Background: The adverse relation between dietary trans fatty acid (TFA) intake and coronary artery disease risk is well established. Many countries in the European Union (EU) and worldwide have implemented different policies to reduce the TFA intake of their populations.Objective: The aim of this study was to assess the added value of EU-level action by estimating the cost-effectiveness of 3 possible EU-level policy measures to reduce population dietary TFA intake. This was calculated against a reference situation of not implementing any EU-level policy .Design: We developed a mathematical model to compare different policy options at the EU level: 1) to do nothing beyond the current state (reference situation), 2) to impose mandatory TFA labeling of prepackaged foods, 3) to seek voluntary agreements toward further reducing industrially produced TFA (iTFA) content in foods, and 4) to impose a legislative limit for iTFA content in foods.Results: The model indicated that to impose an EU-level legal limit or to make voluntary agreements may, over the course of a lifetime (85 y), avoid the loss of 3.73 and 2.19 million disability-adjusted life-years (DALYs), respectively, and save >51 and 23 billion euros when compared with the reference situation. Implementing mandatory TFA labeling can also avoid the loss of 0.98 million DALYs, but this option incurs more costs than it saves compared with the reference option.Conclusions: The model indicates that there is added value of an EU-level action, either via a legal limit or through voluntary agreements, with the legal limit option producing the highest additional health benefits. Introducing mandatory TFA labeling for the EU common market may provide some additional health benefits; however, this would likely not be a cost-effective strategy. TFA intake can be of industrial  or natural (ruminant food sources) origin  origin . The det) origin  and are ) origin –5. Other) origin , 6. Most) origin . Many co) origin ; these a) origin .In the European Union (EU), dietary TFA intake has been decreasing since the 1980–1990s, from as high as 4.3 E% in elderly Dutch men in 1985 , 11. The−1 · y−1. Another study suggests that introducing a legal limit on iTFAs in England would prevent ∼7200 deaths from CAD  between 2015 and 2020, providing the greatest health benefits and reduction in the inequality gap when compared with improved TFA labeling or TFA removal from restaurants and fast foods –related events and deaths as well as cost savings] –18. Restst foods , 22, thiWe developed a computer-simulated, Markov, state-transition model with the use of Excel (Microsoft Office 2010). This type of model is appropriate because Markov models are suitable for changing systems . In this case, the different states are the conditions in which an individual can be, such as “well,” with “CAD” or “history of CAD,” or “dead” see , 24.Supplemental Tables 1–3). The model was applied to the EU population and accounts for all costs and effects applicable or resulting from the following policy options over the course of a lifetime (85 y) (1) Reference situation (no action at the EU level): The reference situation is described by the highest cumulative TFA intake  in all of the 4 options, and therefore it also entails the highest risk of CAD. Nevertheless, even for this case of “no action at EU level,” in the model we assume a continued decrease in TFA consumption that leads to a removal of iTFAs from the food supply over 10 y due to continuous innovation in the industry and efforts at the national or regional levels. In terms of costs, there are no added public costs from implementing this policy option; all costs result from CAD-associated morbidity and loss of productivity.2) Voluntary agreements: With this option policy makers actively seek agreements at the EU level, such as with the food industry and retailers to introduce measures that reduce TFA amounts in foods and/or between EU member states, to agree on a common framework toward reducing TFAs in foods and diets similarly to the EU salt reduction framework (ramework . In this3) Mandatory TFA labeling: With this strategy the existing rules for the nutrition declaration on foods as governed by EU regulation 1169/2011 would be changed to require the disclosure of the TFA contents in all prepackaged foods. This provides an incentive for food reformulation toward reducing or replacing iTFAs, but only for prepackaged foods. Because this option requires legislative action, in addition to CAD-associated public costs, other non–CAD-related public costs are also considered. These are linked to the implementation of the legislation (mass media costs), worksite interventions, consumer education, and nutrition counseling as well as food inspection (4) Legal limit of iTFA content in foods: This option sees a restriction in the use of iTFAs in the food supply through a legislative limit, such as that already introduced by some EU member states . This measure results in a fast removal of iTFAs in all of the EU food supply and represents therefore the lowest cumulative TFA consumption of all 4 options. The model assumes the total removal of iTFAs in 2 y. This strategy implies, in addition to CAD-associated public costs, other costs that are not associated with CAD such as public costs for food inspection programs.The TFA intake, defined as E%, as a starting point for the model (“today”) was calculated as described in e (85 y) :1) ReferThe model simulates how people are moving in yearly cycles through 4 health states, as shown in Health care costs: These costs stem from the use of health resources . The costs are based on the European Cardiovascular Disease Statistics 2012 .Non–health care costs: This group of costs includes all non–health care costs related to the disease, namely loss of productivity and informal care. The costs are based on the European Cardiovascular Disease Statistics 2012 .Costs of policy-associated measures: Each policy option (apart from option 1 “reference situation”) incurs costs related to the execution of measures needed for their successful implementation. All of the costs considered to account for the burden of TFAs have been adjusted for inflation to 2011 prices (in €); currency exchanges, when applicable, were calculated on the basis of 1 January 2011 exchange rates to the euro. The model considers 3 types of costs  School-based interventions costs: These include training teachers and food service staff and additional curricular activities, but exclude changes in food provision services.2) Worksite interventions costs: These include costs from activities subsidized by the public sector and held in worksites by employers.3) Mass media campaigns costs: These include broadcasting advertisements on national and local radio and television channels and for designing, producing, and distributing flyers and leaflets.4) Physician counseling costs: These costs include counseling provided by physicians to targeted individuals.5) Program of food inspection costs: These include the administration, planning, enforcement, and resources needed to manage food inspection.The following types of costs were considered see also . The firThe model calculates, for each option, CAD events and mortality in yearly cycles over a period of 85 y. It is based on current estimates of iTFA intake  was used, including the weightings as reported in the Global Burden of Disease 2010 study  .The model also includes the probabilities of having a CAD event for the first time, of having another CAD event after the first one, of death at any time and of death because of a CAD event .Scenario 2 assumes an initial overall average iTFA intake of 0.45 E% (assuming that the situation in countries where no estimates were identified is somewhat worse than in our estimate).Supplemental Table 4 and after subtracting an estimated 0.5 E% contribution from ruminant TFAs :Population iTFA intakes in the 3 scenarios diminish in a similar manner as the base case in each of the 4 policy options. A summary of the initial iTFA population intakes for each scenario is provided in In addition, this economic evaluation includes, next to the deterministic analysis, which uses a single value for costs and for effects in the model calculations, a probabilistic sensitivity analysis (PSA). The PSA applies probabilistic distributions to every variable in the model. Each of the distributions used in the PSA were chosen following the current trends and literature recommendations  . These pThe resulting health effects and costs linked to each of the 4 policy options from the deterministic base case analysis are presented in The results shown in To compare the policy options, the difference in costs and DALYs of policy options 2–4 compared with the reference situation (option 1) were calculated and are presented in A policy option is considered dominant if it can save both costs and DALYs when compared with the reference situation. This is the case for the legal TFA limits and voluntary agreements policy options for the base case and in every scenario considered for our model . The legBecause of the uncertainty associated with the wide distribution of some of the model variable values and data  compared with not taking action at the EU level. Note also that although our analysis is focused on the EU market, it is likely that any action at the EU level would also affect the presence of iTFAs in foods and iTFA intake in other non-EU countries.Despite a variety of uncertainties associated with some of the data included in the model, the PSA suggests that these results are robust because both policy options are dominant (saving costs and DALYs) in 100% of the trials in the PSA and the CIs are quite narrow. The same occurred for every scenario tested, except for the voluntary agreements in scenario 1, which assumed the lowest initial population iTFA intake of only 0.15 E% (90.6% probability). Although important, the cost-effectiveness of a particular policy option is not the only variable to be considered by policy makers to implement new policies. For example, our model focused on public expenditure and did not contemplate any potential costs incurred by the industry or other players when limiting iTFA content in foods. This is common practice in public health economic evaluations . In addiAlthough our model applied a lifetime horizon (85 y) for calculating health effects and costs, others used shorter time horizons –18. The Introducing mandatory TFA labeling at the EU level, regardless of the scenario analyzed, also has the potential to avert DALYs but not costs. In general, the differences in costs between the different policy options are driven by the costs related to the incidence of CAD. However, in cases in which the difference in CAD incidence between an alternative option and the reference situation is low, such as the case for mandatory TFA labeling, the leading costs then stem from the costs of the measures .With regard to the differences in DALYs, these are mostly due to the number of CAD events and related premature deaths, as well as the number of years living with disability and the number of years lost because of premature death. Small resulting differences in CAD are particularly likely in scenario 1, in which the lowest initial iTFA intakes are assumed.In this study, we assumed a rather rapid removal of iTFAs from the EU food supply over 10 y, including in the reference situation in which no EU-level action is taken . This rather optimistic assumption for the reference situation means that we used a conservative approach in our conclusions on the EU-level action policy options. Health benefits would be even larger and cost savings more likely should iTFA intakes decrease more slowly than what is assumed in the reference situation in which no EU-level action is taken.As discussed previously, some of the model variables or input data used here are rather uncertain and several assumptions had to be made. There are 3 major sources of potential errors: the estimated current TFA intake, the wide variability observed for many variables between countries, and the lack of data in some instances, such as the lack of data on the number of CAD events per year . To address the concerns related to the accuracy of estimated iTFA intake values entered in the model, we used different scenarios with different values for initial iTFA intake. In all of the scenarios and for all policy options it was assumed that, with time, iTFA intakes will eventually decrease to 0 E%. This seems reasonable given the current estimates of iTFA intakes of ∼0.01 E% for Denmark, where a legal limit on iTFA contents of foods has been in place for 10 y (see Supplemental Tables 1 and 2 and reference The results presented here should be interpreted as a comparison between different policy options rather than considering absolute costs, DALYs, or deaths per option. Previous work has shown the benefits of TFA intake reduction . RecentlIn conclusion, the results of this study suggest additional health benefits and reductions in public spending when taking EU-level action toward reducing population iTFA intakes. Although now introducing a mandatory TFA labeling scheme may not be a cost-effective solution in the EU, both a legal limit on the iTFA content in foods and voluntary agreements toward removing PHOs from foods produce large-enough reductions in CAD morbidity and mortality that the related reductions in costs outweigh the costs linked to the implementation of these strategies. Finally, introducing a legal limit at the EU level would produce the greatest health benefits of all of the options included in this study.


A symmetric full cell using such electrodes demonstrates a stable potential window of 3.0 V in an aqueous electrolyte, as well as high energy and power performance, nearly 100% coulombic efficiency and 85% energy efficiency after 25,000 charge–discharge cycles. The interplay between hydroxylated interphase on the surface and the unique bivalence structure of Mn5O8 suppresses the gas evolution reactions, offers a two-electron charge transfer via Mn2+/Mn4+ redox couple, and provides facile pathway for Na-ion transport via intra-/inter-layer defects of Mn5O8.Aqueous electrochemical energy storage devices have attracted significant attention owing to their high safety, low cost and environmental friendliness. However, their applications have been limited by a narrow potential window (∼1.23 V), beyond which the hydrogen and oxygen evolution reactions occur. Here we report the formation of layered Mn 5O8 pseudocapacitor electrode with bivalence structure that expands the potential window to deliver high energy and power performance.Rechargeable aqueous electrochemical energy storage is a promising technology but suffers from a narrow potential window. Here the authors report a surface hydroxylated Mn Rechargeable aqueous electrochemical energy storage (EES) devices, especially ones using earth-abundant and non-toxic materials, have shown great promise for many applications owing to their high safety, low cost and environmental friendlinesset al. reported a water-in-salt electrolyte, by dissolving concentrated Li-bis(trifluoromethane sulfonyl)imide salt in water. This electrolyte system introduces a desirable SEI that enables an aqueous Li-ion full cell operation at 2.3 V for more than 1,000 cyclesExtensive efforts have been made to break this limit on the potential window while retaining the benefits of aqueous energy storage systems. One approach is to adjust the pH of the electrolyte to suppress HER and/or OER5O8 electrode. As probed by synchrotron-based surface-sensitive soft X-ray spectroscopy (sXAS), an ice-like surface hydroxyl layer is formed after cycling. Density functional theory (DFT) calculations show that the interplay between the hydroxylated interphase and the unique bivalence (Mn2+2Mn4+3O8) layered structure suppresses the HER and OER with a stable potential window of 3.0 V. The system exhibits a two-electron charge transfer via Mn2+/Mn4+ redox couple, and provides facile pathway for Na-ion transport at high rate. The 3.0 V aqueous symmetric full cell exhibits a high energy density, 23 Wh kg−1 at a rate of 550 C, with nearly 100% coulombic efficiency and 85% energy efficiency after 25,000 charge–discharge cycles.Here we report a high-rate high-voltage aqueous Na-ion full cell system based on a surface hydroxylated Mn5O8 is the only bivalent manganese oxide that has a layered structure5O8 has abundant interlayer and intralayer defects for facile ionic transport, its capability for energy storage has rarely been reported. In this work, Mn5O8 nanoparticles were synthesized by heating Mn3O4 nanoparticles at 270 °C for 2 h in the open air. X-ray and neutron pair distribution function (PDF) analyses, shown in 5O8  and 4.5 eV (Mn4+), respectively. The integrated intensity ratio of Mn2+ and Mn4+ is calculated to be around 1: 2, close to that of theoretical value from Mn5O8 at near surface.Mnn in 5O8 , which c2+ sites . Apart ffraction  and PDF tructure 5. X-ray ocessing . XPS dat5O8-based electrodes. The Mn5O8 material was mixed with carbon black at mass ratio of 7: 3. The CV measurements were conducted using a three-electrode half-cell using a 0.1 M Na2SO4 electrolyte and a mercury sulfate electrode (MSE) as reference electrode. The CVs of carbon black show Mn5O8 material is the major contributor to the overall capacitance  and 0.8 V , demonstrating the high resistance to gas evolution reactions. To our knowledge, this is the first demonstration for a stable 2.5 V potential window in an aqueous Na-ion half-cell. Moreover, the resistive capability of the Mn5O8 electrode towards HER and OER can also be demonstrated by analyzing the reaction kinetics of HER and OER using TAFEL plots  with scan rates and peak separation between anodic and cathodic scans. The peak separation increases with scan rates but in a considerably less extent compared with battery materials. The increased peak separation is typically attributed to a higher overpotential that is required to transport the ions at faster rates. Therefore, the less-distinct peak separation here indicates that our system has the potential for high-power capability with facile Na-ion transport5O8 electrodes were also conducted in Na2SO4-based electrolytes with different pH values, showing very similar behaviour in the CVs and gravimetric capacitances at the various scan rates (an rates .i) obeys the power law relationship with scan rate (v) at a given potential, and can be expressed as a combination of surface-controlled capacitive effects (i1=k1v) and diffusion-controlled Na-ion intercalation (i2=k2v½) (ref. Current–voltage relationship provides insight into the charge-storage mechanism. Assuming that the current (v½) ref. :i/v½ versus v½ curves at a given potential, allows for the calculation of k1 and k2. Thus, the contributions from capacitive charge and diffusion-limited redox charge during the CV cycling are extracted quantitatively  to the Mn5O8 nanoparticles. Mn3O4 is chosen since it has a bivalent structure (Mn2+Mn3+2O4) analogous to that of Mn5O8 (Mn2+2Mn4+3O8). 5O8 nanoparticles have a greater contribution from capacitive storage compared with other materials. The results suggest that Mn5O8 nanoparticles behave as a pseudocapacitive material due to high ratio of capacitive contribution for the charge stored, as well as its unique layered structure for facile Na-ion transport.Plotting tatively . Figure 5O8. Each electrode was prepared by depositing Mn5O8 (5 mg) and carbon black (1.25 mg) on carbon paper current collector (1.77 cm2), yielding a thickness of ∼80 μm. With 1 M Na2SO4 electrolyte, Mn5O8 cell displays a stable potential window of 3.0 V after 2,000 charge and discharge cycles, the largest stable potential window ever reported for an aqueous storage device. Nearly linear potential-capacity curves, characteristic of a pseudocapacitive response, are observed  to 0.75 s , and electrode capacities change from 116 to 20 mAh g−1 accordingly. −1, coloumbic efficiency of Mn5O8 electrode remains nearly 100%, and energy efficiency increases from 80 to 97%. 5O8 full cells at a 3.0 V potential window from 5 to 50 A g−1. At 20 A g−1, the Mn5O8 cell shows excellent coloumbic efficiency (∼100%) and energy efficiency (∼85%), and a plausible electrode capacity of 61 mAh g−1 after 25,000 cycles  in the prolonged cycles, could also be attributed to a parasitic reaction from either hydrogen or oxygen evolution occurring at low rates. The resulting gas evolution could deteriorate the Mn5O8 electrode and cause the loss of capacity at prolonged cycles. It has also been pointed out that though electrode material contains Mn3O4 impurity phase (<20%), it shows very sluggish electro-kinetics of HER and OER as shown in TAFEL plots and slopes (−1), parasitic reaction from gas evolution, possibly catalysed by Mn3O4 impurity phase, may occur in near-equilibrium condition and deteriorate the electrode materials.Low-rate performance of Mn rapidly . This replateaus , concurrplateaus . The facd slopes , indicat5O8 electrode materials show relatively low discharge capacities in the initial cycles, and then the capacities continuously increase before showing steady fades for the rest of the cycles. Although similar behaviours have been reported in several electrode systems895O8 electrode may undergo a similar prolonged-conditioning process during the initial cycling, reflected by the overall electrical resistance of the button-cell as calculated by i–R drop during the discharge processes. Our data show that the cell resistances decrease during the initial cycles and eventually become relatively stable and then start to increase again  and tested for neutron scattering under an inert gas environment, light water (H2O) residuals that might be introduced from moisture in the air showed much strong scattering, so that neutron PDF analysis was unable to identify any interphase on surface of Mn5O8 materials  signal from the after-cycling data to obtain the surface hydroxyl signal. The spectrum of the obtained surface hydroxyl signal is plotted in 22–K sXAS not only evidently shows that a hydroxyl layer is formed on the surface of the Mn5O8 electrodes after cycling, it also indicates a striking ordering of the absorbed molecules, with perfectly aligned H-bonds along O–O direction, like that in ice, but with a much longer distance between the O atoms. Although the underlying mechanism of the formation of hydroxylated interphase is still unclear, we believe it is largely due to the interaction between Mn5O8 surface and water during the electrochemical cycling since no hydroxylated interphase is observed in the pristine Mn5O8 material (2+ terminated (100) surface of Mn5O8 has a lower surface energy (0.87 J m−2) than Mn4+ terminated (100) surface (1.80 J m−2). Therefore, the interaction between Mn2+ and water during the electrochemical cycling might account for the formation of hydroxylated interphase on the Mn2+ terminated (100) surface of Mn5O8.Due to the great sensitivity of the sXAS lineshape to the hydroxyl structure, the O–K spectra reveals a very interesting molecular arrangement of the hydroxyl groups on the surface of Mnmaterial . DFT calL-edge sXAS measurements  and Mn4+ (Li2MnO3) valences. The spectra show that the Mn5O8 electrode material is composed of Mn4+ (641 and 643.5 eV), Mn2+ (640 eV) and a trace amount of Mn3+, supporting the coexistence of Mn5O8 phase and trace Mn3O4 phases. The changes of peak intensity at 640 eV (Mn2+), 641 eV (Mn4+) and 643.5 eV (Mn4+) fingerprints the evolution of oxidation states of Mn with cycling. The Mn5O8 electrode at a reduced state (−1.7 V) displays a high-intensity ratio of Mn2+ to Mn4+, while this ratio decreases at the oxidized state (0.8 V), suggesting the transition from Mn2+ to Mn4+ during oxidation processes. It is therefore evident that a two-charge transfer reaction via Mn2+/Mn4+ redox couple can be achieved in the layered Mn5O8 system, indicating a great potential for high-capacity energy storage.Second, the charge-storage mechanism is revealed by the Mn urements . Mn-L sX5O8 towards HER and OER is obtained through a comparative DFT calculation on the surfaces of hydroxylated Mn5O8, pure Mn5O8 and Mn3O4. The Mn2+ terminated (100) surface of Mn5O8 is considered for the study, since our calculation predicted that Mn2+ terminated (100) surface had a lower surface energy (0.87 J m−2) than Mn4+ terminated (100) surface (1.80 J m−2). 5O8. The limiting potential at which all reaction steps become exothermic is determined to be 1.86, 1.64 and 1.68 V for hydroxylated Mn5O8, Mn5O8 and Mn3O4, respectively, indicating hydroxylated interphase increases the energy barriers for water splitting. The activation energy of each intermediate reaction steps during the HER and OER on the three surfaces was showed (5O8 (1.41 eV)>Mn5O8 (0.45 eV)>Mn3O4 (∼0 eV). Hydroxylated Mn5O8 possesses an activation energy ∼1 eV higher than that of Mn3O4, implying that the reaction rate at room temperature (300 K) is at least 17 orders of magnitude slower according to the Arrhenius equation. Therefore, the DFT calculations directly support the experimental observation of high overpotential (>0.6 V) towards HER and OER for the hydroxylated Mn5O8. It was recently reported that Mn5O8 nanoparticles showed very good OER activity in a neutral electrolyte in the presence of Mn3+ species3+ (from Mn3O4 or Mn2O3 phase) or Mn2+/Mn4+ (from Mn5O8), on the gas evolution reactions.Furthermore, the mechanistic understanding of the resistive capability of surface-hydroxylated Mns showed . The act5O8 electrodes. Gas evolution reactions are effectively inhibited through the interplay between the Mn2+ terminated surface and hydroxylated interphase. In addition, the superior power performance of Mn5O8 cell can be attributed to the unique layered, bivalence structure of Mn5O8 that enables facile Na-ion transport. In general, the ion transport through the electrode and electrolyte interface is critical for the rate performance of energy storage devices. Typical approaches to mitigate the transport barrier include decreasing the dimension of electrode materials to nanoscale, designing functional layered structure for facile ion intercalation, coating secondary conductive material on electrode surface, or developing cation-disordered electrode materials with decreased energy barrier for ion transport6830312+ terminated (100) surface of Mn5O8 has plenty of unoccupied sites, and hence provides natural tunnels for Na-ion transport along <100> direction (black arrows) (5O8 modifies the interaction between Na-ion with Mn5O8 by facilitating the electrostatic interaction between Na-ion and hydroxyl oxygen (red arrow). Although the electrochemical properties of Mn5O8 have been largely overlooked since its structure was determined in 1965  . Through2·4H2O  dissolved in 14 ml deionized water (DI H2O)  at room temperature under an open environment. A 20 ml syringe was used to controllably inject a solution of 0.3 M NaOH  into MnCl2 solution at a constant flow rate of 0.167 ml min−1 for 50 min using a computer-controlled syringe pump . The resulting dark-brown precipitate was permitted to ripen for an additional 30 min at room temperature with stirring. The product was then washed thoroughly with DI H2O and ethanol, vacuum-dried and then heated at 270 °C for 2 h.Synthesis was conducted in a magnetically-stirred 50 ml reaction batch of 25.3 mM MnCl5O8 product were studied via a three-electrode half-cell, which contained a rotating glassy carbon electrode (Pine Instrument Company) as the working electrode, a platinum wire counter electrode and mercury sulfate (saturated K2SO4) reference electrode (MSE). The electrochemical half-cell was powered by single-channel electrochemical workstation . The electrode material was prepared by grinding 7 mg Mn5O8 active material with 3 mg carbon black  and then dissolving the mixture in DI H2O to a concentration of 0.5 mg ml−1. The resulted solution was subsequently sonicated until manganese oxide was evenly dispersed in the solution and 10 μl suspension taken by pipette  was drop-cast onto the glassy carbon working electrode (0.5 cm in diameter and 0.196 cm2 in geometric surface area). Afterwards, the working electrode was dried in vacuum with a loading of Mn5O8 (3.5 μg) and carbon black (1.5 μg). Electrochemical tests were conducted in a 250 ml flat-bottom flask that contained 100 ml 0.1 M Na2SO4 electrolyte  in argon-purged H2O with a rotating rate of 500 r.p.m. CV was conducted at voltage scan rates from 1 to 1,000 mV s−1 for six segments (three cycles) within a potential range of −1.7 to 0.8 V (versus MSE).The anodic electrochemical properties of the Mn2 in area) of Toray carbon paper , which was coated with ∼5 mg Mn5O8 and 1.25 mg carbon black through drop casting. The opposing electrodes were separated via cellulose-based porous filter paper (Whatman). And each button-cell contains 200 μl 1 M Na2SO4 electrolyte . A stainless steel plate was placed on one side of the cell and in conjunction with a tightening bolt to ensure good electrical contact. Chronopotentiometry measurements were conducted at constant current densities from 0.5 to 50 A g−1 (with respective to each electrode for full cell measurements).Symmetric two-electrode button-cells were fabricated for long-term durability and stability analyses. Each electrode for full cell tests consisted of a disk .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-17, Supplementary Tables 1-3, Supplementary Notes 1-2, Supplementary Methods and Supplementary References


This work is based on the premise that overstimulation of N-methyl-d-aspartate receptors (NMDARs) may drive neurodegeneration and that this may be responsible for neuronal cell death associated with Zika virus infection of the central nervous system (CNS). Thus, blockage of the NMDAR channel activity with FDA-approved memantine or other antagonists may reduce neurological complications associated with Zika virus infection. Repurposing a preapproved drug and targeting the host represent intriguing strategies and yet require more analysis prior to moving into clinical trials.Zika virus caught the world by surprise by its rapid spread and frightening disease outcomes. This major epidemic motivated many scientists to focus their attention on controlling this emerging pathogen. As many as 45 vaccine candidates are being developed, but progress in the antiviral arena has been slower. In a recent article (mBio 8:e00350-17, 2017,  N-methyl-d-aspartate receptor (NMDAR) antagonist for the treatment of moderate-to-severe Alzheimer’s disease. The underlying hypothesis for the study was that Zika virus infection, similarly to Alzheimer’s disease and other neurodegenerative diseases, causes an overstimulation of NMDAR, leading to glutamate excitotoxicity and neuronal death. Blocking hyperactivation of NMDAR would therefore reduce rates of Zika virus-induced cell death and help ameliorate neuronal symptoms during infection.Zika virus, an exemplar reemerging virus, poses an enormous global threat that is aggravated by its unique sexual and mosquito vector transmission. Zika virus was discovered in 1947 but remained quiescent for more than half a century, with only a few reported cases of mild human infections. The virus resurfaced recently and initiated severe epidemics in South America and the Pacific islands, where an increase in the incidence of neonatal microcephaly and of autoimmune Guillain-Barré syndrome was observed. Much of what we know about the virus has been gathered in the wake of the epidemic. Data from clinical studies, animal models, and cell culture experiments have proven beyond reasonable doubt that Zika virus is neurotropic and causes a wide spectrum of neurodevelopmental defects in the human fetus, now grouped under the umbrella of congenital Zika syndrome. These include, but are not limited to, microcephaly, ventriculomegaly, hydrocephalus, intracranial calcifications, arthrogryposis, fetal brain disruption sequence, intrauterine fetal growth restriction, and a range of visual, auditory, motor, and cognitive deficits . The gro+) and potassium (K+) ions  along their electrochemical gradients. This generates a local depolarization or excitatory postsynaptic potential; if the required threshold is achieved, an action potential is generated that transmits across the axon of the postsynaptic neuron. The members of the other group of glutamate receptors, the metabotropic receptors, are not ion channels themselves but are G protein-coupled receptors that indirectly modulate the activity of ion channels through a signaling cascade. There are three ionotropic and eight metabotropic glutamate receptor families with distinct pharmacological, functional, localization, and kinetic profiles. NMDAR belongs to the ionotropic group of glutamate receptors along with the members of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainate receptor (KR) families. The families are named according to their differentiating pharmacological agonists. These receptors exist as tetrameric combinations of different subunits: NMDAR , AMPAR (GluA1-4), and KR (GluK1-5). NMDAR is unique and differs from the other ionotropic glutamate receptor families in (i) its requirement for a coagonist, glycine, for activation and (ii) its dependence on voltage for activity—at resting membrane potential, nonpermeant magnesium ion (Mg2+) from the extracellular milieu blocks the NMDAR ion channel. Depolarization of adequate strength and duration via activation of fast AMPAR channels is required to expel Mg2+ from NMDAR to allow passage of permeant cations; (iii) unlike KR and AMPAR channels containing the GluA2 subtype that have no or minor calcium conductance, NMDAR permits passage of Ca2+ in addition to Na+ and K+ ions. Ca2+ influx into the postsynaptic cells triggers a signaling cascade that serves to strengthen (long-term potentiation) or weaken (long-term depression) synapses. Regulating the number and conductance of AMPAR channels and controlling the presynaptic glutamate release via retrograde nitric oxide signaling are two examples of the ways in which the NMDAR-mediated Ca2+ signaling governs synaptic plasticity  cycle. Glutamate is converted to glutamine in astrocytes by glutamine synthetase and released from cells via a glutamine transporter. The reverse happens in neurons (conversion of glutamine to glutamate by glutaminase) followed by packaging of glutamate into synaptic vesicles by vesicular glutamate transporters (vGLUT). The concentrations of glutamate in the cytoplasm of neurons and astrocytes are about 10 and 2 mM, respectively, and can be as high as 100 mM in synaptic vesicles. The concentration of glutamate is very low (<1 μM) in the cerebrospinal fluid and is anticipated to be even lower in the synaptic cleft. Presynaptic depolarization transiently increases the extracellular glutamate levels, but the neurotransmitter is rapidly and efficiently cleared by excitatory amino acid transporters 1 (EAAT1) to EAAT5, with EAAT1 and EAAT2 localized on astrocytes. Astrocytes are essential for maintaining the appropriate levels of extracellular glutamate. Astrocytes can also release glutamate in the extracellular milieu by several methods that are poorly understood, including those associated with Ca2+-dependent channels, exocytosis, volume-sensitive organic anion channels (during astrocyte swelling), and reverse activity of EAATs (upon astrocyte damage)  .2+ overload in neurons, which can initiate apoptosis or necrosis in cells  receptor-deficient, immunocompromised adult mice (IFN-α/βR−/−) yielded mixed results. While the drug treatment was able to suppress the increase of intraocular pressure and the histopathological signs of neurodegeneration and microgliosis observed during Zika virus infection, it failed to prevent loss of body weight or the increase of inflammatory markers and cytokines in the brain. Those authors propose a possible use of memantine in conjunction with antivirals to manage neuronal symptoms during Zika virus infection. Memantine is an uncompetitive inhibitor that is believed to selectively inhibit the activity of a pathological-chronically active NMDAR, while the physiological functioning of NMDAR is left unaffected. Properties such as strong voltage dependence, moderate affinity, and high on/off rates make it superior to both Mg2+ and any tight-binding antagonist (such as MK-801)  . Further+/K+ pumps that maintain resting potential) in these cells. Costa et al. reported a dramatic increase in the number of activated tissue-resident macrophages in the brain  during Zika virus infection, a process termed "microgliosis." Activated microglia can also release glutamate in the extracellular milieu via the cystine-glutamate antiporter (Xc−). Xc− usually imports one molecule of glutamate into cells for every cystine molecule that is pushed out; the directionality of flow of these molecules may be reversed should excess cystine be required to synthesize the glutathione needed to counteract reactive oxygen species (ROS) produced in activated microglia (The work by Costa and colleagues is relevant from both the translational and fundamental perspectives. It is currently unknown how Zika virus induces neuronal cell death. If glutamate excitotoxicity is one of the mechanisms, the issue then is that of how Zika virus infection perturbs glutamate homeostasis. icroglia . Furthericroglia . Investiin vitro by memantine and other NMDAR antagonists administered in various doses. While the possibility of protection of bystander cells mediated by NMDAR antagonists is easier to envision, it is not clear how Zika virus-infected neurons are spared cell death. NMDAR channels are also believed to be present on the presynaptic membranes, but their significance during physiology and pathology is not well understood. The role of pre- and postsynaptic NMDAR in Zika virus pathogenesis requires further investigation.The glutamate signal is received by neighboring neurons, many of which may not be infected, and bystander cells can thus die as a result of excitotoxicity during Zika virus infection; measuring the incidence of this bystander cell death would provide valuable information regarding Zika virus pathology. An intriguing observation in the study was the complete rescue of cell death of Zika virus-infected primary neurons An important assumption by the authors is that Zika virus infection is a “neurodegenerative” disease, and they have accordingly subscribed to the use of an adult mouse model for their studies. However, Zika virus has been shown to perturb neurodevelopment by primarily infecting neuronal progenitor cells . It appe


Virologists. You might know a couple of them, but unless you are a virologist yourself, the probability that you have collaborated with one in the past is low. The community is relatively small, but they pack a heavy punch and are expected to play a leading role in the research into pathogens that lies ahead. You may ask why we think virologists are our future. Suffice it to say that it is not just because they have invented technologies that belong to the space age, including use of viruses as vehicles to shuttle genes into cells, organicBioinformaticians. You may know a couple of them, but usually you “just” need one to do proper math for your -omics experiments. Nowadays, nearly everyone in the life sciences has used BLAST  at leastIt is possibly not the problem of virologists but of bioinformaticians themselves. Although it was a virus that was the first organism to have its genome completely sequenced, bioinfoClearly virologists are well positioned to tackle some of the major disease threats that will inevitably face humans in the decades to come—but they are currently not the best equipped. In order to give virologists weapons, i.e., bioinformatic tools, we recently founded the European Virus Bioinformatics Center (EVBC).As alluded to above, this Center was developed based on our belief that we lack bioinformatical tools specifically designed for virology. We urgently need to develop algorithms for multiple genome alignments. It sounds trivial, but we still have no tool that can align, for example, 500,000 full-length coronaviruses . We cannot visualize such alignments that are clearly needed to rapidly identify mutational hotspots and compensatory mutations.Can you believe that there is not even a unified database for viruses? We only have good databases for Influenza (EpiFlu ), HIV 1, and humThese examples emphasize that we need to bring virologists and bioinformaticians together. Again, this seems obvious and, perhaps, trivial. But in reality, they speak different languages. We urgently need young scientists that understand both virology and bioinformatics so that they can bridge the gap between these two complementary yet different disciplines.The EVBC aims to develop bioinformatical tools for nearly all areas: (1) for detection of viruses, e.g., from high-throughput sequencing data; (2) virus assembly; (3) quasispecies reconstruction; (4) intraviral interactions; (5) virus entry, i.e., protein—protein interaction; (6) virus—host interactions; (7) phylogeny/cophylogeny; and (8) therapy. For the latter two areas, we have already established tools for HIV and Influenza A, but a large number of outstanding research questions remain. Finally, the EVBC will initiate and coordinate ring trials, undergraduate courses, graduate summer schools, and courses for principal investigators.evbc.uni-jena.de.Interested? Take a look here:


Aedes and Culex mosquitoes, is the most commonly transmitted arbovirus in Australia. A serosurvey of blood donors in French Polynesia during 2011–2013 suggested that RRV circulated without being detected. We report RRV circulation in French Polynesia based on further screening of blood samples collected during 2014–2015.Ross River virus (RRV), spread by  Togaviridae, is an arbovirus transmitted by Aedes and Culex mosquito species (Ross River virus (RRV), an alphavirus of the family In French Polynesia (FP), a territory of the Pacific region with ≈270,000 inhabitants, dengue virus (DENV) was the only arbovirus detected until identification of the Zika virus (ZIKV), then chikungunya virus (CHIKV), causing outbreaks during 2013–2014 and 2014–2015, respectively (We tested 3 groups of participants randomly sampled in FP for the presence of anti-RRV IgG by using recombinant antigen-based indirect ELISA (patent no.WO2012076715A1) and microsphere multiplex immunoassay (patent no. WO2013083847A2) (RRV seropositivity rates among participants sampled in the 5 archipelagos in 2014 ranged 16% to 49% (average 35%), and were significantly different between the Society and Marquesas (p = 0.036), Tuamotu and Marquesas (p = 0.001), and Tuamotu and Austral-Gambier (p = 0.002) Islands . In the Although no RRV outbreaks were reported in the PICTs after 1980, identification of RRV infections among travelers returning from Fiji between 1997–2009 suggested subsequent circulation of the virus in the Pacific (In this study, we also provide evidence that RRV circulated in all archipelagos. The overall seroprevalence rate of 35% found in the general population in 2014 is consistent with the rate of 34.40% previously obtained in blood donors (Increasing evidence that RRV circulated silently in several PICTs, in the absence of marsupial animal reservoirs (


The complete genome sequences of four foot-and-mouth disease viruses of South African territories 1 (SAT 1) serotype are reported. These viruses originate from an outbreak in Nigeria in 2015 and belong to the novel SAT 1 topotype X from the west and central African virus pool. Aphthovirus, family Picornaviridae that causes a highly contagious vesicular disease in cloven-hooved animals. The virus is classified into 7 immunologically distinct serotypes , each containing numerous genetically and geographically distinct evolutionary lineages or topotypes (Foot-and-mouth disease virus (FMDV) is a positive-sense, single-stranded RNA virus of the genus Here, we report the complete genome sequences of four FMDV SAT 1 viruses that were isolated from epithelial tissue samples from cattle showing typical FMD lesions. All samples were collected from cattle herds located in Plateau State (Nigeria) during the 2015 outbreak. Total RNA was extracted from cell culture supernatant with a NucleoSpin RNA virus kit (Macherey Nagel) and treated with Baseline-ZERO DNase (Epicentre) to remove host DNA. cDNA was synthesized according to the manufacturer’s instructions using SuperScript IV reverse transcriptase (Thermo Fisher Scientific), an anchored oligo(dT) primer, and an FMDV-specific internal primer. Sequencing libraries were prepared using a Nextera XT kit (Illumina) as described by the user’s manual. The fragment length distributions of the libraries were verified on a Bioanalyzer System (Agilent Technologies), and libraries were quantified using a KAPA library quantification kit (Kapa Biosystems). Sequencing was performed on a MiSeq system using MiSeq reagent kit v3 .De novo assembly of the L fragment was performed using IVA (version 1.0.8) (Raw reads were trimmed using Trimmomatic (version 0.36) with a quality cutoff of 15, sliding window of 4 bp, and minimum length cutoff of 50 bp  tract could not be determined. The genomes were predicted to contain a single open reading frame (ORF) of 7,017 nt encoding a polyprotein of 2,338 amino acids.MF678823 to MF678826.The nucleotide sequences for FMDV SAT1/NIG/1/15, SAT1/NIG/2/15, SAT1/NIG/3/15, and SAT1/NIG/4/15 have been deposited in GenBank under accession numbers


The ideal protein expression system should provide recombinant proteins in high quality and quantity involving low production costs only. However, especially for complex therapeutic proteins like monoclonal antibodies many challenges remain to meet this goal and up to now production of monoclonal antibodies is very costly and delicate. Particularly, emerging disease outbreaks like Ebola virus in Western Africa in 2014–2016 make it necessary to reevaluate existing production platforms and develop robust and cheap alternatives that are easy to handle.Phaeodactylum tricornutum to produce monoclonal IgG antibodies against the nucleoprotein of Marburg virus, a close relative of Ebola virus causing severe hemorrhagic fever with high fatality rates in humans. Sequences for both chains of a mouse IgG antibody were retrieved from a murine hybridoma cell line and implemented in the microalgal system. Fully assembled antibodies were shown to be secreted by the alga and antibodies were proven to be functional in western blot, ELISA as well as IFA studies just like the original hybridoma produced IgG. Furthermore, synthetic variants with constant regions of a rabbit IgG and human IgG with optimized codon usage were produced and characterized.In this study, we engineered the microalga This study highlights the potential of microalgae as robust and low cost expression platform for monoclonal antibodies secreting IgG antibodies directly into the culture medium. Microalgae possess rapid growth rates, need basically only water, air and sunlight for cultivation and are very easy to handle.The online version of this article (doi:10.1186/s12934-017-0745-2) contains supplementary material, which is available to authorized users. Antibodies are important tools in diagnostics as well as in therapy and monoclonal antibodies currently represent one of the best-selling classes of biologics with US sales reaching 20.3 billion dollar in 2011 , 5. HencE. coli cells is feasible representing a break through for low cost production of aglycosylated antibodies  until staining for no more than 5 days. Primary antibodies were used in a concentration of 85 ng/µl.


In the study of lime as the basic component of historical mortars and plasters, four lime putties prepared from various kinds of lime of various granulometry and by various ways of preparation were evaluated. The rheological properties and micro-morphologic changes, growing of calcite crystals, and rate of carbonation were monitored. The lime putty prepared from lump lime achieves the best rheological properties, yield stress 214.7 Pa and plastic viscosity 2.6 Pa·s. The suitability of this lime putty was checked by testing the development of calcium hydroxide and calcite crystals using scanning electron microscopy and environmental scanning electron microscopy. The disordered crystals of calcium hydroxide exhibit better carbonation resulting in the large crystals of calcite; therefore, the mortar prepared from the lump lime has the highest flexural strength and compressive strength 0.8/2.5 MPa, its carbonation is the fastest and exhibits the longest durability. Also, thanks to the micro-morphological characterization of samples in their native state by means of environmental scanning electron microscopy, the new way of lime putty preparation by mixing was proven. The preparation consists in the mechanical crash of the lime particles immediately after hydration. This enables the properties of putty prepared from lump lime to be nearly reached. Lime putty is obtained by the hydration of calcined limestone in an excess of water according to the following equation:In the past lime putty was traditional material with a wide application in construction. It used to be prepared from lump lime and it featured good workability, plasticity and durability. The traditional preparation of lime putty was replaced by modern chemical-technological processes. Lime putty is essential for the restoration of historic plaster, wall painting, sgraffito etc2 is 16.9/33.2. Lime putty is an aqueous suspension of calcium hydroxide particles. A small proportion of calcium carbonate can be present due to incomplete calcination of limestone or reaction with carbon dioxide. The process of hydration of CaO is dependent upon the reactivity of quicklime, which is influenced by the temperature of calcination of CaCO3. CaO is released from limestone and the pore structure of grains is generated during calcination. The physical properties of quicklime, such as porosity, density, grain size and hydration rate, are dependent on the temperature of calcination. Furthermore, the process of CaO hydration is dependent on the distribution of particle size, the quantity and quality of the added water, the temperature of the water, the presence of impurities in the quicklime, and the presence of ions in the mixing water3. The specific surface area is the most reliable parameter which can be used to evaluate the reactivity of quicklime. The higher specific surface area of quicklime is the reason for its higher reactivity7. The burning of CaO leads to changes in the microstructure and these differences in the microstructure of CaO crystals burnt at different temperatures result in different hydration activity of CaO8. The hydration process of quicklime is also affected by the particle size of CaO. The finer particles are more sintered and thus they cause a low reactivity of quicklime. The coarser particles will contain some amount of unburned CaCO3, which is an inert material in quicklime9. Various methods and the temperature of hydration of CaO lead to obtaining lime putty with a different morphology, size, and size distribution of calcium hydroxide crystals. The calcium hydroxide crystals with a size of approximately 100 –110 nm originate during the hydration in the excess of water and at a lower temperature5. Calcium hydroxide hydrated in the excess of water exhibits crystals which have clearly visible edges. Calcium hydroxide hydrated in a smaller amount of water is without the sharp edges of crystal and tends to form agglomerates10.It is a strongly exothermic reaction which leads to the enlargement of volume. The ratio of change in the particles’ volume of CaO to Ca(OH)13. The microstructure of calcium hydroxide is changed during the maturing of lime putty15 and the Ca(OH)2 crystals diminish. The Ca(OH)2 crystals with a size of between 37–50 nm occur in freshly hydrated lime - the crystals have a size of 34 nm at the age of two months and 28 nm at the age of 14 months. The specific surface area of the calcium hydroxide increases with this reduction in the size of the calcium hydroxide crystals16. Furthermore, the calcium hydroxide crystals change their form from large prismatic crystals to submicrometre plate crystals and secondary nucleation of nanometre plate crystals17. The change in the original prismatic crystals into smaller submicrometre plate crystals with a higher specific surface area leads to an increased adsorption of water and thereby increases plasticity and workability17. Atzeni et al.16 examined the effect of the maturation of lime putty on its viscosity. They prepared three lime putties with different times of maturation. Observation of the lime putties by TEM revealed that the calcium hydroxide crystals reduce in size over time. The oldest lime putty showed the greatest shear resistance and it was even more viscous than the three-month-old lime putty as well as the non-matured lime putty. The non-matured lime putty contains the agglomerates of calcium hydroxide and these agglomerates are covered by a thin film of water, which acts as a lubricant, and so this lime putty is extremely fluid. The prolonged ageing of lime putty leads to a higher viscosity, which is caused by the change in its microstructure and the specific surface area of calcium hydroxide during the ageing process. Analogous to the investigation of lime, Paiva et al.18 prepared lime mortars with a different maturation period. The mortars matured for seven days were more viscous in their fresh state, showed higher strengths at the age of 90 days, and exhibited a lower capillary absorption coefficient than the mortars without maturation. Margalha et al.19 found that slaked lump lime provides a binder with a higher proportion of active lime than powdered lime. The mortars prepared from the micronized lime show a higher porosity with larger pores, leading to a reduction in their mechanical strengths. The mortars prepared from lump lime exhibit higher strengths, but they require over-ageing treatment because of their shrinking.The long-term maturing of lime putty in an excess of water has a beneficial effect on its quality. Such stored lime putty has better plasticity, workability, and it retains sufficient water to prepare plaster of a high durability19.The results of the measurement of the plastic viscosity and the yield stress of lime putties SL-A, SL-B, SL-C, and SL-B1 during their maturation at the age of 3, 30, 60, and 90 days are shown in Figs 2.aq), which is already discernible after three days of hydration (maturation) Figs Figures 21. Thus, there are no artefacts distorting the microstructural interpretation.The HR-ESEM images of lime putty SL-A without preparation of the sample and sputtering at the age of 3 and 30 days are shown in Fig. The same specific differences in the sample morphology may also be caused due to the samples drying and coating of a metal layer for SEM observation. Conversely, observation in ESEM does not require the above processes and the samples are observed naturally wet and without any modifications. The surface morphology of the crystals observed using the ESEM are somewhat smooth and free of sharp edges and micro-cracks see Fig. 2 and humidity (70%). The length of the side of the calcite crystals was measured. The regular crystals which grew separately were selected for measurement. The images in Fig. Figure 2 environment are demonstrated in Fig. 2 and 65.7% CaCO3) compared to lime B (20.0% Ca(OH)2 and 68.7% CaCO3). Sample C-SL-C, prepared from lump lime, carbonates very quickly (2.8% Ca(OH)2 and 85.8% CaCO3). Sample C-SL-B1 contained less calcium hydroxide and, actually, more calcium carbonate (8.9% Ca(OH)2 and 80.0% CaCO3) than lime putty C-SL-B due to the process of activation.The results of differential thermal analysis of carbonated lime putties C-SL-A, C-SL-B, C-SL-C, and C-CL-B1 after 14 days of storage in a COThe effect of lime putty preparation on the carbonation process was also assessed by determining the strengths of lime mortars. The results of the tensile and compressive strengths measurements of lime mortars M-A, M-B, M-C, and M-B1 at the age of 28 days are illustrated in Table The effect of the method of preparation of lime putty is also noticeable because mortar M-B1 achieved higher strengths than mortar M-B. Both of these lime mortars were prepared from lime with the same granulometry, but lime putty SL-B1 was mixed, unlike lime putty SL-B.The aim of the article was to analyse the development of the lime putties in the dependence on time by means of selected methods giving accurate results. The found results exactly characterize the lime putties’ properties and correspond with the results published by other authors. The authors of the article consider a high potential in the micro-morphological characterization of the lime putties in their native state, so without any sample treatment with minimum occurrence of artefacts, by means of ESEM. The article presents an evaluation of the results obtained by means of classic SEM and ESEM for the objective arbitration.The use of lump lime or mixing a suspension of lime for the preparation of lime putty leads to the formation of incompletely regular calcium hydroxide crystals and a high amount of hydrogel formation. It results in a faster formation of calcium carbonate, the growth of larger calcite crystals, and higher strengths of lime mortars.The values of plastic viscosity and yield stress increase with the increasing period of maturation. The use of aged limes leads to obtaining an intermediate product of higher quality for the preparation of lime mortars.The process of particle disruption during the mixing of lime putty helps accelerate the hydrogel formation as well as increasing plastic viscosity and yield stress.The use of lump lime or the mechanical disruption of lime particles is the most suitable for the properties of lime putty and, consequently, for the preparation of lime mortars.Matured lime putties prepared from lump lime and by mixing lime putty are the most suitable for the preparation of mortar.Four lime putties prepared in different ways from quicklimes with varying granulometry were analysed in the dependence on the maturation time. During the maturation of lime putties, the regular hexagonal crystals of calcium hydroxide are transformed into smaller irregular crystals which are coated with a layer of hydrogel. The rheological properties are dependent on the amount of hydrogel produced during the ageing of lime putties.The most important contribution of the article in the field of conservation and restoration of historical mortars consists in the new way of lime putties preparation by means of mechanical crush of the lime particles. Mortars produced from such prepared lime putty achieved similar properties as mortars made from the matured lime putty prepared from lump lime. An advantage of the new way of lime putty preparation is the use of commonly available powder lime without the necessity of using more difficult to obtain lump lime. The presented results exhibited that mortars with higher strengths can be prepared from common powder lime. These mortars will be more durable with a higher resistance to environmental influences and contaminated atmosphere.Lime A with a grain size of under 90 µm, lime B with a grain size of under 200 µm, and lump lime with a grain size from 10 mm to 63 mm  were used to prepare the lime putties. Lime A and lime B are mixtures of lime burned partially (70%) in the co-current regenerative furnace at 1000 °C and in the countercurrent shaft furnace (30%) at 1300 °C. Lime C is a mixture of lime burned in the co-current regenerative furnace at 1100 °C (50%) and in the countercurrent shaft furnace at 1300 °C (50%). The content of total CaO/active CaO in lime A was 96.05/93.74, in lime B 94.08/88.34, and in lime C 95.87/92.33. The reactivity of lime expressed as the time taken to reach a temperature of 60 °C, according to EN 459-2, was 2.3 min for lime A, 2.6 min for lime B, and 3.5 min for lime C.The granulometry of lime A and lime B is shown in Table 2 and air drying. The samples were then kept in closed bottles at a temperature of 20 ± 1 °C. Samples SL-A, SL-B, SL-C, and SL-B1 were stored in a box with 70% humidity and a 20% concentration of CO2 for 14 days for accelerated carbonation. Lime mortars M-A, M-B, M-C, and M-B1 were prepared from the lime putties and sand with a ratio of 1:3. The lime mortars were placed in moulds with dimensions of 40 × 40 × 160 mm. Specimens were stored under laboratory conditions at a temperature of 20 ± 1 °C and a relative humidity of 50 ± 5% after their removal from the moulds.Lime putties SL-A, SL-B, and SL-C were prepared by mixing 200 g of lime and 640 ml of deionized water. Lime putty SL-B1 was prepared by mixing 200 g of lime and 640 ml of deionized water and this lime putty was mixed  for 5 minutes after its hydration. The fresh lime putties were immediately stored in plastic bottles and they were supplemented with water to avoid reaction with CO−1 to 200 sec−1 over 2 minutes. The values of yield stress and plastic viscosity were determined according to the Bingham model,τ [Pa] is the shear stress,τ0 is the yield stress, μ [Pa·s] is the plastic viscosity, and −1] is the shear strain rate22. Temperature of the measurement apparatus and the laboratory were kept constant at 20 (±0.5) °C.The rheological properties of lime putties SL-A, SL-B, SL-C, and SL-B1after 3, 30, 60, and 90 days’ maturation were monitored by a Discovery Hybrid Rheometer HR-1 . The rheological properties of the lime putties were determined from the curves of flow with the increasing and decreasing shear rate in the range from 0 sec2, and the samples were sputtered.Lime putty samples SL-A, SL-B, SL-C, and SL-B1 were observed in the high resolution scanning electron microscope (HR-SEM) JSM6700F JEOL  after 3, 30, 60, and 90 days. The samples of the lime putties were prepared for observation in the SEM in the following manner: the samples were rinsed with 10 ml of acetone to remove water and 10 ml of diethyl ether for drying. Next, the samples were dried in a nitrogen atmosphere in order to eliminate the influence of COThe sample of lime putty SL-A was observed in the environmental scanning electron microscope (ESEM) AQUASEM II  after 3 and 30 days. The sample was studied directly without sputtering.The different development of the calcite crystals in carbonated lime putties C-SL-A, C-SL-B, C-SL-C a C-SL- B1 was monitored with the high resolution environmental scanning electron microscope (HR-ESEM) QUANTA 650 FEG . The samples were studied directly without sputtering. The length of the side of the calcite crystals was measured using the Scandium software.2. The used instrument exhibits accuracy with an error ±10%. The measurement was carried out under a nitrogen atmosphere.The differential thermal analysis of carbonated lime putties C-SL-A, C-SL-B, C-SL-C, and C-SL-B1 was performed using the thermal analyser NETSCH STA 2500 after 14 days of being stored in a box with 70% humidity and a 20% concentration of COThe flexural strength and compressive strength of lime mortars M-A, M-B, M-C, and M-B1 were determined at 28 days according to EN 1015-11.All presented data was calculated as average value from the repeated five measurements and standard deviation and repeatability limit (probability level 95%) were calculated.


Zika virus infection during pregnancy can lead to congenital Zika syndrome. Implementation of screening programs and interpretation of test results can be particularly challenging during ongoing local mosquitoborne transmission. We conducted a retrospective chart review of 2,327 pregnant women screened for Zika virus in Miami–Dade County, Florida, USA, during 2016. Of these, 86 had laboratory evidence of Zika virus infection; we describe 2 infants with probable congenital Zika syndrome. Delays in receipt of laboratory test results (median 42 days) occurred during the first month of local transmission. Odds of screening positive for Zika virus were higher for women without health insurance or who did not speak English. Our findings indicate the increase in screening for Zika virus can overwhelm hospital and public health systems, resulting in delayed receipt of results of screening and confirmatory tests and the potential to miss cases or delay diagnoses. Zika virus infection during pregnancy can lead to congenital Zika syndrome . The Florida Department of Health (FLDOH) and its contracting laboratory, LabCorp , modified the CDC guidelines by using Zika virus real-time reverse transcription PCR (rRT-PCR) and IgM to screen blood (IgM and rRT-PCR) and urine (rRT-PCR) samples simultaneously, regardless of symptoms or time from potential Zika virus exposure. All samples were collected at a regular obstetrics visit and sent in daily batches to the local FLDOH laboratory, which triaged them.Before local transmission began in July 2016, laboratory testing of pregnant women was based on Zika virus exposure history . After documented local transmission, we routinely offered laboratory screening for Zika virus to all pregnant women. If a woman had not been tested for Zika virus during her pregnancy, she was offered testing on arrival to labor and delivery. When a woman had any laboratory evidence of Zika virus infection during her pregnancy or when a congenital malformation in her neonate prompted evaluation for congenital Zika syndrome, urine, serum, or other relevant samples were sent from the neonatology inpatient service directly to the FLDOH, which tested samples directly or forwarded them for testing to LabCorp. The ordering clinician did not determine which entity tested the samples or the type of testing done.If rRT-PCR or IgM testing yielded positive results at LabCorp or FLDOH, specimens were forwarded to CDC for plaque-reduction neutralization testing (PRNT). Women and infants who were eligible for the US Zika Pregnancy Registry were reported by FLDOH  in maternal serum. This change in care was done as part of clinical management. Women with rRT-PCR–positive serum or urine were considered to have acute Zika virus infection. Women with positive Zika virus IgM were presumed to have Zika virus infection until PRNT results were returned, after which we followed the CDC guidelines  where PRNT was not recommended and relied on the IgM results  with 95% CIs. We excluded from the χ2 and logistic regression analyses women whose test results were still pending. We also excluded women in the Native American and other race/ethnicity categories from the χ2 test and logistic regression model between race/ethnicity and result status because no women in these groups tested positive for Zika virus. We also excluded from individual statistical analysis patients with missing data.We performed statistical analyses using SAS University Edition . Descriptive statistics were presented as means ± SDs or medians according to the statistical distribution of continuous data and as number of patients and percentages for categorical parameters. To examine the association between insurance status, primary language, race/ethnicity, and clinical result status among the pregnant women, we used χDuring 2016, a total of 2,327 pregnant women were tested for Zika virus . Based oOf the 2,327 women screened, 1,999 (85.9%) had no laboratory evidence of Zika virus during pregnancy . Eight . Of the Of women with acute Zika virus infection, rRT-PCR documented prolonged Zika virus in 6 women. Two of these had 1 positive rRT-PCR time point; for the rest, time from first to last positive rRT-PCR result were 13, 55, 48, 19, 25, and 13 days. For the remainder of our analysis, pregnant women with evidence acute Zika virus infection, women with evidence of Zika virus (timing of infection undetermined), and women with presumptive Zika virus  were analyzed together as having any laboratory evidence of Zika virus infection during pregnancy because their clinical management during pregnancy and the subsequent testing of the infants was the same.>2 previous pregnancies  had gnancies . Fifty-tgnancies . Twenty-We assessed outcomes for 52 infants of women with laboratory evidence of Zika virus during pregnancy and 1 infant with laboratory-confirmed Zika virus for whom maternal rRT-PCR and IgM were negative for Zika virus . Two infNone of the antenatal ultrasounds for 66 women for whom they were documented showed intracranial calcifications. For 2 infants, intracranial calcifications noted after birth were not detected before delivery . For 5  met crit2 test). Uninsured patients had higher odds of receiving a positive Zika virus test result  than did women with public insurance. A total of 65.3% of pregnant women who had laboratory evidence of Zika virus infection had public insurance, 15.8% had private insurance, and the remaining 18.9% were uninsured . We foun2 test). However, the ORs for each race/ethnicity compared with non-Hispanic white patients were not significant .Language preference was significantly associated with Zika virus test result (p = 0.0001). Patients speaking Spanish and Haitian Creole had higher odds of receiving a positive Zika virus test result than English speakers . Race/ethnicity also was significantly associated with Zika virus test result p = 0.0013 by χWe report on the clinical outcomes, challenges in testing, and social factors associated with screening positive for Zika virus infection during pregnancy. Of the 52 infants born to women with evidence of Zika virus infection, 2 (3.7%) had evidence of probable congenital Zika virus infection, both from first trimester Zika virus infections. Difficulty in estimating the true percentage of infants affected by Zika virus is challenging because current testing might not provide laboratory evidence of fetal Zika virus infection after delivery (,,,,In the cohort we report, delays in receipt of results of Zika virus screening occurred during the first half of 2016. The longest delays occurred in before local mosquitoborne transmission began; delays decreased as the laboratories and public health agencies became accustomed to an increased number of laboratory tests. In the context of pregnancy, delays in result reporting may affect decisions about continuation and termination of pregnancy (,,,Recent clinical guidelines recommend including rRT-PCR in screening during each trimester of pregnancy to add specificity in detecting Zika virus. Zika virus IgM is detectable for longer than previously anticipated; the median time from seroconversion to IgM negative is 122 days and as long as 210 days (As part of the public health response to local transmission of Zika virus in 2016, Florida state authorities made access to Zika virus screening free for all pregnant women through FLDOH. Review of the cohort reported here suggests that removal of financial barriers to screening were important; 18.9% of the women in this study had no insurance and had increased odds of testing positive for Zika virus during pregnancy. We consider removal of financial barriers to screening as an important adjunct to provider counseling. Similarly, pregnant women in this cohort who primarily spoke Spanish or Haitian Creole had increased odds of positive Zika virus screening during pregnancy. These 2 findings are relevant to the design and implementation of public awareness campaigns.The findings of our study are subject to several limitations. The high rate of loss to follow-up was due in part to screening only at delivery or late during pregnancy, resulting in discharge before receipt of results. Reengagement with this patient population has been difficult. Because our study was a retrospective chart review, we relied on accurate documentation of symptoms potentially attributable to Zika virus, which possibly limited detection of women who might have been symptomatic. Also, as tertiary care centers, we frequently receive patients who were initially managed at outlying clinics, and complete records were often fragmented, particularly in terms of PRNT results. In addition, because not all infants were tested for Zika virus at birth or were fully evaluated, we might not have accurately represented the impact of congenital Zika syndrome in this cohort. The strengths of this study include the large number of pregnant women screened for Zika virus in a diverse patient population. The wide socioeconomic strata represented by these women enabled identification of factors associated with the odds of screening positive for Zika virus during pregnancy.,Among the multiple patient management and counseling issues our study raises are the caveats in laboratory result interpretation and the need for initial counseling that provides the most current understanding of Zika virus infection during pregnancy. In addition, our study provides lessons for other regions at risk for local transmission. Specifically, the increase in screening for Zika virus can overwhelm hospital and public health systems, resulting in delayed receipt of results of screening and confirmatory tests. Similarly, delay in penetration of screening guidelines to the medical community may result in lack of screening during pregnancy, which can lead to missed cases or delayed diagnoses. Because the understanding of the effect of Zika virus infection during pregnancy and the guidelines regarding testing interpretation are rapidly evolving, clinicians need to be well-versed on the current national guidelines for Zika virus testing (


Enveloped viruses, such as HIV, Ebola and Influenza, are among the most deadly known viruses. Cellular membrane penetration of enveloped viruses is a critical step in the cascade of events that lead to entry into the host cell. Conventional ensemble fusion assays rely on collective responses to membrane fusion events, and do not allow direct and quantitative studies of the subtle and intricate fusion details. Such details are accessible via single particle investigation techniques, however. Here, we implement nano-infrared spectroscopic imaging to investigate the chemical and structural modifications that occur prior to membrane fusion in the single archetypal enveloped virus, influenza X31. We traced in real-space structural and spectroscopic alterations that occur during environmental pH variations in single virus particles. In addition, using nanospectroscopic imaging we quantified the effectiveness of an antiviral compound in stopping viral membrane disruption  during environmental pH variations. Many enveloped viruses continue to be a persistent health threat to human populations. To enter a cell, viruses attach to host-cell receptors. Detailed understanding of how viral entry proteins interact with their host-cell receptors and how the viral membrane envelope undergoes changes that lead to entry offer opportunities for the development of novel therapeutics and vaccines. For example, influenza virus (IFV) has been used as a prototype enveloped virus to study virus entry into the host cell. Hemagglutinin (HA) is a major surface glycoprotein embedded in the IFV membrane envelope. HA is responsible for IFV attachment to the host cell receptor and is involved in mediating membrane fusion during virus entry. Previous studies have led to a generally accepted model for the fusion mechanism between the target and viral membranes . In thisDetailed understanding the complex sequential fusion mechanism at a single virus level offers a tremendous opportunity to design antiviral compounds tuned to interrupt the cascade of events leading to viral infection. Conventional ensemble fusion assays relay on collective responses of fusion events and as a result cannot allow direct quantification of some of the subtle fusion details that are more accessible via studies of single isolated particles. Appropriate single virus particle interrogation tools that not only offer high spatial resolution but also possess the capability to probe mechanical/chemical properties and effects of environmental changes could facilitate detailed understanding of viral membrane fusion. For example, the viral infection process causes chemical and structural composition alterations of both the viral and the host cellular membranes at the molecular level, and these changes can be probed by molecular-specific infrared spectroscopic techniques. The diffraction limit is a bottleneck for laser based spectroscopy investigations of single viruses due to their nanoscale size. Scattering type scanning near-field optical microscope (s-SNOM) is not limited by diffraction, and provides the ability to undertake (infrared) spectroscopic studies of objects with a spatial resolution down to 10 nm, allowing high resolution in situ spectroscopic chemical and structural identification and mapping of a single virus and its interaction with environmental triggers and a cell. s-SNOM functions as a local probe via scattering of optical fields from a nano-localized probe tip, either at single frequency or using broadband light sources covering the broad infrared (IR) fingerprint frequency range see . Simulta3) images are shown at 1088  and phase (φ3) images taken at two laser frequencies (1225 and 1665 cm-1 and pH = 5&7). Other even lower unphysiological pH exposures studies are given in the -1 image, which represents the amide I protein absorption , shows a uniform protein distribution across the viruses. In contrast, the 1225 cm-1 image, that represents phosphate absorption, is fragmented, more and more so at lower pH. These differences in the 1665 and 1225 cm-1 images likely indicate disintegration of the lipid bilayer as well as RNA at low pH. This direct visualization of the effect of low pH on single virus particles clearly demonstrates viral membrane disruption in the absence of a host cell membrane, an intriguing observation that offers better insight into our current understanding of viral membrane fusion.We first investigate the structural and spectral alterations in four virus particles as they are exposed to lower pH environment. -1) of the same set of viruses before compound 136 treatment and neutral pH values  that inhibits influenza virus fusion at picomolar concentrations in plaque reduction assays , 13. Com6 that inTo monitor the modification of chemical composition of a virus due to exposure to lower pH environment, we obtained infrared absorption spectra of single viruses before and after acid exposure. These single virus spectra before acid exposure are shown in -1 are the well-known amide I and amide II absorption bands, respectively. These bands are predominantly due to the HA proteins. These bands may represent different protein secondary structures [-1 is often associated with β-sheet types of protein secondary structure that are abundant in the head of HA [-1 in the IR spectra is well known to be associated with membrane lipids [In the IR spectra shown in ructures , or possad of HA . The lipad of HA , 18. Thee lipids , in parte lipids . On the e lipids .-1 region are observed, with the overall intensity of this set of features being less than the amide I absorption band. It is interesting to compare this observation to IR spectra of whole cells [-1 region. Since the bands in the 1290–1050 cm-1 region are associated mainly with RNA/lipids, this implies that the ratio of RNA/lipids to protein derived from the IR spectra is less than 1.0 in influenza virus similar to that found in other biological cell types.In the near-field IR spectra in le cells  which al-1 region [2-) and RNA (PO2-) stretching vibrations are known to occur near 1230 and 1085 cm-1, respectively. We note that both of these asymmetric and symmetric stretching vibrations could contribute to the broad unresolved spectrum [Lipids , carbohy1 region . As a respectrum . For bot-1 both before and after acid treatment. However, both the amide I & II band intensities significantly reduced after acid treatment, which may indicate some, not unexpected, protein unfolding  upon treatment. The broad bands in the 1290–1050 cm-1 region do not seem to be significantly altered in these spectra. However, hyperspectral image taken [-1 region differs significantly from point to point on the virus surface  and after exposure to acidic solution at pH 5 (in red) are plotted in ge taken  using IR surface . The hypIn this work, we have undertaken spectroscopy and imaging experiments on single influenza virus particles in order to investigate the chemical and structural modifications of the viral protein and lipid bilayer during various environmental pH variations and upon interaction with antiviral compounds. A remarkable finding of our work is that we directly show that if the environment pH is lowered, disruption of viral membrane could occur without the presence of a targeting membrane, contrary to the current viral fusion model, which requires virus binding to a host cell membrane for forming the fusion pore by fusion of two membranes to release the viral genome . The fus2. The media were collected after 48 hours post infection. After clarification to remove cell debris, the virus was pelleted through a 33% sucrose cushion at 85,000 x g. The virus pellet was resuspended in PBS and pelleted again through a 30% glycerol cushion at 15,000 x g. The virus pellet was resuspended in PBS and stored at 4°C before usage. The virus particles are then drop casted on pre-cleaned Si wafer for s-SNOM experiments. After sitting for 15 min, unbound particles were washed off with water. For changes of condition, the particle deposited wafer was incubated with water at various pH values adjusted with a stock solution of 1M HCL, or 1 μM of compound 136 in 1% DMSO.To prepare the influenza virus Madin-Darby canine kidney (MDCK) cells  were maintained with EMEM media complemented with 4% fetal bovine serum. Confluent MDCK cells were infected with influenza A virus (STRAIN X-31) at a MOI of 1.0 in EMEM media containing 2 μg/mL TPCK-treated trypsin, and the infected cells were incubated at 37°C, 5% COhttp://www.neaspec.com/). The probe tip in s-SNOM functions as a local probe via scattering of the nanolocalized optical fields from a probe tip either at single frequency or using broadband light sources covering wide IR finger print frequency range.[f~280 kHz. For single frequency nanoimaging, it is irradiated by focused quantum cascade lasers (QCLs) (http://www.daylightsolutions.com/) at 45° with respect to the sample surface using a parabolic mirror (PM). The scattered signal from the tip-sample interface region is demodulated at high harmonics of the tip resonance frequency  and detected by phase-modulation (pseudo-heterodyne) interferometry using a beam splitter (BS) and a vibrating mirror. s-SNOM nanoimaging results in simultaneous topography, IR amplitude and phase images that offer complementary information. The heights of the viruses were measured from the topography image. The near-field phase, being directly proportional to the imaginary part of the permittivity, represents vibrational absorption of a sample [cy range.–12 The pa sample . For obtS1 Fig(a) Topography of a virus particle and (b) 3rd harmonic near-field amplitude image taken at 1045 cm-1 showing contrast difference between the outer viral envelop and the inner structure of the virus. (c) Topography and amplitude line profiles. Scale bar is 100 nm.(PDF)Click here for additional data file.S2 FigTopography (black & white), scale bar 100 nm and near-field amplitude (A3) and phase (φ3) images of four virus particles at progressively lower pH (left to right). Spectral images taken at (a) 1225 cm-1. The columns i-v in the topography represent successively decreasing pH from neutral (i) to pH 2 (v).(PDF)Click here for additional data file.S3 FigGeometric, height (black points) and surface area (blue points), evolution plot of influenza x-31 virus particles as a function of decreasing pH from neutral (i) to pH 2 (v).(PDF)Click here for additional data file.


Infection with herpes simplex virus-1 (HSV-1) brings numerous changes in cellular gene expression. Levels of most host mRNAs are reduced, limiting synthesis of host proteins, especially those involved in antiviral defenses. The impact of HSV-1 on host microRNAs (miRNAs), an extensive network of short non-coding RNAs that regulate mRNA stability/translation, remains largely unexplored. Here we show that transcription of the miR-183 cluster  is selectively induced by HSV-1 during productive infection of primary fibroblasts and neurons. ICP0, a viral E3 ubiquitin ligase expressed as an immediate-early protein, is both necessary and sufficient for this induction. Nuclear exclusion of ICP0 or removal of the RING  finger domain that is required for E3 ligase activity prevents induction. ICP0 promotes the degradation of numerous host proteins and for the most part, the downstream consequences are unknown. Induction of the miR-183 cluster can be mimicked by depletion of host transcriptional repressors zinc finger E-box binding homeobox 1 (ZEB1)/δ-crystallin enhancer binding factor 1 (δEF1) and zinc finger E-box binding homeobox 2 (ZEB2)/Smad-interacting protein 1 (SIP1), which we establish as new substrates for ICP0-mediated degradation. Thus, HSV-1 selectively stimulates expression of the miR-183 cluster by ICP0-mediated degradation of ZEB transcriptional repressors. MicroRNAs (miRNAs) function as post-transcriptional regulators of gene expression, limiting protein expression through a combination of mRNA destabilization and translational inhibition . The majSeveral virus families produce their own viral miRNAs, with the herpesviruses providing the most prominent examples . In the In broad strokes, two general strategies have been uncovered by which viruses alter host miRNA–mRNA networks. The first strategy is to express viral miRNAs that mimic existing cellular miRNAs, allowing the virus to exploit an evolutionarily inflexible network of binding sites in target mRNAs. For example, Kaposi’s sarcoma-associated herpesvirus (KSHV), avian Marek’s disease virus-1 and 2 (MDV-1 and MDV-2), EBV, rhesus lymphocryptovirus, and bovine leukemia virus, a retrovirus, encode mimics of miRNAs, miR-155, or miR-29 that promote B-cell tumorigenesis ,20,21. TSuppression of host gene expression, otherwise known as host shutoff, is especially profound in HSV-1 infections and shapes the viral replication program by sharpening the transitions between different kinetic classes of temporally transcribed mRNAs ,25. Mechn12 (∆ICP4) virus from Dr. Neal DeLuca  . T. T68]. TTo determine whether miR-183/96/182 induction requires HSV-1 gene expression, NHDFs were infected with either untreated or ultraviolet (UV)-irradiated wild type HSV-1 GFP-Us11 virus B. FibrobTo identify the viral protein(s) responsible, we performed infection studies using HSV-1 mutant viruses lacking key IE and E genes required for progression through the productive replication cycle. First a virus, HP66, lacking much of the viral DNA polymerase gene (HSV-1 ΔDNA Pol)  was usedn12 (ΔICP4) virus reduced but did not entirely abolish induction  virus showed a reduced ability to induce miR-183/96/182 transcription, despite expression of wild type ICP0 (n12) viruses. As shown in n12 mutant compared to wild type virus (n12) virus.During the immediate-early phase of the HSV-1 productive replication cycle, ICP0 accumulates in the nucleus of infected cells but is later redistributed to the cytoplasm ,74,75. Iype ICP0 F. To conpe virus E. This sTo further examine the role of ICP0 nuclear localization, infections were performed using the HSV-1 mutant virus D8 A, in whiMany ICP0 functions involve the targeting of host proteins for accelerated degradation by the proteasome . In manyAside from the C3HC4 RING domain and NLS, other regions of ICP0 are involved in substrate recognition, often through direct protein–protein interactions ,83,84,85Transcriptional regulation of the miR-183/96/182 cluster is complex and dependent on cell context . Two potThe impact on HSV-1 infection on ZEB1 abundance in HFFs was assessed by immunoblotting B. With aThe reduction in ZEB1 levels could be recapitulated by transfection of a plasmid expressing wild type ICP0 fused to enhanced GFP  cycle. However, this temporal assignment may not be so clear-cut. There is genetic evidence that ICP0 is expressed in murine sensory neurons during the establishment of latency, such that mutations that either increase or decrease ICP0 expression lead to changes in the structure of viral chromatin and in the expression of both productive-cycle genes and the latency-associated transcript (LAT) RNA ,101,102.At this time, we can only speculate on the biological impact of microRNA upregulation with respect to HSV-1. The miR-183 family targets a diverse set of host mRNAs, some of which have the potential influence viral replication by changing the behavior or resilience to stress of the infected cells . We haveInterestingly, HSV-1 is not the only herpesvirus to alter expression of the miR-183/96/182 cluster or interact with the ZEB proteins. HCMV, a β-herpesvirus, alters the abundance of several host miRNAs during its productive infection cycle, with miR-96, miR-182, and miR-183 being the most strongly upregulated ,112. β-HDuring type II and III latency, the γ-herpesvirus EBV selectively downregulates miR-183/96/182 expression ,115,116.Collectively, these studies reveal a recurring interaction between representatives of the three major herpesvirus subfamilies and a conserved host regulatory network involving the ZEB factors and ZEB-regulated genes such as the miR-183 cluster. More study is needed to understand the functional consequences for each virus and this may depend on the infected tissue, input from other signaling pathways, and whether the virus is replicating productively or maintaining latency. For HSV-1, we have defined a mechanism for manipulation the ZEB factors via the viral E3 ligase and shown that even in the context of a robust host shut off, HSV-1 can selectively modify the profile of host miRNAs.


Given the speed with which viruses transmitted by the respiratory route spread globally, epidemics caused by these viruses pose great threats to global public health. Recent examples include the global outbreak of severe acute respiratory syndrome caused by a coronavirus in 2003–04 (http://transmission2017.med.hku.hk/). The Croucher Foundation, an independent private Hong Kong foundation, also provided financial support for this conference.As part of its 130th anniversary celebration, the Li Ka Shing Faculty of Medicine of the University of Hong Kong has provided financial support for several international conferences in its priority research areas, including respiratory virus transmission and control. An international conference on this topic was held June 19–21, 2017, in Hong Kong, attended by >190 participants from 19 countries , or by experimental approach . This conference aimed to cut across these divisions to bring together researchers working with different respiratory viruses and using different experimental approaches.Mycobacterium tuberculosis were reviewed.The conference was organized around 4 themes . An intePresentations provided data on age-specific contact patterns between humans and other animals and their relevance for mathematical modeling of transmission dynamics. Data from large community-based studies on virus transmission were also presented. Such cohort studies may allow defining of a “contagious phenotype.” One theme of interest was the potential for deep sequencing data to permit estimation of the number of viral particles transmitted from one person to another (the transmission bottleneck) and how this bottleneck might vary for different reasons. Other discussions included the potential use of deep sequencing approaches to study virus evolution, viral fitness, and antiviral resistant mutations, as well as the role of environmental factors such as temperature, humidity, and environmental pollutants  on viral transmission. Social and ecologic factors examined included crowdedness  and school holidays.A novel in vitro experimental approach that allows delivery of aerosolized virus onto human ciliated epithelial cells followed by monitoring changes of physiologic parameters  on these cells was described. There was particular interest in the use of experimental animal models for research on virus transmission, and data were provided on the advantages and disadvantages of different experimental animal models for influenza, RSV, morbilliviruses, and parainfluenza viruses. As an extension of previous work that identified the importance of influenza transmission through respiratory droplets in the ferret model  in China, were presented. Refining and validating experimental animals and ex vivo and in vitro models for assessing risk of emerging respiratory pathogens remain an area for future research.A section of the conference focused on the study of airborne virus particles. The performance and advantages of different air samplers were discussed. Data on measurement of virus-laden particles in air in different settings were presented, including experimental animal studies, between humans in community and healthcare settings, and at the animal–human interface, such as live poultry markets in Asia and swine barns and county fairs in the United States.The final topic in the conference was control of respiratory pathogens. Patterns of aerosols generated by procedures and devices in hospitals were discussed, together with engineering solutions to minimize such risks. Viral and environmental factors that lead to generation of infectious droplets in live poultry markets and slaughtering facilities were investigated, along with options for reducing such risks. The effect of vaccines on reducing virus transmission was also discussed.The conference was very well received by delegates, many of whom expressed enthusiasm for the theme and the active interdisciplinary discussions that were a feature of the meeting. There was near unanimous support for this to be the start of a series of meetings with multidisciplinary participation, perhaps at 2–3-year intervals. The International Society for Influenza and Other Respiratory Virus Diseases (ISIRV) provided some logistical support for this conference; a potential development is the creation of a special interest group within ISIRV for conference participants and other researchers to discuss these shared interests.