Comparing Methods to Genetically Engineer Bacteriophage and Increase Host Range.

INTRODUCTION: Antibacterial resistance is an emerging problem in military medicine. Disruptions to the health care systems in war-torn countries that result from ongoing conflict can potentially exacerbate this problem and increase the risk to U.S. forces in the deployed environment. Therefore, novel therapies are needed to mitigate the impact of these potentially devastating infections on military operations. Bacteriophages are viruses that infect and kill bacteria. They can be delivered as therapeutic agents and offer a promising alternative to traditional antibiotic chemotherapy. There are several potential benefits to their use, including high specificity and comparative ease of use in the field setting. However, the process of engineering phages for military medical applications can be a laborious and time-consuming endeavor. This review examines available techniques and compares their efficacy. MATERIALS AND METHODS: This review evaluates the scientific literature on the development and application of four methods of bacteriophage genome engineering and their consideration in the context of military applications. Preffered Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed for a systematic review of available literature that met criteria for analysis and inclusion. The research completed for this review article originated from the United States Military Academy's library "Scout" search engine, which compiles results from 254 available databases (including PubMed, Google Scholar, and SciFinder). Particular attention was focused on identifying useful mechanistic insight into the nature of the engineering technique, the ease of use, and the applicability of the technique to countering the problem of antimicrobial resistance in the military setting. RESULTS: A total of 52 studies were identified that met inclusion criteria following PRISMA guidelines. The bioengineering techniques analyzed included homologous recombination (12 articles), in vivo recombineering (9 articles), bacteriophage recombineering of electroporated DNA (7 articles), and the CRISPR-Cas system (10 articles). Rates of success and fidelity varied across each platform, and comparative benefits and drawbacks are considered. CONCLUSIONS: Each of the phage engineering techniques addressed herein varies in amount of effort and overall success rate. CRISPR-Cas-facilitated modification of phage genomes presents a highly efficient method that does not require a lengthy purification and screening process. It therefore appears to be the method best suited for military medical applications.

Conduction Band Replicas in a 2D Moiré Semiconductor Heterobilayer.

Stacking monolayer semiconductors creates moiré patterns, leading to correlated and topological electronic phenomena, but measurements of the electronic structure underpinning these phenomena are scarce. Here, we investigate the properties of the conduction band in moiré heterobilayers of WS2/WSe2 using submicrometer angle-resolved photoemission spectroscopy with electrostatic gating. We find that at all twist angles the conduction band edge is the K-point valley of the WS2, with a band gap of 1.58 ± 0.03 eV. From the resolved conduction band dispersion, we deduce an effective mass of 0.15 ± 0.02 me. Additionally, we observe replicas of the conduction band displaced by reciprocal lattice vectors of the moiré superlattice. We argue that the replicas result from the moiré potential modifying the conduction band states rather than final-state diffraction. Interestingly, the replicas display an intensity pattern with reduced 3-fold symmetry, which we show implicates the pseudo vector potential associated with in-plane strain in moiré band formation.

Virtual consultations for oral surgery patients.

BACKGROUND: Following Covid-19, many departments have incorporated teledentistry into practice. As new consultation methods are introduced, it's imperative that patients have as similar an experience with virtual consultations to ensure informed decision-making. This project evaluated patients' perceptions of video consultations and determined if patients seen virtually received the same standard of information by auditing compliance with sending patient information leaflets (PILs) following video consultation. METHOD: The department's PILs were used to create an inclusion list for patients requiring a PIL. A retrospective audit assessed the notes of 100 video consultations for records of if PILs were sent and by what method. The department's PILs were digitalised and a clinical mailbox introduced enabling clinicians to email patients a PIL hyperlink. The audit was repeated for 88 video consultations. Patient and staff feedback was gathered via online surveys. RESULTS: Initially, 51% of cases met the criteria requiring a PIL and 16% of patients were sent PILs. Following mailbox introduction, 53% of cases met the criteria and 94% were sent PILs, 100% via email. Patient and staff feedback was positive regarding video consultations and digital PILS. Technical difficulties were reported in 44% of cases. CONCLUSIONS: Patients perceive virtual consultations to be a positive change and the introduction of a mailbox enhances video consultations in an efficient and cost-effective manner. Patient information can be standardised, via digital PILs, regardless of consultation type. As departments implement post-pandemic changes, utilisation of a mailbox can provide multiple improvements to care.

Field-Dependent Band Structure Measurements in Two-Dimensional Heterostructures.

In electronic and optoelectronic devices made from van der Waals heterostructures, electric fields can induce substantial band structure changes which are crucial to device operation but cannot usually be directly measured. Here, we use spatially resolved angle-resolved photoemission spectroscopy to monitor changes in band alignment of the component layers, corresponding to band structure changes of the composite heterostructure system, that are produced by electrostatic gating. Our devices comprise graphene on a monolayer semiconductor, WSe2 or MoSe2, atop a boron nitride dielectric and a graphite gate. Applying a gate voltage creates an electric field that shifts the semiconductor bands relative to those in the graphene by up to 0.2 eV. The results can be understood in simple terms by assuming that the materials do not hybridize.

Low Doses of Mycotoxin Mixtures below EU Regulatory Limits Can Negatively Affect the Performance of Broiler Chickens: A Longitudinal Study.

Several studies have reported a wide range of severe health effects as well as clinical signs, when livestock animals are exposed to high concentration of mycotoxins. However, little is known regarding health effects of mycotoxins at low levels. Thus, a long-term feeding trial (between May 2017 and December 2019) was used to evaluate the effect of low doses of mycotoxin mixtures on performance of broiler chickens fed a naturally contaminated diet. In total, 18 successive broiler performance trials were carried out during the study period, with approximately 2200 one-day-old Ross-308 chicks used for each trial. Feed samples given to birds were collected at the beginning of each trial and analysed for multi-mycotoxins using a validated LC-MS/MS method. Furthermore, parameters including feed intake, body weight and feed efficiency were recorded on a weekly basis. In total, 24 mycotoxins were detected in samples analysed with deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FBs), apicidin, enniatins (ENNs), emodin and beauvericin (BEV), the most prevalent mycotoxins. Furthermore, significantly higher levels (however below EU guidance values) of DON, ZEN, FBs, BEV, ENNs and diacetoxyscirpenol (DAS) were detected in 6 of the 18 performance trials. A strong positive relationship was observed between broilers feed efficiency and DON (R2 = 0.85), FBs (R2 = 0.53), DAS (R2 = 0.86), ZEN (R2 = 0.92), ENNs (R2 = 0.60) and BEV (R2 = 0.73). Moreover, a three-way interaction regression model revealed that mixtures of ZEN, DON and FBs (p = 0.01, R2 = 0.84) and ZEN, DON and DAS (p = 0.001, R2 = 0.91) had a statistically significant interaction effect on the birds' feed efficiency. As farm animals are often exposed to low doses of mycotoxin mixtures (especially fusarium mycotoxins), a cumulative risk assessment in terms of measuring and mitigating against the economic, welfare and health impacts is needed for this group of compounds.

Atomic reconstruction in twisted bilayers of transition metal dichalcogenides.

Van der Waals heterostructures form a unique class of layered artificial solids in which physical properties can be manipulated through controlled composition, order and relative rotation of adjacent atomic planes. Here we use atomic-resolution transmission electron microscopy to reveal the lattice reconstruction in twisted bilayers of the transition metal dichalcogenides, MoS2 and WS2. For twisted 3R bilayers, a tessellated pattern of mirror-reflected triangular 3R domains emerges, separated by a network of partial dislocations for twist angles θ < 2°. The electronic properties of these 3R domains, featuring layer-polarized conduction-band states caused by lack of both inversion and mirror symmetry, appear to be qualitatively different from those of 2H transition metal dichalcogenides. For twisted 2H bilayers, stable 2H domains dominate, with nuclei of a second metastable phase. This appears as a kagome-like pattern at θ ≈ 2°, transitioning at θ → 0 to a hexagonal array of screw dislocations separating large-area 2H domains. Tunnelling measurements show that such reconstruction creates strong piezoelectric textures, opening a new avenue for engineering of 2D material properties.

Visualizing electrostatic gating effects in two-dimensional heterostructures.

The ability to directly monitor the states of electrons in modern field-effect devices-for example, imaging local changes in the electrical potential, Fermi level and band structure as a gate voltage is applied-could transform our understanding of the physics and function of a device. Here we show that micrometre-scale, angle-resolved photoemission spectroscopy1-3 (microARPES) applied to two-dimensional van der Waals heterostructures4 affords this ability. In two-terminal graphene devices, we observe a shift of the Fermi level across the Dirac point, with no detectable change in the dispersion, as a gate voltage is applied. In two-dimensional semiconductor devices, we see the conduction-band edge appear as electrons accumulate, thereby firmly establishing the energy and momentum of the edge. In the case of monolayer tungsten diselenide, we observe that the bandgap is renormalized downwards by several hundreds of millielectronvolts-approaching the exciton energy-as the electrostatic doping increases. Both optical spectroscopy and microARPES can be carried out on a single device, allowing definitive studies of the relationship between gate-controlled electronic and optical properties. The technique provides a powerful way to study not only fundamental semiconductor physics, but also intriguing phenomena such as topological transitions5 and many-body spectral reconstructions under electrical control.

Structural and dynamical description of the enzymatic reaction of a phosphohexomutase.

Enzymes are known to adopt various conformations at different points along their catalytic cycles. Here, we present a comprehensive analysis of 15 isomorphous, high resolution crystal structures of the enzyme phosphoglucomutase from the bacterium Xanthomonas citri. The protein was captured in distinct states critical to function, including enzyme-substrate, enzyme-product, and enzyme-intermediate complexes. Key residues in ligand recognition and regions undergoing conformational change are identified and correlated with the various steps of the catalytic reaction. In addition, we use principal component analysis to examine various subsets of these structures with two goals: (1) identifying sites of conformational heterogeneity through a comparison of room temperature and cryogenic structures of the apo-enzyme and (2) a priori clustering of the enzyme-ligand complexes into functionally related groups, showing sensitivity of this method to structural features difficult to detect by traditional methods. This study captures, in a single system, the structural basis of diverse substrate recognition, the subtle impact of covalent modification, and the role of ligand-induced conformational change in this representative enzyme of the α-D-phosphohexomutase superfamily.

Indirect to Direct Gap Crossover in Two-Dimensional InSe Revealed by Angle-Resolved Photoemission Spectroscopy.

Atomically thin films of III-VI post-transition metal chalcogenides (InSe and GaSe) form an interesting class of two-dimensional semiconductors that feature a strong variation of their band gap as a function of the number of layers in the crystal and, specifically for InSe, an expected crossover from a direct gap in the bulk to a weakly indirect band gap in monolayers and bilayers. Here, we apply angle-resolved photoemission spectroscopy with submicrometer spatial resolution (µARPES) to visualize the layer-dependent valence band structure of mechanically exfoliated crystals of InSe. We show that for one-layer and two-layer InSe the valence band maxima are away from the Γ-point, forming an indirect gap, with the conduction band edge known to be at the Γ-point. In contrast, for six or more layers the band gap becomes direct, in good agreement with theoretical predictions. The high-quality monolayer and bilayer samples enable us to resolve, in the photoluminescence spectra, the band-edge exciton (A) from the exciton (B) involving holes in a pair of deeper valence bands, degenerate at Γ, with a splitting that agrees with both µARPES data and the results of DFT modeling. Due to the difference in symmetry between these two valence bands, light emitted by the A-exciton should be predominantly polarized perpendicular to the plane of the two-dimensional crystal, which we have verified for few-layer InSe crystals.

Asp263 missense variants perturb the active site of human phosphoglucomutase 1.

The enzyme phosphoglucomutase 1 (PGM1) plays a central role in glucose homeostasis. Clinical studies have identified mutations in human PGM1 as the cause of PGM1 deficiency, an inherited metabolic disease. One residue, Asp263, has two known variants associated with disease: D263G and D263Y. Biochemical studies have shown that these mutants are soluble and well folded, but have significant catalytic impairment. To better understand this catalytic defect, we determined crystal structures of these two missense variants, both of which reveal a similar and indirect structural change due to the loss of a conserved salt bridge between Asp263 and Arg293. The arginine reorients into the active site, making interactions with residues responsible for substrate binding. Biochemical studies also show that the catalytic phosphoserine of the missense variants is more stable to hydrolysis relative to wild-type enzyme. The structural perturbation resulting from mutation of this single amino acid reveals the molecular mechanism underlying PGM1 deficiency in these missense variants. DATABASE: Structural data are available in the PDB under the accession numbers 5JN5 and 5TR2.

Induced Structural Disorder as a Molecular Mechanism for Enzyme Dysfunction in Phosphoglucomutase 1 Deficiency.

Human phosphoglucomutase 1 (PGM1) plays a central role in cellular glucose homeostasis, mediating the switch between glycolysis and gluconeogenesis through the conversion of glucose 1-phosphate and glucose 6-phosphate. Recent clinical studies have identified mutations in this enzyme as the cause of PGM1 deficiency, an inborn error of metabolism classified as both a glycogen storage disease and a congenital disorder of glycosylation. Reported here are the first crystal structures of two disease-related missense variants of PGM1, along with the structure of the wild-type enzyme. Two independent glycine-to-arginine substitutions (G121R and G291R), both affecting key active site loops of PGM1, are found to induce regions of structural disorder, as evidenced by a nearly complete loss of electron density for as many as 23 aa. The disordered regions are not contiguous in sequence to the site of mutation, and even cross domain boundaries. Other structural rearrangements include changes in the conformations of loops and side chains, some of which occur nearly 20 Šaway from the site of mutation. The induced structural disorder is correlated with increased sensitivity to proteolysis and lower-resolution diffraction, particularly for the G291R variant. Examination of the multi-domain effects of these G➔R mutations establishes a correlation between interdomain interfaces of the enzyme and missense variants of PGM1 associated with disease. These crystal structures provide the first insights into the structural basis of enzyme dysfunction in PGM1 deficiency and highlight a growing role for biophysical characterization of proteins in the field of precision medicine.