Phosphorylation and sulfation share a common biosynthetic pathway, but extend biochemical and evolutionary diversity of biological macromolecules in distinct ways.

Phosphate and sulfate groups are integral to energy metabolism and introduce negative charges into biological macromolecules. One purpose of such modifications is to elicit precise binding/activation of protein partners. The physico-chemical properties of the two groups, while superficially similar, differ in one important respect-the valency of the central (phosphorus or sulfur) atom. This dictates the distinct properties of their respective esters, di-esters and hence their charges, interactions with metal ions and their solubility. These, in turn, determine the contrasting roles for which each group has evolved in biological systems. Biosynthetic links exist between the two modifications; the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate being formed from adenosine triphosphate (ATP) and adenosine phosphosulfate, while the latter is generated from sulfate anions and ATP. Furthermore, phosphorylation, by a xylosyl kinase (Fam20B, glycosaminoglycan xylosylkinase) of the xylose residue of the tetrasaccharide linker region that connects nascent glycosaminoglycan (GAG) chains to their parent proteoglycans, substantially accelerates their biosynthesis. Following observations that GAG chains can enter the cell nucleus, it is hypothesized that sulfated GAGs could influence events in the nucleus, which would complete a feedback loop uniting the complementary anionic modifications of phosphorylation and sulfation through complex, inter-connected signalling networks and warrants further exploration.

Outcomes and interventions in patients transported to hospital with ongoing CPR after out-of-hospital cardiac arrest - An observational study.

INTRODUCTION: The main objective was to present characteristics and outcome of patients without sustained field return of spontaneous circulation (ROSC) transported to hospital with ongoing cardiopulmonary resuscitation (CPR). Our secondary objectives were to investigate hospital-based interventions and the performance of the universal Termination of Resuscitation-rule (uTOR). METHODS: In this retrospective observational cohort study, out-of-hospital cardiac arrest (OHCA) patients arriving to the emergency department of a university hospital in Sweden during a six-year period (2010-2015) were identified using a prospectively recorded hospital-based registry. Additional data were retrieved from medical records and from the Swedish cardiopulmonary resuscitation registry. RESULTS: Among 409 patients transported with ongoing CPR, 7 survived to hospital discharge (1.7%). Hospital-based interventions against a suspected cause of arrest were attempted during ongoing resuscitation in 34 patients (8.3%), of whom 3 survived to hospital discharge. The remaining 4 survivors had spontaneous in-hospital ROSC. Survivors presented with either a shockable rhythm (n = 4) or pulseless electrical activity (n = 3). The uTOR identified non-survivors with a positive predictive value (PPV) of 98.4% and a specificity of 71.4% for termination. CONCLUSION: Survival after OHCA where sustained prehospital ROSC is not achieved is rare and available in-hospital interventions are rarely utilised. No patient with asystole as the first recorded rhythm survived. The uTOR identified non-survivors with a PPV of 98.4% but showed poor specificity.

Postmastectomy Radiation Therapy Planning After Immediate Implant-based Reconstruction Using the European Society for Radiotherapy and Oncology-Advisory Committee in Radiation Oncology Practice Consensus Guidelines for Target Volume Delineation.

AIMS: To evaluate how common radiation therapy techniques perform in the setting of the new European Society for Radiotherapy and Oncology-Advisory Committee in Radiation Oncology Practice (ESTRO-ACROP) delineation recommendations for immediate breast reconstruction (IBR). MATERIALS AND METHODS: Seven Danish radiation therapy centres and six international European centres participated in this project. Two breast cancer cases (one left-sided and one right-sided) with a retropectoral implant were chosen for radiation therapy planning using deep-inspiration breath-hold. Target volumes were delineated according to ESTRO-ACROP delineation recommendations. The centres were asked to plan the cases using any radiation therapy technique according to the Danish Breast Cancer Group plan objectives. RESULTS: In total, 35 treatment plans were collected. Half of the submitted plans, for both the left-sided and the right-sided case, used the field-in-field (FiF) technique (nine for each), a quarter used volumetric arc radiation therapy (VMAT; five for right-sided, four for left-sided) and the remaining quarter was a mix of inverse intensity-modulated radiation therapy (IMRT), helicoidal therapy and hybrid (combined open fields and VMAT) techniques. Mean clinical target volume doses were in the range 99-102% of the prescribed dose. The median FiF mean heart dose (MHD) for right-sided radiation therapy was 1 Gy (range 0.8-3.7) and 5.2 Gy for left-sided radiation therapy (range 2.2-6.5). For right-sided radiation therapy, the median VMAT MHD was 3.42 Gy, for IMRT was 2.3 Gy and for helicoidal therapy was 5.1 Gy. For left-sided radiation therapy, the median VMAT MHD was 6.3 Gy, for IMRT was 7.8 Gy and for helicoidal therapy was 7.3 Gy. CONCLUSIONS: Different radiation therapy techniques could be used to plan radiation therapy in the setting of IBR. FiF provided good coverage with acceptable organ at risk doses. The best dose distribution results as a trade-off between the objectives of target volume coverage and high-dose organ at risk inclusion. The radiation therapy technique affects the interplay between these objectives.

Extracellular histones stimulate collagen expression in vitro and promote liver fibrogenesis in a mouse model via the TLR4-MyD88 signaling pathway.

BACKGROUND: Liver fibrosis progressing to liver cirrhosis and hepatic carcinoma is very common and causes more than one million deaths annually. Fibrosis develops from recurrent liver injury but the molecular mechanisms are not fully understood. Recently, the TLR4-MyD88 signaling pathway has been reported to contribute to fibrosis. Extracellular histones are ligands of TLR4 but their roles in liver fibrosis have not been investigated. AIM: To investigate the roles and potential mechanisms of extracellular histones in liver fibrosis. METHODS: In vitro, LX2 human hepatic stellate cells (HSCs) were treated with histones in the presence or absence of non-anticoagulant heparin (NAHP) for neutralizing histones or TLR4-blocking antibody. The resultant cellular expression of collagen I was detected using western blotting and immunofluorescent staining. In vivo, the CCl4-induced liver fibrosis model was generated in male 6-week-old ICR mice and in TLR4 or MyD88 knockout and parental mice. Circulating histones were detected and the effect of NAHP was evaluated. RESULTS: Extracellular histones strongly stimulated LX2 cells to produce collagen I. Histone-enhanced collagen expression was significantly reduced by NAHP and TLR4-blocking antibody. In CCl4-treated wild type mice, circulating histones were dramatically increased and maintained high levels during the duration of fibrosis-induction. Injection of NAHP not only reduced alanine aminotransferase and liver injury scores, but also significantly reduced fibrogenesis. Since the TLR4-blocking antibody reduced histone-enhanced collagen I production in HSC, the CCl4 model with TLR4 and MyD88 knockout mice was used to demonstrate the roles of the TLR4-MyD88 signaling pathway in CCl4-induced liver fibrosis. The levels of liver fibrosis were indeed significantly reduced in knockout mice compared to wild type parental mice. CONCLUSION: Extracellular histones potentially enhance fibrogenesis via the TLR4-MyD88 signaling pathway and NAHP has therapeutic potential by detoxifying extracellular histones.

Out-of-hospital cardiac arrest termination of resuscitation with ongoing CPR: An observational study.

INTRODUCTION: Termination of resuscitation guidelines for out-of-hospital cardiac arrest can identify patients in whom continuing resuscitation has little chance of success. This study examined the outcomes of patients transferred to hospital with ongoing CPR. It assessed outcomes for those who would have met the universal prehospital termination of resuscitation criteria (no shocks administered, unwitnessed by emergency medical services, no return of spontaneous circulation). METHODS: A retrospective cohort study of consecutive adult patients who were transported to hospital with ongoing CPR was conducted at three hospitals in the West Midlands, UK between September 2016 and November 2017. Patient characteristics, interventions and response to treatment (ROSC, survival to discharge) were identified. RESULTS: 227 (median age 69 years, 67.8% male) patients were identified. 89 (39.2%) met the universal prehospital termination of resuscitation criteria. Seven (3.1%) were identified with a potentially reversible cause of cardiac arrest. After hospital arrival, patients received few specialist interventions that were not available in the prehospital setting. Most (n = 210, 92.5%) died in the emergency department. 17 were admitted (14 to intensive care), of which 3 (1.3%) survived to hospital discharge. There were no survivors (0%) in those who met the criteria for universal prehospital termination of resuscitation. CONCLUSION: Overall survival amongst patients transported to hospital with ongoing CPR was very poor. Application of the universal prehospital termination of resuscitation rule, in patients without obvious reversible causes of cardiac arrest, would have allowed resuscitation to have been discontinued at the scene for 39.2% of patients who did not survive.

Machine learning "red dot": open-source, cloud, deep convolutional neural networks in chest radiograph binary normality classification.

AIM: To develop a machine learning-based model for the binary classification of chest radiography abnormalities, to serve as a retrospective tool in guiding clinician reporting prioritisation. MATERIALS AND METHODS: The open-source machine learning library, Tensorflow, was used to retrain a final layer of the deep convolutional neural network, Inception, to perform binary normality classification on two, anonymised, public image datasets. Re-training was performed on 47,644 images using commodity hardware, with validation testing on 5,505 previously unseen radiographs. Confusion matrix analysis was performed to derive diagnostic utility metrics. RESULTS: A final model accuracy of 94.6% (95% confidence interval [CI]: 94.3-94.7%) based on an unseen testing subset (n=5,505) was obtained, yielding a sensitivity of 94.6% (95% CI: 94.4-94.7%) and a specificity of 93.4% (95% CI: 87.2-96.9%) with a positive predictive value (PPV) of 99.8% (95% CI: 99.7-99.9%) and area under the curve (AUC) of 0.98 (95% CI: 0.97-0.99). CONCLUSION: This study demonstrates the application of a machine learning-based approach to classify chest radiographs as normal or abnormal. Its application to real-world datasets may be warranted in optimising clinician workload.

Potential role for Streptococcus gordonii-derived hydrogen peroxide in heme acquisition by Porphyromonas gingivalis.

Streptococcus gordonii, an accessory pathogen and early colonizer of plaque, co-aggregates with many oral species including Porphyromonas gingivalis. It causes α-hemolysis on blood agar, a process mediated by H2 O2 and thought to involve concomitant oxidation of hemoglobin (Hb). Porphyromonas gingivalis has a growth requirement for heme, which is acquired mainly from Hb. The paradigm for Hb heme acquisition involves the initial oxidation of oxyhemoglobin (oxyHb) to methemoglobin (metHb), followed by heme release and extraction through the actions of K-gingipain protease and/or the HmuY hemophore-like protein. The ability of S. gordonii to mediate Hb oxidation may potentially aid heme capture during co-aggregation with P. gingivalis. Hemoglobin derived from zones of S. gordonii α-hemolysis was found to be metHb. Generation of metHb from oxyHb by S. gordonii cells was inhibited by catalase, and correlated with levels of cellular H2 O2 production. Generation of metHb by S. gordonii occurred through the higher Hb oxidation state of ferrylhemoglobin. Heme complexation by the P. gingivalis HmuY was employed as a measure of the ease of heme capture from metHb. HmuY was able to extract iron(III)protoporphyrin IX from metHb derived from zones of S. gordonii α-hemolysis and from metHb generated by the action of S. gordonii cells on isolated oxyHb. The rate of HmuY-Fe(III)heme complex formation from S. gordonii-mediated metHb was greater than from an equivalent concentration of auto-oxidized metHb. It is concluded that S. gordonii may potentially aid heme acquisition by P. gingivalis by facilitating metHb formation in the presence of oxyHb.

Photochemical model evaluation of 2013 California wild fire air quality impacts using surface, aircraft, and satellite data.

The Rim Fire was one of the largest wildfires in California history, burning over 250,000 acres during August and September 2013 affecting air quality locally and regionally in the western U.S. Routine surface monitors, remotely sensed data, and aircraft based measurements were used to assess how well the Community Multiscale Air Quality (CMAQ) photochemical grid model applied at 4 and 12 km resolution represented regional plume transport and chemical evolution during this extreme wildland fire episode. Impacts were generally similar at both grid resolutions although notable differences were seen in some secondary pollutants (e.g., formaldehyde and peroxyacyl nitrate) near the Rim fire. The modeling system does well at capturing near-fire to regional scale smoke plume transport compared to remotely sensed aerosol optical depth (AOD) and aircraft transect measurements. Plume rise for the Rim fire was well characterized as the modeled plume top was consistent with remotely sensed data and the altitude of aircraft measurements, which were typically made at the top edge of the plume. Aircraft-based lidar suggests O3 downwind in the Rim fire plume was vertically stratified and tended to be higher at the plume top, while CMAQ estimated a more uniformly mixed column of O3. Predicted wildfire ozone (O3) was overestimated both at the plume top and at nearby rural and urban surface monitors. Photolysis rates were well characterized by the model compared with aircraft measurements meaning aerosol attenuation was reasonably estimated and unlikely contributing to O3 overestimates at the top of the plume. Organic carbon was underestimated close to the Rim fire compared to aircraft data, but was consistent with nearby surface measurements. Periods of elevated surface PM2.5 at rural monitors near the Rim fire were not usually coincident with elevated O3.

In vitro investigations on the effects of semi-synthetic, sulphated carbohydrates on the immune status of cultured common carp (Cyprinus carpio) leucocytes.

The rapid emergence of drug resistance, unfavourable immunosuppression and mounting evidence to suggest the deleterious accumulation of drug breakdown residues within animal tissues has driven a strong desire to move away from these current methods of disease control. Some natural products such as ß-glucan, which are extracted from, for example, plants and fungi, are able to modulate the immune system and increase protection against diseases. However, these products are heterogeneous and their effects can be variable thus limiting their applicability and reliability. Carbohydrates were modified via chemical sulphation and these semi-synthetic, sulphated carbohydrates analysed for their immunological activity utilising carp pronephric cells and a carp leucocyte cell line (CLC). A sulphated ß(1,4)-glucan, methyl hydroxyethyl cellulose sulphate (MHCS), demonstrated a stimulatory effect on fish immune cells. MHCS induced a range of bioactive effects in carp leucocyte cells whilst not affecting cell viability when cells were exposed for 24 h at concentrations of 1-150 µgml-1. MHCS stimulated the innate immune system where a significant increase in respiratory burst activity was observed at concentrations 25-250 µgml-1 in comparison to control (sterile water), cellulose ether, MacroGard® and zymosan. Also, under in mock bacterial and viral infection conditions i.e. Lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (Poly(I:C)), MHCS enhanced the immune responses of pronephric cells by stimulating the respiratory burst activity at concentrations 50 and 150 µgml-1. MHCS also enhanced the expression of cytokines including interleukin 1 beta (IL1ß), tumor necrosis factor alpha 1 and 2 (TNFα 1,2), interferons alpha 2 (IFN α2) and inducible nitric oxide synthase (iNOS) in carp pronephric cells. It is proposed that this new semi-synthetic carbohydrate is a potential candidate for the development of a new generation of immunostimulants and adjuvants for use in vaccination strategies in aquaculture.

Determining the mechanical properties of a radiochromic silicone-based 3D dosimeter.

New treatment modalities in radiotherapy (RT) enable delivery of highly conformal dose distributions in patients. This creates a need for precise dose verification in three dimensions (3D). A radiochromic silicone-based 3D dosimetry system has recently been developed. Such a dosimeter can be used for dose verification in deformed geometries, which requires knowledge of the dosimeter's mechanical properties. In this study we have characterized the dosimeter's elastic behaviour under tensile and compressive stress. In addition, the dose response under strain was determined. It was found that the dosimeter behaved as an incompressible hyperelastic material with a non-linear stress/strain curve and with no observable hysteresis or plastic deformation even at high strains. The volume was found to be constant within a 2% margin at deformations up to 60%. Furthermore, it was observed that the dosimeter returned to its original geometry within a 2% margin when irradiated under stress, and that the change in optical density per centimeter was constant regardless of the strain during irradiation. In conclusion, we have shown that this radiochromic silicone-based dosimeter's mechanical properties make it a viable candidate for dose verification in deformable 3D geometries.

Coupling of vinculin to F-actin demands Syndecan-4 proteoglycan.

Syndecans are heparan sulfate proteoglycans characterized as transmembrane receptors that act cooperatively with the cell surface and extracellular matrix proteins. Syn4 knockdown was performed in order to address its role in endothelial cells (EC) behavior. Normal EC and shRNA-Syn4-EC cells were studied comparatively using complementary confocal, super-resolution and non-linear microscopic techniques. Confocal and super-resolution microscopy revealed that Syn4 knockdown alters the level and arrangement of essential proteins for focal adhesion, evidenced by the decoupling of vinculin from F-actin filaments. Furthermore, Syn4 knockdown alters the actin network leading to filopodial protrusions connected by VE-cadherin-rich junction. shRNA-Syn4-EC showed reduced adhesion and increased migration. Also, Syn4 silencing alters cell cycle as well as cell proliferation. Moreover, the ability of EC to form tube-like structures in matrigel is reduced when Syn4 is silenced. Together, the results suggest a mechanism in which Syndecan-4 acts as a central mediator that bridges fibronectin, integrin and intracellular components (actin and vinculin) and once silenced, the cytoskeleton protein network is disrupted. Ultimately, the results highlight Syn4 relevance for balanced cell behavior.

Feedback and motor skill acquisition using a haptic dental simulator.

AIM: To investigate the effect of qualitatively different types of pedagogical feedback (FB) on the training, transfer and retention of basic manual dexterity dental skills using a virtual reality (VR) haptic dental simulator. METHODS: Sixty-three participants (M = 22.7 years; SD = 3.4 years), with no previous dental training, were randomly allocated to one of three groups (n = 21 each). Group 1 received device-only feedback during the training phase, that is the visual display of the simulator (DFB); Group 2 received verbal feedback from a qualified dental instructor (IFB); and Group 3 received a combination of instructor and device feedback (IDFB). Participants completed four tasks during which feedback was given according to group allocation as well as two skills transfer tests. Skill retention was examined immediately after training, at 1 week and at 1 month post-test. RESULTS: Statistically significant differences were found between the groups in overall performance (P < 0.001) and error (P = 0.006). Post hoc comparisons revealed the IDFB group produced substantially better performance and fewer errors in comparison with DFB and IFB training. This difference translated to improved performance in skill retention and generalisation of knowledge to novel tasks. CONCLUSION: These data indicate that the acquisition and retention of basic dental motor skills in novice trainees is best optimised through a combination of instructor and visual display (VR)-driven feedback. The results have implications for the utility and implementation of VR haptic technology in dental education.

Should prospective dental students be screened for colour vision deficits?

A perennial question in dental education is: what skills and aptitudes should be assessed in prospective dental students? Intellectual capacity and manual dexterity understandably rank highly, but are there minimum thresholds for visual perception that applicants need be able to demonstrate before they enter the profession? We have recently flagged this issue with regard to the thresholds of stereoscopic acuity required for a dentist when operating on teeth. In the present article, we highlight the issue of identifying a minimum acceptable level of colour vision.

Subscapularis and deltoid preserving anterior approach for reverse shoulder arthroplasty.

HYPOTHESIS: We hypothesize that performing a RSA using an anterior approach without cutting the subscapularis tendon and the deltoid muscle could provide patients with superior short-term clinical outcomes and immediate active range of motion (ROM) without immobilization. METHODS: Between August 2013 and June 2015, all patients who had a primary RSA were considered potentially eligible for inclusion in this prospective study. RESULTS: No immediate intra- or postoperative complications were noted. A statistically significant improvement of VAS (from 6.7 to 1; P<.001), SANE (from 34 to 80; P<.001), and elevation (from 103° to 128°; P=.02) was observed. In some cases, patients who had pseudoparalysis preoperative were able to achieve full anterior elevation few days after the operation. DISCUSSION: Using a subscapularis and deltoid preserving anterior approach is an option for patients requiring RSA. Leaving this tendon intact and preserving the deltoid minimize the need for immediate postoperative immobilization and allow for faster recovery of shoulder ROM, without risking the concern of humeral anterior dislocation. Overall duration of hospital stay as well as length of postoperative physical therapy may be minimized, with substantial long-term economic gain. Longer follow-up and comparison with standard approaches is necessary in the future. LEVEL OF EVIDENCE OF THE STUDY: Level IV, case series with no comparative group.

Eliminating the dose-rate effect in a radiochromic silicone-based 3D dosimeter.

Comprehensive dose verification, such as 3D dosimetry, may be required for safe introduction and use of advanced treatment modalities in radiotherapy. A radiochromic silicone-based 3D dosimetry system has recently been suggested, though its clinical use has so far been limited by a considerable dose-rate dependency of the dose response. In this study we have investigated the dose-rate dependency with respect to the chemical composition of the dosimeter. We found that this dependency was reduced with increasing dye concentration, and the dose response was observed to be identical for dosimeters irradiated with 2 and 6 Gy min(-1) at concentrations of 0.26% (w/w) dye and 1% (w/w) dye solvent. Furthermore, for the optimized dosimeter formulation, no dose-rate effect was observed due to the attenuation of the beam fluence with depth. However, the temporal stability of the dose response decreased with dye concentration; the response was reduced by (62 ± 1)% within approximately 20 h upon irradiation, at the optimal chemical composition and storage at room temperature. In conclusion, this study presents a chemical composition for a dose-rate independent silicone dosimeter which has considerably improved the clinical applicability of such dosimeters, but at the cost of a decreased stability.

Monovalent maleimide functionalization of gold nanoparticles via copper-free click chemistry.

A single maleimide was installed onto the self-assembled monolayer of gold nanoparticles by copper-free click chemistry. Simple covalent biofunctionalisation is demonstrated by coupling fibroblast growth factor 2 and an oligosaccharide in a 1 : 1 stoichiometry by thiol-Michael addition.

The influence of contemporary and historic landscape features on the genetic structure of the sand dune endemic, Cirsium pitcheri (Asteraceae).

Narrow endemics are at risk from climate change because of their restricted habitat preferences, lower colonization ability and dispersal distances. Landscape genetics combines new tools and analyses that allow us to test how both past and present landscape features have facilitated or hindered previous range expansion and local migration patterns, and thereby identifying potential limitations to future range shifts. We have compared current and historic habitat corridors in Cirsium pitcheri, an endemic of the linear dune ecosystem of the Great Lakes, to determine the relative contributions of contemporary migration and post-glacial range expansion on genetic structure. We used seven microsatellite loci to characterize the genetic structure for 24 populations of Cirsium pitcheri, spanning the center to periphery of the range. We tested genetic distance against different measures of geographic distance and landscape permeability, based on contemporary and historic landscape features. We found moderate genetic structure (Fst=0.14), and a north-south pattern to the distribution of genetic diversity and inbreeding, with northern populations having the highest diversity and lowest levels of inbreeding. High allelic diversity, small average pairwise distances and mixed genetic clusters identified in Structure suggest that populations in the center of the range represent the point of entry to the Lake Michigan and a refugium of diversity for this species. A strong association between genetic distances and lake-level changes suggests that historic lake fluctuations best explain the broad geographic patterns, and sandy habitat best explains local patterns of movement.

Heparan sulphate, its derivatives and analogues share structural characteristics that can be exploited, particularly in inhibiting microbial attachment

Heparan sulphate (HS) and the related polysaccharide, heparin, exhibit conformational and charge arrangement properties, which provide a degree of redundancy allowing several seemingly distinct sequences to exhibit the same activity. This can also be mimicked by other sulphated polysaccharides, both in overall effect and in the details of interactions and structural consequences of interactions with proteins. Together, these provide a source of active compounds suitable for further development as potential drugs. These polysaccharides also possess considerable size, which bestows upon them an additional useful property: the capability of disrupting processes comprising many individual interactions, such as those characterising the attachment of microbial pathogens to host cells. The range of involvement of HS in microbial attachment is reviewed and examples, which include viral, bacterial and parasitic infections and which, in many cases, are now being investigated as potential targets for intervention, are identified.