominant crystallographic orientation. ReaxFF molecular dynamics (MD) simulation demonstrates that, while a mixture of Sn/Se stoichiometries forms initially, SnSe stabilizes as the cluster size evolves. The MD results further show that the excess selenium coalesces into Se clusters that weakly interact with the surface of the SnSe particles, leading to the limited stoichiometric change. Raman spectroscopy corroborates this model showing the initial formation of SnSe2 transitioning into SnSe as experimental film growth progresses. Transmission electron microscopy measurements taken on films deposited with growth rates above 0.25 Å s-1 show a thin layer of SnSe2 that disrupts the crystallographic orientation of the SnSe films. Therefore, using the conditions for self-limiting SnSe growth while avoiding the formation of SnSe2 was found to increase the lateral scale of the SnSe layers. Overall, self-limiting stoichiometry provides a promising avenue for maintaining growth of large lateral-scale SnSe for device fabrication.

A bio-coupling approach using a dextran-binding domain to immobilize an engineered streptavidin to Sephadex for easy preparation of affinity matrix.

An engineered streptavidin, SAVSBPM18 with reversible biotin binding capability, has been successfully applied to purify biotinylated and streptavidin-binding peptide (SBP) tagged proteins. To simplify the preparation for the SAVSBPM18 affinity matrix without chemical conjugation, two bio-coupling approaches were developed based on a 14-kDa dextran-binding domain (DBD) from a Leuconostoc mesenteroides dextransucrase. The first approach offers simplicity for bio-coupling by creating a direct fusion, SAVSBPM18-Linker-DBD. Purification of the fusion from crude extract and its immobilization to Sephadex can be consolidated in one-step. The second approach aims at flexibility. A SnoopCatcher (SC) was fused to DBD to create SC-Linker-DBD. This fusion can covalently capture any recombinant proteins tagged with a SnoopTag (ST) including SAVSBPM18-Linker-ST via the formation of an isopeptide bond at the interface through the SnoopCatcher-SnoopTag interaction. Although monomeric DBD binds to dextran with nanomolar affinity, DBD tetramerized via streptavidin (SAVSBPM18-Linker-ST·SC-Linker-DBD) showed an even tighter binding to Sephadex. The majority of the fluorescently labelled DBD tetramers were retained on the Sephadex surface even after four months. Affinity columns generated using either approach effectively purified both SBP-tagged and biotinylated proteins. These columns are reusable and functional even after a year of frequent use.

Randomised, double-blind, parallel group, placebo-controlled study to evaluate the analgesic efficacy and safety of VVZ-149 injections for postoperative pain following laparoscopic colorectal surgery.

INTRODUCTION: In spite of advances in understanding and technology, postoperative pain remains poorly treated for a significant number of patients. In colorectal surgery, the need for developing novel analgesics is especially important. Patients after bowel surgery are assessed for rapid return of bowel function and opioids worsen ileus, nausea and constipation. We describe a prospective, double-blind, parallel group, placebo-controlled randomised controlled trial testing the hypothesis that a novel analgesic drug, VVZ -149, is safe and effective in improving pain compared with providing opioid analgesia alone among adults undergoing laparoscopic colorectal surgery. METHODS AND ANALYSIS: Based on sample size calculations for primary outcome, we plan to enrol 120 participants. Adult patients without significant medical comorbidities or ongoing opioid use and who are undergoing laparoscopic colorectal surgery will be enrolled. Participants are randomly assigned to receive either VVZ-149 with intravenous (IV) hydromorphone patient-controlled analgesia (PCA) or the control intervention (IV PCA alone) in the postoperative period. The primary outcome is the Sum of Pain Intensity Difference over 8 hours (SPID-8 postdose). Participants receive VVZ-149 for 8 hours postoperatively to the primary study end point, after which they continue to be assessed for up to 24 hours. We measure opioid consumption, record pain intensity and pain relief, and evaluate the number of rescue doses and requests for opioid. To assess safety, we record sedation, nausea and vomiting, respiratory depression, laboratory tests and ECG readings after study drug administration. We evaluate for possible confounders of analgesic response, such as anxiety, depression and catastrophising behaviours. The study will also collect blood sample data and evaluate for pharmacokinetic and pharmacodynamic relationships. ETHICS AND DISSEMINATION: Ethical approval of the study protocol has been obtained from Institutional Review Boards at the participating institutions. Trial results will be disseminated through scientific conference presentations and by publication in scientific journals. TRIAL REGISTRATION NUMBER: NCT02489526; pre-results.

Prospective Validation of the National Field Triage Guidelines for Identifying Seriously Injured Persons.

BACKGROUND: The national field trauma triage guidelines have been widely implemented in US trauma systems, but never prospectively validated. We sought to prospectively validate the guidelines, as applied by out-of-hospital providers, for identifying high-risk trauma patients. STUDY DESIGN: This was an out-of-hospital prospective cohort study from January 1, 2011 through December 31, 2011 with 44 Emergency Medical Services agencies in 7 counties in 2 states. We enrolled injured patients transported to 28 acute care hospitals, including 7 major trauma centers (Level I and II trauma hospitals) and 21 nontrauma hospitals. The primary exposure term was Emergency Medical Services' use of one or more field triage criteria in the national field triage guidelines. Outcomes included Injured Severity Score ≥16 (primary) and critical resource use within 24 hours of emergency department arrival (secondary). RESULTS: We enrolled 53,487 injured children and adults transported by Emergency Medical Services to an acute care hospital, 17,633 of which were sampled for the primary analysis; 13.9% met field triage guidelines, 3.1% had Injury Severity Score ≥16, and 1.7% required early critical resources. The sensitivity and specificity of the field triage guidelines were 66.2% (95% CI, 60.2-71.7%) and 87.8% (95% CI, 87.7-88.0%) for Injury Severity Score ≥16 and 80.1% (95% CI, 65.8-89.4%) and 87.3% (95% CI 87.1-87.4%) for early critical resource use. Triage guideline sensitivity decreased with age, from 87.4% in children to 51.8% in older adults. CONCLUSIONS: The national field triage guidelines are relatively insensitive for identifying seriously injured patients and patients requiring early critical interventions, particularly among older adults.

Strain engineering strategies for improving whole-cell biocatalysis: engineering Escherichia coli to overproduce xylitol as an example.

This chapter provides an overview of key tools and methodologies available to practitioners of biocatalysis interested in using microorganisms to carry out biotransformations and describes specific examples of applying genetic modification strategies for strain design. We focus on the use of the polymerase chain reaction (PCR) for gene amplification, plasmid DNA for recombinant gene cloning and expression, and homologous recombination and phage transduction for modifying chromosomal DNA. Specifically we use Escherichia coli as the host organism, and the overproduction of xylitol by reduction of xylose represents the biotransformation of interest.

Improved NADPH supply for xylitol production by engineered Escherichia coli with glycolytic mutations.

Escherichia coli engineered to uptake xylose while metabolizing glucose was previously shown to produce high levels of xylitol from a mixture of glucose and xylose when expressing NADPH-dependent xylose reductase from Candida boidinii (CbXR) (Cirino et al., Biotechnol Bioeng. 2006;95:1167-1176). We then described the effects of deletions of key metabolic pathways (e.g., Embden-Meyerhof-Parnas and pentose phosphate pathway) and reactions (e.g., transhydrogenase and NADH dehydrogenase) on resting-cell xylitol yield (Y RPG: moles of xylitol produced per mole of glucose consumed) (Chin et al., Biotechnol Bioeng. 2009;102:209-220). These prior results demonstrated the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions. This study describes strain modifications that improve coupling between glucose catabolism (oxidation) and xylose reduction using two fundamentally different strategies. We first examined the effects of deleting the phosphofructokinase (pfk) gene(s) on growth-uncoupled xylitol production and found that deleting both pfkA and sthA (encoding the E. coli-soluble transhydrogenase) improved the xylitol Y RPG from 3.4 ± 0.6 to 5.4 ± 0.4. The second strategy focused on coupling aerobic growth on glucose to xylitol production by deleting pgi (encoding phosphoglucose isomerase) and sthA. Impaired growth due to imbalanced NADPH metabolism (Sauer et al., J Biol Chem. 2004;279:6613-6619) was alleviated upon expressing CbXR, resulting in xylitol production similar to that of the growth-uncoupled precursor strains but with much less acetate secretion and more efficient utilization of glucose. Intracellular nicotinamide cofactor levels were also quantified, and the magnitude of the change in the NADPH/NADP+ ratio measured from cells consuming glucose in the absence vs. presence of xylose showed a strong correlation to the resulting Y RPG.

Computational design of Candida boidinii xylose reductase for altered cofactor specificity.

In this study we introduce a computationally-driven enzyme redesign workflow for altering cofactor specificity from NADPH to NADH. By compiling and comparing data from previous studies involving cofactor switching mutations, we show that their effect cannot be explained as straightforward changes in volume, hydrophobicity, charge, or BLOSUM62 scores of the residues populating the cofactor binding site. Instead, we find that the use of a detailed cofactor binding energy approximation is needed to adequately capture the relative affinity towards different cofactors. The implicit solvation models Generalized Born with molecular volume integration and Generalized Born with simple switching were integrated in the iterative protein redesign and optimization (IPRO) framework to drive the redesign of Candida boidinii xylose reductase (CbXR) to function using the non-native cofactor NADH. We identified 10 variants, out of the 8,000 possible combinations of mutations, that improve the computationally assessed binding affinity for NADH by introducing mutations in the CbXR binding pocket. Experimental testing revealed that seven out of ten possessed significant xylose reductase activity utilizing NADH, with the best experimental design (CbXR-GGD) being 27-fold more active on NADH. The NADPH-dependent activity for eight out of ten predicted designs was either completely abolished or significantly diminished by at least 90%, yielding a greater than 10(4)-fold change in specificity to NADH (CbXR-REG). The remaining two variants (CbXR-RTT and CBXR-EQR) had dual cofactor specificity for both nicotinamide cofactors.

Transcriptional effects of CRP* expression in Escherichia coli.

BACKGROUND: Escherichia coli exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native crp gene with a catabolite repression mutant (referred to as crp*) enables co-utilization of glucose and other sugars in E. coli. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of E. coli W3110 (expressing wild-type CRP) to that of mutant strain PC05 (expressing CRP*) in the presence and absence of glucose. RESULTS: The glucose effect is significantly suppressed in strain PC05 relative to strain W3110. The expression levels of glucose-sensitive genes are generally not altered by glucose to the same extent in strain PCO5 as compared to W3110. Only 23 of the 80 genes showing significant differential expression in the presence of glucose for strain PC05 are present among the 418 genes believed to be directly regulated by CRP. Genes involved in central carbon metabolism (including several TCA cycle genes) and amino acid biosynthesis, as well as genes encoding nutrient transport systems are among those whose transcript levels are most significantly affected by CRP* expression.We present a detailed transcription analysis and relate these results to phenotypic differences between strains expressing wild-type CRP and CRP*. Notably, CRP* expression in the presence of glucose results in an elevated intracellular NADPH concentration and reduced NADH concentration relative to wild-type CRP. Meanwhile, a more drastic decrease in the NADPH/NADP+ ratio is observed for the case of CRP* expression in strains engineered to reduce xylose to xylitol via a heterologously expressed, NADPH-dependent xylose reductase. Altered expression levels of transhydrogenase and TCA cycle genes, among others, are consistent with these observations. CONCLUSION: While the simplest model of CRP*-mediated gene expression assumes insensitivity to glucose (or cAMP), our results show that gene expression in the context of CRP* is very different from that of wild-type in the absence of glucose, and is influenced by the presence of glucose. Most of the transcription changes in response to CRP* expression are difficult to interpret in terms of possible systematic effects on metabolism. Elevated NADPH availability resulting from CRP* expression suggests potential biocatalytic applications of crp* strains that extend beyond relief of catabolite repression.

Analysis of NADPH supply during xylitol production by engineered Escherichia coli.

Escherichia coli strain PC09 (DeltaxylB, cAMP-independent CRP (crp*) mutant) expressing an NADPH-dependent xylose reductase from Candida boidinii (CbXR) was previously reported to produce xylitol from xylose while metabolizing glucose [Cirino et al. (2006) Biotechnol Bioeng 95(6): 1167-1176]. This study aims to understand the role of NADPH supply in xylitol yield and the contribution of key central carbon metabolism enzymes toward xylitol production. Studies in which the expression of CbXR or a xylose transporter was increased suggest that enzyme activity and xylose transport are not limiting xylitol production in PC09. A constraints-based stoichiometric metabolic network model was used to understand the roles of central carbon metabolism reactions and xylose transport energetics on the theoretical maximum molar xylitol yield (xylitol produced per glucose consumed), and xylitol yields (Y(RPG)) were measured from resting cell biotransformations with various PC09 derivative strains. For the case of xylose-proton symport, omitting the Zwf (glucose-6-phosphate dehydrogenase) or PntAB (membrane-bound transhydrogenase) reactions or TCA cycle activity from the model reduces the theoretical maximum yield from 9.2 to 8.8, 3.6, and 8.0 mol xylitol (mol glucose)(-1), respectively. Experimentally, deleting pgi (encoding phosphoglucose isomerase) from strain PC09 improves the yield from 3.4 to 4.0 mol xylitol (mol glucose)(-1), while deleting either or both E. coli transhydrogenases (sthA and pntA) has no significant effect on the measured yield. Deleting either zwf or sucC (TCA cycle) significantly reduces the yield from 3.4 to 2.0 and 2.3 mol xylitol (mol glucose)(-1), respectively. Expression of a xylose reductase with relaxed cofactor specificity increases the yield to 4.0. The large discrepancy between theoretical maximum and experimentally determined yield values suggests that biocatalysis is compromised by pathways competing for reducing equivalents and dissipating energy. The metabolic role of transhydrogenases during E. coli biocatalysis has remained largely unspecified. Our results demonstrate the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions, and suggest that the pool of reduced cofactors available for biotransformation is not readily interchangeable via transhydrogenase.

Comparison between Escherichia coli K-12 strains W3110 and MG1655 and wild-type E. coli B as platforms for xylitol production.

Escherichia coli W3110 was previously engineered to produce xylitol from a mixture of glucose plus xylose by expressing xylose reductase (CbXR) and deleting xylulokinase (DeltaxylB), combined with either plasmid-based expression of a xylose transporter (XylE or XylFGH) (Khankal et al., J Biotechnol, 2008) or replacing the native crp gene with a mutant (crp*) that alleviates glucose repression of xylose transport (Cirino et al., Biotechnol Bioeng 95:1167-1176, 2006). In this study, E. coli K-12 strains W3110 and MG1655 and wild-type E. coli B were compared as platforms for xylitol production from glucose-xylose mixtures using these same strategies. The engineered strains were compared in fed-batch fermentations and as non-growing resting cells. Expression of CRP* in the E. coli B strains tested was unable to enhance xylose uptake in the presence of glucose. Xylitol production was similar for the (crp*, DeltaxylB)-derivatives of W3110 and MG1655 expressing CbXR (average specific productivities of 0.43 g xylitol g cdw(-1 ) h(-1) in fed-batch fermentation). In contrast, results varied substantially between different DeltaxylB-derivative strains co-expressing either XylE or XylFGH. The differences in genetic background between these host strains can therefore profoundly influence metabolic engineering strategies.

Role of xylose transporters in xylitol production from engineered Escherichia coli.

Escherichia coli W3110 was previously engineered to co-utilize glucose and xylose by replacing the wild-type crp gene with a crp* mutant encoding a cAMP-independent CRP variant (Cirino et al., 2006 [Cirino, P.C., Chin, J.W., Ingram, L.O., 2006. Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. Biotechnol. Bioeng. 95, 1167-1176.]). Subsequent deletion of the xylB gene (encoding xylulokinase) and expression of xylose reductase from Candida boidinii (CbXR) resulted in a strain which produces xylitol from glucose-xylose mixtures. In this study we examine the contributions of the native E. coli xylose transporters (the d-xylose/proton symporter XylE and the d-xylose ABC transporter XylFGH) and CRP* to xylitol production in the presence of glucose and xylose. The final batch xylitol titer with strain PC09 (Delta xylB and crp*) is reduced by 40% upon deletion of xylG and by 60% upon deletion of both xyl transporters. Xylitol production by the wild-type strain (W3110) expressing CbXR is not reduced when xylE and xylG are deleted, demonstrating tight regulation of the xylose transporters by CRP and revealing significant secondary xylose transport. Finally, plasmid expression of XylE or XylFGH with CbXR in PC07 (Delta xylB and wild-type crp) growing on glucose results in xylitol titers similar to that achieved with PC09 and provides an alternative strategy to the use of CRP*.

Engineering Escherichia coli for xylitol production from glucose-xylose mixtures.

The range of value-added chemicals produced by Escherichia coli from simple sugars has been expanded to include xylitol. This was accomplished by screening the in vivo activity of a number of heterologous xylitol-producing enzymes. Xylose reductases from Candida boidinii (CbXR), Candida tenuis (CtXR), Pichia stipitis (PsXR), and Saccharmoyces cerivisiae (ScXR), and xylitol dehydrogenases from Gluconobacter oxydans (GoXDH) and Pichia stipitis (PsXDH) were all functional in E. coli to varying extents. Replacement of E. coli's native cyclic AMP receptor protein (CRP) with a cyclic AMP-independent mutant (CRP*) facilitated xylose uptake and xylitol production from mixtures of glucose and xylose, with glucose serving as the growth substrate and source of reducing equivalents. Of the enzymes tested, overexpression of NADPH-dependent CbXR produced the highest concentrations of xylitol in shake-flask cultures (approximately 275 mM in LB cultures, approximately 180 mM using minimal medium). Expression of CbXR in strain PC09 (crp*, DeltaxylB) in a 10-L controlled fermentation containing minimal medium resulted in production of approximately 250 mM xylitol (38 g/L), with concomitant utilization of approximately 150 mM glucose. The ratio of moles xylitol produced (from xylose) per mole glucose consumed was improved to > 3.7:1 using metabolically active "resting" cells.

WEDS: a Web services-based environment for distributed simulation.

Web services have the potential to radically enhance the ability of researchers to make use of distributed computing resources, but jargon and a plethora of standards make their use almost impossible for the scientist without prior experience of the necessary technologies. A powerful and simple WSRF-based middleware scheme is presented, designed to let scientists remotely deploy single or multiple instances of a pre-existing code across multiple resources, and giving steering, visualization and workflow functionality with only simple modifications to program code. It is hoped that the development and implementation of such a toolkit will be relevant not only to the problem of deploying workstation-class codes in real time, but also the move towards more tractable alternatives to the Globus toolkit for deployment of processes in a high-performance computing environment.

Large-scale lattice Boltzmann simulations of complex fluids: advances through the advent of computational Grids.

During the last 2.5 years, the RealityGrid project has allowed us to be one of the few scientific groups involved in the development of computational Grids. Since smoothly working production Grids are not yet available, we have been able to substantially influence the direction of software and Grid deployment within the project. In this paper, we review our results from large-scale three-dimensional lattice Boltzmann simulations performed over the last 2.5 years. We describe how the proactive use of computational steering, and advanced job migration and visualization techniques enabled us to do our scientific work more efficiently. The projects reported on in this paper are studies of complex fluid flows under shear or in porous media, as well as large-scale parameter searches, and studies of the self-organization of liquid cubic mesophases.

Structural and dynamical characterization of Hele-Shaw viscous fingering.

Viscous fingering occurs in the interfacial zone between two fluids confined between two plates with a narrow gap (Hele-Shaw geometry) when a highly viscous fluid is displaced by a fluid with relatively low viscosity. Using a mesoscopic approach--the lattice Boltzmann method--we investigate the dynamics of spatially extended Hele-Shaw flow under conditions corresponding to various experimental systems by tuning the 'surface tension' and the reactivity between the two fluids. We discuss the onset of the fingering instability (dispersion relation), analyse the structural properties (characterization of the interface) and the dynamical properties (growth of the mixing zone) of the Hele-Shaw systems, and show the effect of reactive processes on the structure of the interfacial zone.

Rapid cleavage of cyclic tertiary amides of Kemp's triacid: effects of ring structure.

The piperidyl and prolyl amides of Kemp's triacid (7 and 8, respectively) have been prepared and their rates of intramolecular acylolysis measured as a function of pD. The piperidyl derivative 7 reacts approximately four-times faster (e.g., t(1/2)=3 min at 20 degrees C and pD7.7) than the previously reported pyrrolidyl and methylphenethyl amide derivatives, while the prolyl derivative 8 reacts two-times more slowly (e.g., e.g., t(1/2)=30 min at 20 degrees C and pD7.8). Molecular-mechanics calculations indicate that the nonbonded interactions in the piperidyl derivative 7 are distinct from those in the prolyl, pyrrolidyl, and methylphenethyl amide derivatives, a result that supports the suggestion that ground-state pseudoallylic strain contributes to the enormous reactivity of Kemp's triacid tertiary amides. In sum, the results reported indicate that the Kemp's triacid scaffolding provides a general means of activating tertiary amide derivatives.

Genetic alterations in prostate cancer.

Prostate cancer is the number one malignancy among men. The search for causative factors has proven to be difficult and, accordingly, treatment options for advanced prostate cancer remain limited. However, technologic breakthroughs in the fields of genetics and molecular biology have advanced our understanding of the mechanisms involved in prostate carcinogenesis. The aim of this article is to review the most recent evidence for the role of various genetic insults at specific steps in tumor formation and to suggest potential therapeutic targets.

Molecular markers and prostate cancer prognosis.

Prostate cancer is the most common malignancy among American men and is the second-leading cause of cancer-related mortality. Although radical prostatectomy and radiation therapy offer hope for cure for the majority of men with localized tumors, we continue to lack the tools to definitively determine which cancers need to be treated, which cancers will recur after treatment, and which cancers will behave aggressively when they have metastasized. Recent breakthroughs in molecular biology have led to the identification of a number of potential biomarkers for prostate cancer, many of which have been suggested to have prognostic significance. Eventually, combinations of these markers will hopefully enable us to more rationally facilitate counseling and direct management for men with prostate cancer.

Lattice Boltzmann study of spinodal decomposition in two dimensions.

A lattice Boltzmann model using the Shan-Chen prescription for a binary immiscible fluid is described, and the macroscopic equations obeyed by the model are derived. The model is used to quantitatively examine spinodal decomposition of a two-dimensional binary fluid. This model allows examination of the early-time period corresponding to interface formation, and shows agreement with analytical solutions of the linearized Cahn-Hilliard equation, despite the fact that the model contains no explicit free-energy functional. This regime has not, to the knowledge of the authors, been previously observed using any lattice Boltzmann method. In agreement with other models, a scaling law with the exponent 2/3 is observed for late-time domain growth. Breakdown of scaling is also observed for certain sets of simulation parameters.