In-cell NMR: from metabolites to macromolecules.

In-cell NMR of macromolecules has gained momentum over the last ten years as an approach that might bridge the branches of cell biology and structural biology. In this review, we put it in the context of earlier efforts that aimed to characterize by NMR the cellular environment of live cells and their intracellular metabolites. Although technical aspects distinguish these earlier in vivo NMR studies and the more recent in cell NMR efforts to characterize macromolecules in a cellular environment, we believe that both share major concerns ranging from sensitivity and line broadening to cell viability. Approaches to overcome the limitations in one subfield thereby can serve the other one and vice versa. The relevance in biomedical sciences might stretch from the direct following of drug metabolism in the cell to the observation of target binding, and thereby encompasses in-cell NMR both of metabolites and macromolecules. We underline the efforts of the field to move to novel biological insights by some selected examples.

Infectious disease models in zebrafish.

In recent years, the zebrafish (Danio rerio) has developed as an important alternative to mammalian models for the study of hostpathogen interactions. Because they lack a functional adaptive immune response during the first 4-6weeks of development, zebrafish rely upon innate immune responses to protect against injuries and infections. During this early period of development, it is possible to isolate and study mechanisms of infection and inflammation arising from the innate immune response without the complications presented by the adaptive immune response. Zebrafish possess several inherent characteristics that make them an attractive option to study hostpathogen interactions, including extensive sequence and functional conservation with the human genome, optical clarity in larvae that facilitates the high-resolution visualization of host cell-microbe interactions, a fully sequenced and annotated genome, robust forward and reverse genetic tools and techniques (e.g., CRISPR-Cas9 and TALENs), and amenability to chemical studies and screens. Here, we describe methods for studying hostpathogen interactions both through systemic infections and through localized infections that allow analysis of host cell response, migration patterns, and behavior. Each of the methods described can be modified for use in downstream applications that include ecotoxicant studies and chemical screens.

A DN-mda5 transgenic zebrafish model demonstrates that Mda5 plays an important role in snakehead rhabdovirus resistance.

Melanoma Differentiation-Associated protein 5 (MDA5) is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family, which is a cytosolic pattern recognition receptor that detects viral nucleic acids. Here we show an Mda5-dependent response to rhabdovirus infection in vivo using a dominant-negative mda5 transgenic zebrafish. Dominant-negative mda5 zebrafish embryos displayed an impaired antiviral immune response compared to wild-type counterparts that can be rescued by recombinant full-length Mda5. To our knowledge, we have generated the first dominant-negative mda5 transgenic zebrafish and demonstrated a critical role for Mda5 in the antiviral response to rhabdovirus.

Rapid, minimally invasive adult voluntary male circumcision: A randomised trial.

BACKGROUND: Voluntary medical male circumcision (VMMC) is a priority HIV preventive intervention. To facilitate male circumcision scale- up, the World Health Organization is actively seeking circumcision techniques that are quicker, easier, and safer than open surgical methods. OBJECTIVE: To compare conventional open surgical circumcision with suturing with a minimally invasive technique using the Gomco circumcision clamp plus tissue adhesive. METHODS: We conducted a non-blinded randomised controlled trial comprising 200 male volunteers >18 years of age, seen at the outpatient university teaching clinic of the Catholic University of Mozambique. We compared two interventions - open surgical circumcision with suturing v. Gomco instrument plus tissue adhesive. Our primary outcome was intraoperative time and our secondary outcomes included: ease of performance, post-operative pain, adverse events, time to healing, patient satisfaction and cosmetic result. RESULTS: The intraoperative time was less with the Gomco/tissue adhesive technique (mean 12.8 min v. 22.5 min; p<0.001). Adverse events were similar except that wound disruption was greater in the Gomco/tissue adhesive group, with no difference in wound healing at 4 weeks. Levels of satisfaction were high in both groups. The cosmetic result was superior in the Gomco/tissue adhesive group. CONCLUSIONS: This study has important implications for the scale-up of VMMC services. Removing the foreskin with the Gomco instrument and sealing the wound with cyanoacrylate tissue adhesive in adults is quicker, is an easier technique to learn, and is potentially safer than open surgical VMMC. A disposable plastic, Gomco-like device should be produced and evaluated for use in resource-limited settings.

Simulated browsing affects leaf shedding phenology and litter quality of oak and birch saplings.

Herbivore effects on leaf litter can have a strong impact on ecosystem nutrient cycling. Although such effects are well described for insect herbivory, research on the impacts of browsing by mammalian herbivores on leaf litter dynamics and nutrient cycling has been more limited, particularly at the level of the individual plant. Clipping treatments (66% shoot removal twice, plus unclipped) were applied to analyse the effect of browsing on the phenology (start date and pattern of leaf shedding) and leaf litter quality (nitrogen (N), soluble sugars, starch and total non-structural carbohydrate concentrations, plus C : N ratios) of Betula pubescens Ehrh. and Quercus petraea [Matt.] Liebl. saplings. Clipping decreased leaf litter biomass and delayed leaf senescence and shedding, but did not change the phenological timing of litterfall between senescence and shedding. The quality of leaf litter of both species was increased by simulated browsing, through an increase in N and carbohydrate concentrations (mainly soluble sugars) and a decreased C : N ratio. This is the first evidence we are aware of that browsing may cause changes in leaf shedding phenology, delaying the process without altering its pattern. Our results also indicate that simulated browsing increases the quality of leaf litter. However, the potential positive effect of browsing on N cycling through litter quality may be offset by its negative impact on the amount of N shed per tree.

Ground-level ozone differentially affects nitrogen acquisition and allocation in mature European beech (Fagus sylvatica) and Norway spruce (Picea abies) trees.

Impacts of elevated ground-level ozone (O(3)) on nitrogen (N) uptake and allocation were studied on mature European beech (Fagus sylvatica L.) and Norway spruce (Picea abies [L.] Karst.) in a forest stand, hypothesizing that: (i) chronically elevated O(3) limits nutrient uptake, and (ii) beech responds more sensitively to elevated O(3) than spruce, as previously found for juvenile trees. Tree canopies were exposed to twice-ambient O(3) concentrations (2 × O(3)) by a free-air fumigation system, with trees under ambient O(3) serving as control. After 5 years of O(3) fumigation, (15)NH(4)(15)NO(3) was applied to soil, and concentrations of newly acquired N (N(labelled)) and total N (N(total)) in plant compartments and soil measured. Under 2 × O(3), N(labelled) and N(total) were increased in the bulk soil and tended to be lower in fine and coarse roots of both species across the soil horizons, supporting hypothesis (i). N(labelled) was reduced in beech foliage by up to 60%, and by up to 50% in buds under 2 × O(3). Similarly, N(labelled) in stem bark and phloem was reduced. No such reduction was observed in spruce, reflecting a stronger effect on N acquisition in beech in accordance with hypothesis (ii). In spruce, 2 × O(3) tended to favour allocation of new N to foliage. N(labelled) in beech foliage correlated with cumulative seasonal transpiration, indicating impaired N acquisition was probably caused by reduced stomatal conductance and, hence, water transport under elevated O(3). Stimulated fine root growth under 2 × O(3) with a possible increase of below-ground N sink strength may also have accounted for lowered N allocation to above-ground organs. Reduced N uptake and altered allocation may enhance the use of stored N for growth, possibly affecting long-term stand nutrition.

Browsing affects intra-ring carbon allocation in species with contrasting wood anatomy.

Current knowledge on tree carbon (C) allocation to wood is particularly scarce in plants subjected to disturbance factors, such as browsing, which affects forest regeneration worldwide and has an impact on the C balance of trees. Furthermore, quantifying the degree to which tree rings are formed from freshly assimilated vs. stored carbohydrates is highly relevant for our understanding of tree C allocation. We used (13)C labelling to quantify seasonal allocation of stored C to wood formation in two species with contrasting wood anatomy: Betula pubescens Ehrh. (diffuse-porous) and Quercus petraea [Matt.] Liebl. (ring-porous). Clipping treatments (66% shoot removal, and unclipped) were applied to analyse the effect of browsing on C allocation into tree rings, plus the effects on tree growth, architecture, ring width and non-structural carbohydrates (NSCs). The relative contribution of stored C to wood formation was greater in the ring-porous (55-70%) than in the diffuse-porous species (35-60%), although each species followed different seasonal trends. Clipping did not cause a significant depletion of C stores in either species. Nonetheless, a significant increase in the proportion of stored C allocated to earlywood growth was observed in clipped birches, and this could be explained through changes in tree architecture after clipping. The size of C pools across tree species seems to be important in determining the variability of seasonal C allocation patterns to wood and their sensibility to disturbances such as browsing. Our results indicate that the observed changes in C allocation to earlywood in birch were not related to variations in the amount or concentration of NSC stores, but to changes in the seasonal availability of recently assimilated C caused by modifications in tree architecture after browsing.

Why are there local shortfalls in anaesthesia consultant staffing? A case study of operational workforce planning.

PURPOSE: Anecdotally, many hospitals experience shortfalls in anaesthetic consultant staffing. This paper aims to investigate whether these subjective experiences are confirmed objectively. DESIGN/METHODOLOGY/APPROACH: The paper hypothesises that a simple model that estimated service delivery capability using consultant entitlements to annual and other types of leave would not (null hypothesis) accurately predict the magnitude of any shortfall that existed. It also hypothesises that excessive leave-taking was an important cause of any shortfall. A comparison is made between the model predictions for total leave taken and service delivery with results from a real data set from a large university teaching hospital's department of anaesthetics. FINDINGS: The model prediction for leave (median total 45 days absence in a year per consultant, range (30-59)) closely matched the reality (median 41 days (tenth-ninetieth deciles 30-69)). Consequently, both model predictions and the real data for annual elective service delivery agreed: median 228 sessions (193-266) vs 232 (183-266) per consultant respectively. Taking into account likely service delivery by trainees (2,304-4,140 elective sessions in total annually) the predicted shortfall of 2,220 sessions was very close to the true elective service shortfall of 2,148 sessions for the department as a whole over the year. PRACTICAL IMPLICATIONS: Rejecting the null hypothesis, it is concluded that a simple model that estimates elective service delivery using leave entitlements as the main factor can accurately predict actual service capability for a department. There is no evidence that excessive leave-taking occurs. ORIGINALITY/VALUE: The paper computes an estimate that 2.2-2.6 consultants per functional operating theatre are necessary to ensure that staffing matches the elective workload.

Complex coacervate core micro-emulsions.

Complex coacervate core micelles form in aqueous solutions from poly(acrylic acid)-block-poly(acrylamide) (PAAxPAAmy, x and y denote degree of polymerization) and poly(N,N-dimethyl aminoethyl methacrylate) (PDMAEMA150) around the stoichiometric charge ratio of the two components. The hydrodynamic radius, Rh, can be increased by adding oppositely charged homopolyelectrolytes, PAA140 and PDMAEMA150, at the stoichiometric charge ratio. Mixing the components in NaNO3 gives particles in highly aggregated metastable states, whose Rh remain unchanged (less than 5% deviation) for at least 1 month. The Rh increases more strongly with increasing addition of oppositely charged homopolyelectrolytes than is predicted by a geometrical packing model, which relates surface and volume of the particles. Preparation in a phosphate buffer - known to weaken the electrostatic interactions between PAA and PDMAEMA - yields swollen particles called complex coacervate core micro-emulsions (C3-µEs) whose Rh increase is close to that predicted by the model. These are believed to be in the stable state (lowest free energy). A two-regime increase in Rh is observed, which is attributed to a transition from more star-like to crew-cut-like, as shown by self-consistent field calculations. Varying the length of the neutral and polyelectrolyte block in electrophoretic mobility measurements shows that for long neutral blocks (PAA26PAAm405 and PAA39PAAm381) the ζ-potential is nearly zero. For shorter neutral blocks the ζ-potential is around -10 mV. This shows that the C3-µEs have excess charge, which can be almost completely screened by long enough neutral blocks.

Five-year safety and efficacy of a novel polymethylmethacrylate aesthetic soft tissue filler for the correction of nasolabial folds.

BACKGROUND: A novel soft tissue filler composed of polymethylmethacrylate (PMMA) microspheres suspended in a collagen gel matrix containing 0.3% lidocaine (ArteFill, Artes Medical, Inc.) was recently approved by the Food and Drug Administration for the correction of nasolabial folds. A randomized, multicenter, controlled pivotal trial performed in the United States established the safety and efficacy of this medical device throughout a 12-month study period. OBJECTIVE: The objective was to substantiate the long-term 5-year safety and efficacy of this novel soft tissue PMMA filler. METHODS AND MATERIALS: Attempts were made to contact all subjects treated with the PMMA filler that were enrolled in the original pivotal study. Safety was assessed by standard adverse event reporting methods. Efficacy was determined using a validated six-point facial fold assessment photometric grading scale using blinded observers' assessment of standardized photographs. RESULTS: Subjects (n=119) demonstrated significant improvement in nasolabial folds comparing baseline (before any treatment) to 5 years after their last treatment (p<.001). Notably, subjects also demonstrated continued improvement between 6 months after their last treatment and Year 5 (p=.002). No serious unanticipated device-related adverse events were reported. CONCLUSION: This PMMA filler is the first soft tissue filler to demonstrate continued improvement and persistence of correction over a 5-year period posttreatment.

Non-structural carbohydrates and nitrogen dynamics in mediterranean sub-shrubs: an analysis of the functional role of overwintering leaves.

Previous studies have led to contrasting results about the role of overwintering leaves as storage sites, which is related to leaf longevity and life-form. The aim of this study was to evaluate the functional role of the leaves of four species of Mediterranean sub-shrubs, with different leaf phenology, as sources of nitrogen (N) and non-structural carbohydrates (NSC) for shoot growth. The seasonal dynamics of the concentrations and pools of N and NSC were assessed monthly in the leaves and woody organs of each species. Overwintering and spring leaves served as N and NSC sources for shoot growth in the evergreen species analyzed, providing up to 73 % and 324 % of the N demand for spring and autumn growth, respectively. Excess autumn N was stored in woody structures which contributed to the N and NSC requirements of spring growth. In the winter deciduous species, woody organs were the main N source for spring growth, while current photosynthesis from immature brachyblasts seemed to be the main carbon (C) source. Due to their short lifespan, overwintering and spring leaves did not show several translocation processes throughout their life time, their contribution to new growth being made during senescence. The successive exchange of leaf cohorts displayed by Mediterranean sub-shrubs might serve as a mechanism to recycle N and C between consecutive cohorts as plants perform the pheno-morphological changes needed to adapt their morphology to the seasonality of their environment.

A closed queueing network approach to the analysis of patient flow in health care systems.

OBJECTIVES: To model patient flow in health care systems with bed capacity constraints in order to provide a useful decision aid for health service managers. METHODS: We model the patient flow of health care systems using a closed queueing network framework with the assumption that the system is always full. Key performance measures of the health care system are also derived. RESULTS: Using parameters taken from a study of a geriatric department in the UK, we show that the model is useful in helping service managers to gain better understanding of the behaviour of the system. In addition, we demonstrate that the model could help improving decision-making by allowing managers to explore different options and evaluate their impacts on performance. Our findings highlight the importance of policy makers taking into account the interactions between different phases of care. CONCLUSIONS: We have developed a novel approach to modelling the flow of patients through health care systems with constrained bed capacity.

Cytokine alterations in lichen sclerosus: an immunohistochemical study.

BACKGROUND: Although the histology of lichen sclerosus is characteristic, the precise nature of the inflammatory changes and the signals provoking them is uncertain. OBJECTIVES: To delineate the inflammatory changes in lichen sclerosus more accurately by studying cytokine changes. METHODS: An immunohistochemical study of 12 specimens of genital lichen sclerosus and one specimen of extragenital lichen sclerosus was undertaken using monoclonal antibodies to interferon (IFN)-gamma, IFN-gamma receptor, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-2 receptor (CD25), intercellular adhesion molecule-1 (ICAM-1) and its ligand CD11a. Control specimens were seven specimens of normal vulva obtained during gynaecological procedures, three specimens of normal skin, adjacent uninvolved thigh from three of the patients with lichen sclerosus, five specimens of nonvulval psoriasis, four specimens of nonvulval lichen planus and two specimens from chronic wounds. RESULTS: The lichen sclerosus specimens demonstrated slightly increased staining for IFN-gamma within the epidermis compared with the normal vulva and nonvulval skin. There was increased dermal staining for IFN-gamma both within the pale zone of the upper dermis and within the inflammatory zone below this. We confirmed our previous demonstration that in lichen sclerosus HLA-DR immunostaining is increased in association with vascular endothelium, the inflammatory cell infiltrate and around the keratinocytes. The areas of the epidermis with the strongest immunostaining for HLA-DR generally also had the strongest staining for IFN-gamma. In the lichen sclerosus specimens the zone of inflammation also demonstrated increased immunostaining for TNF-alpha, IL-1alpha, IFN-gamma receptor, CD25, CD11a and ICAM-1 while the zone of sclerosus demonstrated a smaller increase in immunostaining for IFN-gamma receptor, TNF-alpha, CD11a and ICAM-1, and the epidermis demonstrated increased staining for ICAM-1. CONCLUSIONS: The increased staining for IFN-gamma, TNF-alpha, IL-1alpha, IFN-gamma receptor, CD25, CD11a and ICAM-1 suggest that the cytokine response in lichen sclerosus shares characteristics of the cytokine response in lichen planus and chronic wounds.

ArteFill: a long-lasting injectable wrinkle filler material--summary of the U.S. Food and Drug Administration trials and a progress report on 4- to 5-year outcomes.

ArteFill, the successor product to Artecoll, is an injectable wrinkle filler composed of polymethylmethacrylate microspheres and bovine collagen, which offers long-lasting and probably permanent augmentation of wrinkles and skin contour deformities. The pivotal U.S. Food and Drug Administration study consisted of 251 subjects at eight centers in the United States who received injections of ArteFill or bovine collagen dermal filler (control) in 1334 wrinkles of the glabella, nasolabial folds, radial upper lip lines, and corners of the mouth. The efficacy data generated by masked observers using a photographic Facial Fold Assessment Scale demonstrated a significant improvement with ArteFill compared with collagen at 6 months (p < 0.001) in the nasolabial folds. In the ArteFill group, 12-month follow-up was obtained for 111 subjects (86.7 percent) and showed persistence of significant wrinkle correction. A subgroup of 69 patients who received ArteFill were recalled 4 to 5 years later. Five patients reported six late adverse events that occurred from 2 to 5 years after the initial injection; four of the adverse events were mild cases of lumpiness and two were severe. The total number of late adverse events was six of 272 (2.2 percent) wrinkles injected. Among the 272 wrinkles evaluated at 5 years, two events (0.7 percent) in one patient were rated as severe (a nodular, minimally inflammatory to noninflammatory reaction in both nasolabial folds). Investigator Facial Fold Assessment ratings at 4 to 5 years were improved from baseline by 1.67 points (p < 0.001).

Detection of infectious haematopoietic necrosis virus and infectious salmon anaemia virus by molecular padlock amplification.

A new method for the molecular detection of the fish pathogens, infectious haematopoietic necrosis virus (IHNV) and infectious salmon anaemia virus (ISAV), is described. By employing molecular padlock probe (MPP) technology combined with rolling circle amplification (RCA) and hyperbranching (Hbr), it is possible to detect RNA target sequence from these viruses at levels comparable with those detected by the polymerase chain reaction (PCR), but without prior reverse transcription. The use of MPP technology combined with RCA and Hbr for the detection of IHNV and ISAV in fish exhibited selectivity comparable with that of PCR while potentially reducing the time and cost required for analysis. The method described was used to detect as few as 10(4) DNA oligonucleotide targets and was sequence-specific at the single base level. Viral RNA could be detected directly, either alone or in the presence of non-viral RNA from fish tissue. This technology is applicable for detecting a variety of microbes, in addition to IHNV and ISAV, and is ideal for further integration into a biosensor platform for on-site diagnosis of pathogen infection in fish.

Detection of Escherichia coli O157:H7 with langasite pure shear horizontal surface acoustic wave sensors.

The toxigenic Escherichia coli O157:H7 bacterium has been connected with hemorrhagic colitis and hemolytic uremic syndrome, which may be characterized by diarrhea, kidney failure and death. On average, O157:H7 causes 73,000 illnesses, 2100 hospitalizations and 60 deaths annually in the United States alone. There is the need for sensors capable of rapidly detecting dangerous microbes in food and water supplies to limit the exposure of human and animal populations. Previous work by the authors used shear horizontal surface acoustic wave (SH SAW) devices fabricated on langasite (LGS) Euler angles (0 degrees, 22 degrees, 90 degrees) to successfully detect macromolecular protein assemblies. The devices also demonstrated favorable temperature stability, biocompatibility and low attenuation in liquid environments, suggesting their applicability to bacterial detection. In this paper, a biosensor test setup utilizing a small volume fluid injection system, stable temperature control and high frequency phase measurement was applied to validate LGS SH SAW biosensors for bacterial detection. The LGS SH SAW delay lines were fabricated and derivatized with a rabbit polyclonal IgG antibody, which selectively binds to E. coli O157:H7, in this case a non-toxigenic test strain. To quantify the effect of non-specific binding (negative control), an antibody directed against the trinitrophenyl hapten (TNP) was used as a binding layer. Test E. coli bacteria were cultured, fixed with formaldehyde, stained with cell-permeant nucleic acid stain, suspended in phosphate buffered saline and applied to the antibody-coated sensing surfaces. The biosensor transmission coefficient phase was monitored using a network analyzer. Phase responses of about 14 degrees were measured for the E. coli detection, as compared to 2 degrees due to non-specific anti-TNP binding. A 30:1 preference for E. coli binding to the anti-O157:H7 layer when compared to the anti-TNP layer was observed with fluorescence microscopy, thus confirming the selectivity of the antibody surface to E. coli.

Do competition and herbivory alter the internal nitrogen dynamics of birch saplings?

Deciduous trees recycle nitrogen within their tissues. The aim of this study was to test the hypothesis that reductions in plant growth, caused by competition and herbivory, reduce the sink strength for N during autumn nutrient withdrawal, and reduce the storage capacity and hence the amount of N remobilized in the following spring. We used (15)N-labelled fertilizer to quantify N uptake, leaf N withdrawal and remobilization. Betula pubescens saplings were grown with either Molinia caerulea or Calluna vulgaris, and subjected to simulated browsing damage. Competition reduced B. pubescens leaf N withdrawal and remobilization, with C. vulgaris having a greater effect than M. caerulea. However, simulated browsing had no significant effect on sapling N dynamics. The patterns of leaf N withdrawal and remobilization closely followed sapling dry mass. We conclude that the effect of competition on sapling mass reduces their N-storage capacity. This reduces sink strength for leaf N withdrawal and the source strength for remobilized N. The ability of saplings to compensate for browsing damage removed any potential effect of browsing on N dynamics.