Breaking Down Silos in the Workplace: A Framework to Foster Collaboration.

INTRODUCTION: Employees are often placed within an organization based on their respective roles or duties, which can lead to vertical and horizontal organizational silos. Organizational silos may restrict information, resources, and stymie progress and innovation. This analysis presents a framework to mitigate silos and overcome communication barriers within an organization by increasing collaboration. METHODS: The project team examined results from the Centers for Disease Control and Prevention (CDC), the National Center for Chronic Disease Prevention and Health Promotion (NCCDPHP) 2020 Employee Viewpoint Survey Results and conducted 19 key informant discussions with NCCDPHP employees. Participants were asked to provide feedback on (1) understanding silos in the workplace and (2) best practices for reducing silos and fostering collaboration. A thematic analysis was conducted to understand organizational silos, the motivation to reduce silos, and identify best practices and strategies. RESULTS: Respondents felt that siloing exists at the division and branch levels; however, 95% of respondents were motivated to reduce silos. Fifty-eight percent of respondents identified that institutional factors such as the organizational structure (n = 8) and red tape/bureaucracy (n = 3) contribute to siloing. Additional behaviors and actions that perpetuate silos were identified, and efforts to reduce silos were categorized to propose a model: Framework to Foster Collaboration for improving organizational collaborative efforts. DISCUSSION AND CONCLUSION: Key themes included inclusion, shared goals and vision, bi-directional communication, and relationship building and developing trust as critical elements for improving collaboration and creating synergy across teams in efforts to reduce silos in the workplace.

Tandem High-Pressure Crystallography-Optical Spectroscopy Unpacks Noncovalent Interactions of Piezochromic Fluorescent Molecular Rotors.

To develop luminescent molecular materials with predictable and stimuli-responsive emission, it is necessary to correlate changes in their geometries, packing structures, and noncovalent interactions with the associated changes in their optical properties. Here, we demonstrate that high-pressure single-crystal X-ray diffraction can be combined with high-pressure UV-visible absorption and fluorescence emission spectroscopies to elucidate how subtle changes in structure influence optical outputs. A piezochromic aggregation-induced emitter, sym-heptaphenylcycloheptatriene (Ph7C7H), displays bathochromic shifts in its absorption and emission spectra at high pressure. Parallel X-ray measurements identify the pressure-induced changes in specific phenyl-phenyl interactions responsible for the piezochromism. Pairs of phenyl rings from neighboring molecules approach the geometry of a stable benzene dimer, while conformational changes alter intramolecular phenyl-phenyl interactions correlated with a relaxed excited state. This tandem crystallographic and spectroscopic analysis provides insights into how subtle structural changes relate to the photophysical properties of Ph7C7H and could be applied to a library of similar compounds to provide general structure-property relationships in fluorescent organic molecules with rotor-like geometries.

Advanced airway management and drug-assisted intubation skills in an advanced critical care practitioner team.

INTRODUCTION: Airway management, including endotracheal intubation, is one of the cornerstones of care of critically ill patients. Internationally, health professionals from varying backgrounds deliver endotracheal intubation as part of their critical care role. This article considers the development of airway management skills within a single advanced critical care practitioner (ACCP) team and uses case series data to analyse the safety profile in performing this aspect of critical care. Skills were acquired during and after the ACCP training pathway. A combination of theoretical teaching, theatre experience, simulation and work-based practice was used. Case series data of all critical care intubations by ACCPs were collected. Audit results: Data collection identified 675 intubations carried out by ACCPs, 589 of those being supervised, non-cardiac arrest intubations requiring drugs. First pass success was achieved in 89.6% of cases. A second intubator was required in 4.3% of cases. Some form of complication was experienced by 42.3% of patients; however, the threshold for complications was set at a low level. CONCLUSIONS: This ACCP service developed a process to acquire advanced airway management skills including endotracheal intubation. Under medical supervision, ACCPs delivered advanced airway management achieving a first pass success rate of 89.6%, which compares favourably with both international and national success rates. Although complications were experienced in 48.3% of patients, when similar complication cut-offs are compared with published data, ACCPs also matched favourably.

Photophysics of Azobenzene Constrained in a UiO Metal-Organic Framework: Effects of Pressure, Solvation and Dynamic Disorder.

Photophysical studies of chromophoric linkers in metal-organic frameworks (MOFs) are undertaken commonly in the context of sensing applications, in search of readily observable changes of optical properties in response to external stimuli. The advantages of the MOF construct as a platform for investigating fundamental photophysical behaviour have been somewhat overlooked. The linker framework offers a unique environment in which the chromophore is geometrically constrained and its structure can be determined crystallographically, but it exists in spatial isolation, unperturbed by inter-chromophore interactions. Furthermore, high-pressure studies enable the photophysical consequences of controlled, incremental changes in local environment or conformation to be observed and correlated with structural data. This approach is demonstrated in the present study of the trans-azobenzene chromophore, constrained in the form of the 4,4'-azobenzenedicarboxylate (abdc) linker, in a UiO topology framework. Previously unobserved effects of pressure-induced solvation and conformational distortion on the lowest energy, nπ* transition are reported, and interpreted the light of crystallographic data. It was found that trans-azobenzene remains non-fluorescent (with a quantum yield less than 10-4 ) despite the prevention of trans-cis isomerization by the constraining MOF structure. We propose that efficient non-radiative decay is mediated by the local, pedal-like twisting of the azo group that is evident as dynamic disorder in the crystal structure.

Evaluation of the safety of inter-hospital transfers of critically ill patients led by advanced critical care practitioners.

INTRODUCTION: Ten thousand inter-hospital transfers of critically ill adults take place annually in the UK. Studies highlight deficiencies in experience and training of staff, equipment, stabilisation before departure, and logistical difficulties. This article is a quality improvement review of an advanced critical care practitioner (ACCP)-led inter-hospital transfer service. METHODS: The tool Standards for Quality Improvement Reporting Excellence was used as the format for the review, combined with clinical audit of advanced critical care practitioner-led transfers over a period of more than 3 years. RESULTS: The transfer service has operated for 8 years; ACCPs conducted 934 critical care transfers of mechanically ventilated patients, including 286 inter-hospital transfers, between January 2017 and September 2020. The acuity of transfer patients was high, 82.2% required support of more than one organ, 49% required more than 50% oxygen. Uneventful transfer occurred in 81.4% of cases; the most common patient-related complication being hypotension, logistical issues were responsible for half of the complications. CONCLUSION: This quality improvement project provides an example of safe and effective advanced practice in an area that is traditionally a medically led domain. ACCPs can provide an alternative process of care for critically ill adults who require external transfer, and a benchmark for audit and quality improvement.

Transcriptome analysis of the uterus of hens laying eggs differing in cuticle deposition.

BACKGROUND: Avian eggs have a proteinaceous cuticle. The quantity of cuticle varies and the deposition of a good cuticle in the uterus (Shell-gland) prevents transmission of bacteria to the egg contents. RESULTS: To understand cuticle deposition, uterus transcriptomes were compared between hens with i) naturally good and poor cuticle and, ii) where manipulation of the hypothalamo-pituitary-gonadal-oviduct axis produced eggs with or without cuticle. The highest expressed genes encoded eggshell matrix and cuticle proteins, e.g. MEPE (OC-116), BPIFB3 (OVX-36), RARRES1 (OVX-32), WAP (OVX-25), and genes for mitochondrial oxidative phosphorylation, active transport and energy metabolism. Expression of a number of these genes differed between hens laying eggs with or without cuticle. There was also a high expression of clock genes. PER2, CRY2, CRY1, CLOCK and BMAL1 were differentially expressed when cuticle deposition was prevented, and they also changed throughout the egg formation cycle. This suggests an endogenous clock in the uterus may be a component of cuticle deposition control. Cuticle proteins are glycosylated and glycosaminoglycan binding genes had a lower expression when cuticle proteins were deposited on the egg. The immediate early genes, JUN and FOS, were expressed less when the cuticle had not been deposited and changed over the egg formation cycle, suggesting they are important in oviposition and cuticle deposition. The uterus transcriptome of hens with good and poor cuticle deposition did not differ. CONCLUSIONS: We have gained insights into the factors that can affect the production of the cuticle especially clock genes and immediate early genes. We have demonstrated that these genes change their expression over the period of eggshell formation supporting their importance. The lack of differences in expression between the uterus of hens laying eggs with the best and worse cuticle suggest the genetic basis of the trait may lie outside the oviduct.

Correlating Pressure-Induced Emission Modulation with Linker Rotation in a Photoluminescent MOF.

Conformational changes of linker units in metal-organic frameworks (MOFs) are often responsible for gate-opening phenomena in selective gas adsorption and stimuli-responsive optical and electrical sensing behaviour. Herein, we show that pressure-induced bathochromic shifts in both fluorescence emission and UV/Vis absorption spectra of a two-fold interpenetrated Hf MOF, linked by 1,4-phenylene-bis(4-ethynylbenzoate) ligands (Hf-peb), are induced by rotation of the central phenyl ring of the linker, from a coplanar arrangement to a twisted, previously unseen conformer. Single-crystal X-ray diffraction, alongside in situ fluorescence and UV/Vis absorption spectroscopies, measured up to 2.1 GPa in a diamond anvil cell on single crystals, are in excellent agreement, correlating linker rotation with modulation of emission. Topologically isolating the 1,4-phenylene-bis(4-ethynylbenzoate) units within a MOF facilitates concurrent structural and spectroscopic studies in the absence of intermolecular perturbation, allowing characterisation of the luminescence properties of a high-energy, twisted conformation of the previously well-studied chromophore. We expect the unique environment provided by network solids, and the capability of combining crystallographic and spectroscopic analysis, will greatly enhance understanding of luminescent molecules and lead to the development of novel sensors and adsorbents.

Genetic variation and potential for genetic improvement of cuticle deposition on chicken eggs.

BACKGROUND: The cuticle is an invisible glycosylated protein layer that covers the outside of the eggshell and forms a barrier to the transmission of microorganisms. Cuticle-specific staining and in situ absorbance measurements have been used to quantify cuticle deposition in several pure breeds of chicken. For brown eggs, a pre-stain and a post-stain absorbance measurement is required to correct for intrinsic absorption by the natural pigment. For white eggs, a post-stain absorbance measurement alone is sufficient to estimate cuticle deposition. The objective of the research was to estimate genetic parameters and provide data to promote adoption of the technique to increase cuticle deposition and reduce vertical transmission of microorganisms. RESULTS: For all pure breeds examined here, i.e. Rhode Island Red, two White Leghorns, White Rock and a broiler breed, the estimate of heritability for cuticle deposition from a meta-analysis was moderately high (0.38 ± 0.04). In the Rhode Island Red breed, the estimate of the genetic correlation between measurements recorded at early and late times during the egg-laying period was ~ 1. There was no negative genetic correlation between cuticle deposition and production traits. Estimates of the genetic correlation of cuticle deposition with shell color ranged from negative values or 0 in brown-egg layers to positive values in white- or tinted-egg layers. Using the intrinsic fluorescence of tryptophan in the cuticle proteins to quantify the amount of cuticle deposition failed because of complex quenching processes. Tryptophan fluorescence intensity at 330 nm was moderately heritable, but there was no evidence of a non-zero genetic correlation with cuticle deposition. This was complicated furthermore by a negative genetic correlation of fluorescence with color in brown eggs, due to the quenching of tryptophan fluorescence by energy transfer to protoporphyrin pigment. We also confirmed that removal of the cuticle increased reflection of ultraviolet wavelengths from the egg. CONCLUSIONS: These results provide additional evidence for the need to incorporate cuticle deposition into breeding programs of egg- and meat-type birds in order to reduce vertical and horizontal transmission of potentially pathogenic organisms and to help improve biosecurity in poultry.

Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity.

Fluorescent nucleobase analogues (FBAs) have many desirable features in comparison to extrinsic fluorescent labels, but they are yet to find application in ultrasensitive detection. Many of the disadvantages of FBAs arise from their short excitation wavelengths (often in the ultraviolet), making two-photon excitation a potentially attractive approach. Pentacyclic adenine (pA) is a recently developed FBA that has an exceptionally high two-photon brightness. We have studied the two-photon-excited fluorescence properties of pA and how they are affected by incorporation in DNA. We find that pA is more photostable under two-photon excitation than via resonant absorption. When incorporated in an oligonucleotide, pA has a high two-photon cross section and emission quantum yield, varying with sequence context, resulting in the highest reported brightness for such a probe. The use of a two-photon microscope with ultrafast excitation and pulse shaping has allowed the detection of pA-containing oligonucleotides in solution with a limit of detection of ∼5 molecules, demonstrating that practical single-molecule detection of FBAs is now within reach.

2-Aminopurine as a fluorescent probe of DNA conformation and the DNA-enzyme interface.

Nearly 50 years since its potential as a fluorescent base analogue was first recognized, 2-aminopurine (2AP) continues to be the most widely used fluorescent probe of DNA structure and the perturbation of that structure by interaction with enzymes and other molecules. In this review, we begin by considering the origin of the dramatic and intriguing difference in photophysical properties between 2AP and its structural isomer, adenine; although 2AP differs from the natural base only in the position of the exocyclic amine group, its fluorescence intensity is one thousand times greater. We then discuss the mechanism of interbase quenching of 2AP fluorescence in DNA, which is the basis of its use as a conformational probe but remains imperfectly understood. There are hundreds of examples in the literature of the use of changes in the fluorescence intensity of 2AP as the basis of assays of conformational change; however, in this review we will consider in detail only a few intensity-based studies. Our primary aim is to highlight the use of time-resolved fluorescence measurements, and the interpretation of fluorescence decay parameters, to explore the structure and dynamics of DNA. We discuss the salient features of the fluorescence decay of 2AP when incorporated in DNA and review the use of decay measurements in studying duplexes, single strands and other structures. We survey the use of 2AP as a probe of DNA-enzyme interaction and enzyme-induced distortion, focusing particularly on its use to study base flipping and the enhanced mechanistic insights that can be gained by a detailed analysis of the decay parameters, rather than merely monitoring changes in fluorescence intensity. Finally we reflect on the merits and shortcomings of 2AP and the prospects for its wider adoption as a fluorescence-decay-based probe.

Understanding avian egg cuticle formation in the oviduct: a study of its origin and deposition.

The cuticle is a unique invisible oviduct secretion that protects avian eggs from bacterial penetration through gas exchange pores. Despite its importance, experimental evidence is lacking for where, when, and what is responsible for its deposition. By using knowledge about the ovulatory cycle and oviposition, we have manipulated cuticle deposition to obtain evidence on these key points. Cuticle deposition was measured using staining and spectrophotometry. Experimental evidence supports the location of cuticle deposition to be the shell gland pouch (uterus), not the vagina, and the time of deposition to be within the final hour before oviposition. Oviposition induced by arginine vasotocin or prostaglandin, the penultimate and ultimate factors for the induction of oviposition, produces an egg with no cuticle; therefore, these factors are not responsible for cuticle secretion. Conversely, oviposition induced by GNRH, which mimics the normal events of ovulation and oviposition, results in a normal cuticle. There is no evidence that cuticle deposition differs at the end of a clutch and, therefore, there is no evidence that the ovulatory surge of progesterone affects cuticle deposition. Overall, the results demonstrate that the cuticle is a specific secretion and is not merely an extension of the organic matrix of the shell. Cuticle deposition was found to be reduced by an environmental stressor, and there is no codependence of the deposition of pigment and cuticle. Defining the basic facts surrounding cuticle deposition will help reduce contamination of hen's eggs and increase understanding of the strategies birds use to protect their eggs.

A DFT study of 2-aminopurine-containing dinucleotides: prediction of stacked conformations with B-DNA structure.

The fluorescence properties of dinucleotides incorporating 2-aminopurine (2AP) suggest that the simplest oligonucleotides adopt conformations similar to those found in duplex DNA. However, there is a lack of structural data for these systems. We report a density functional theory (DFT) study of the structures of 2AP-containing dinucleotides (deoxydinucleoside monophosphates), including full geometry optimisation of the sugar-phosphate backbone. Our DFT calculations employ the M06-2X functional for reliable treatment of dispersion interactions and include implicit aqueous solvation. Dinucleotides with 2AP in the 5'-position and each of the natural bases in the 3'-position are examined, together with the analogous 5'-adenine-containing systems. Computed structures are compared in detail with typical B-DNA base-step parameters, backbone torsional angles and sugar pucker, derived from crystallographic data. We find that 2AP-containing dinucleotides adopt structures that closely conform to B-DNA in all characteristic parameters. The structures of 2AP-containing dinucleotides closely resemble those of their adenine-containing counterparts, demonstrating the fidelity of 2AP as a mimic of the natural base. As a first step towards exploring the conformational heterogeneity of dinucleotides, we also characterise an imperfectly stacked conformation and one in which the bases are completely unstacked.

Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil.

Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3'-5' proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase-DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase.

Restriction endonuclease TseI cleaves A:A and T:T mismatches in CAG and CTG repeats.

The type II restriction endonuclease TseI recognizes the DNA target sequence 5'-G^CWGC-3' (where W = A or T) and cleaves after the first G to produce fragments with three-base 5'-overhangs. We have determined that it is a dimeric protein capable of cleaving not only its target sequence but also one containing A:A or T:T mismatches at the central base pair in the target sequence. The cleavage of targets containing these mismatches is as efficient as cleavage of the correct target sequence containing a central A:T base pair. The cleavage mechanism does not apparently use a base flipping mechanism as found for some other type II restriction endonuclease recognizing similarly degenerate target sequences. The ability of TseI to cleave targets with mismatches means that it can cleave the unusual DNA hairpin structures containing A:A or T:T mismatches formed by the repetitive DNA sequences associated with Huntington's disease (CAG repeats) and myotonic dystrophy type 1 (CTG repeats).

Observation of unpaired substrate DNA in the flap endonuclease-1 active site.

The structure- and strand-specific phosphodiesterase flap endonuclease-1 (FEN1), the prototypical 5'-nuclease, catalyzes the essential removal of 5'-single-stranded flaps during replication and repair. FEN1 achieves this by selectively catalyzing hydrolysis one nucleotide into the duplex region of substrates, always targeting the 5'-strand. This specificity is proposed to arise by unpairing the 5'-end of duplex to permit the scissile phosphate diester to contact catalytic divalent metal ions. Providing the first direct evidence for this, we detected changes induced by human FEN1 (hFEN1) in the low-energy CD spectra and fluorescence lifetimes of 2-aminopurine in substrates and products that were indicative of unpairing. Divalent metal ions were essential for unpairing. However, although 5'-nuclease superfamily-conserved active-site residues K93 and R100 were required to produce unpaired product, they were not necessary to unpair substrates. Nevertheless, a unique arrangement of protein residues around the unpaired DNA was detected only with wild-type protein, suggesting a cooperative assembly of active-site residues that may be triggered by unpaired DNA. The general principles of FEN1 strand and reaction-site selection, which depend on the ability of juxtaposed divalent metal ions to unpair the end of duplex DNA, may also apply more widely to other structure- and strand-specific nucleases.

Time-resolved fluorescence of 2-aminopurine in DNA duplexes in the presence of the EcoP15I Type III restriction-modification enzyme.

EcoP15I is a Type III DNA restriction and modification enzyme of Escherichia coli. We show that it contains two modification (Mod) subunits for sequence-specific methylation of DNA and one copy of a restriction endonuclease (Res) subunit for cleavage of DNA containing unmethylated target sequences. Previously the Mod2 dimer in the presence of cofactors was shown to use nucleotide flipping to gain access to the adenine base targeted for methylation (Reddy and Rao, J. Mol. Biol. 298 (2000) 597-610.). Surprisingly the Mod2 enzyme also appeared to flip a second adenine in the target sequence, one which was not subject to methylation. We show using fluorescence lifetime measurements of the adenine analogue, 2-aminopurine, that only the methylatable adenine undergoes flipping by the complete Res1Mod2 enzyme and that this occurs even in the absence of cofactors. We suggest that this is due to activation of the Mod2 core by the Res subunit.

Dental handpiece contamination: a proteomics and surface analysis approach.

Dental handpieces (DHPs) become biofouled internally with patient derived material that is difficult to access for removal and inactivation. This study undertook a quantitative and qualitative investigation of protein contamination of internal components from three different types of DHP: the turbine, slow speed contra-angle and surgical. Eluates from the high speed turbine, low speed spray channels and surgical gear were assayed for protein using an orthophthaldehyde assay. Eluates concentrated by Amicon ultrafiltration were also analysed by SDS-PAGE, mass spectroscopy, Western blotting and ELISA. The surfaces of handpiece components were also investigated by SEM, EFSCAN and EDAX microscopy. Surgical gears contained highest levels of protein (403 µg), followed by low speed spray channels (17.7 µg) and the high speed turbine (<5 µg). Mass spectroscopy of surgical gears demonstrated mostly serum derived proteins. Decontamination of the DHPs using an automated washer disinfector and handpiece irrigator showed a significant reduction in residual protein levels.

Photonic crystal fibres for chemical sensing and photochemistry.

In this review, we introduce photonic crystal fibre as a novel optofluidic microdevice that can be employed as both a versatile chemical sensor and a highly efficient microreactor. We demonstrate that it provides an excellent platform in which light and chemical samples can strongly interact for quantitative spectroscopic analysis or photoactivation purposes. The use of photonic crystal fibre in photochemistry and sensing is discussed and recent results on gas and liquid sensing as well as on photochemical and catalytic reactions are reviewed. These developments demonstrate that the tight light confinement, enhanced light-matter interaction and reduced sample volume offered by photonic crystal fibre make it useful in a wide range of chemical applications.

Enzyme-promoted base flipping controls DNA methylation fidelity.

A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme-substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.HhaI has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein-DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.

Branchpoint expansion in a fully complementary three-way DNA junction.

Branched nucleic acid molecules serve as key intermediates in DNA replication, recombination, and repair; architectural elements in RNA; and building blocks and functional components for nanoscience applications. Using a combination of high-resolution single-molecule FRET, time-resolved spectroscopy, and molecular modeling, we have probed the local and global structure of a DNA three-way junction (3WJ) in solution. We found that it adopts a Y-shaped, pyramidal structure, in which the bases adjacent to the branchpoint are unpaired, despite the full Watson-Crick complementarity of the molecule. The unpairing allows a nanoscale cavity to form at the junction center. Our structure accounts for earlier observations made of the structure, flexibility, and reactivity of 3WJs. We anticipate that these results will guide the development of new DNA-based supramolecular receptors and nanosystems.