Molecular nanotechnology is a rapidly developing field, and tremendous progress has been made in developing synthetic molecular machines. One long-sought after nanotechnology is systems able to achieve the assembly-line like production of molecules. Here we report the discovery of a rudimentary synthetic molecular assembler that produces polymers. The molecular assembler is a supramolecular aggregate of bifunctional surfactants produced by the reaction of two phase-separated reactants. Initially self-reproduction of the bifunctional surfactants is observed, but once it reaches a critical concentration the assembler starts to produce polymers instead of supramolecular aggregates. The polymer size can be controlled by adjusting temperature, reaction time, or introducing a capping agent. There has been considerable debate about molecular assemblers in the context of nanotechnology, our demonstration that primitive assemblers may arise from simple phase separated reactants may provide a new direction for the design of functional supramolecular systems. The feasibility of molecular assemblers as a device to control chemical reactions by positioning molecules with atomic precision is a matter of debate in the literature. Here the authors describe of a rudimentary synthetic molecular assembler, supramolecular aggregate of bifunctional surfactants produced by the reaction of two phase-separated reactants that produces polymers. Macroscopic devices often serve as inspiration for molecular analogs of hinges4, rotors7, switches8, and even complex structures such as cars10. Using molecular machines, functions may be performed11, such as transporting chemicals12, macroscopic movement15, and catalysis16. Although practically still at the proof of concept stage, the array of processes that can be catalyzed by molecular machines is impressive and includes polymerization18 and asymmetric synthesis20.Molecular nanotechnology is a rapidly developing field that has, for several decades now, explored the possibilities of miniaturizing technology found on the macroscopic scale. One area of nanotechnology that has made tremendous progress is developing molecular machines; assemblies that can undergo controlled directional movement driven by external stimuli21. Popularity later grew thanks to Eric Drexler, who introduced the concept of a molecular assembler to nanotechnology; a device able to guide chemical reactions by positioning reactive molecules with atomic precision 22. It was proposed that such an assembler would be able to create any desired molecule with high selectivity and efficiency. In order to produce substantial amounts of product, Drexler envisioned that the molecular assembler should be able to self-replicate. After an adequate amount of assembler has been formed, it could be re-programmed to produce the required product. Drexler’s vision of a molecular assembler later became the subject of a heated and well-publicized debate between Drexler and Richard Smalley, who questioned the feasibility of such an assembler23. Although it is widely accepted that the type of molecular assembler envisioned by Drexler cannot be created, some artificial systems come close to this concept18. In addition, nature’s interpretation of a molecular assembler can be found in the translation process of mRNA into a protein by the ribosome. Information encoded in the DNA of cells is transcribed to mRNA, to which the ribosome can attach. While moving along the mRNA strand, the ribosome translates the encoded information into a growing peptide chain. This is done by base-pairing the mRNA to tRNA fragments, which have the required amino acids attached to them. This results in a step-wise growth of the polypeptide chain with an error rate of only 10−8 24. Although limited to polypeptides, this model of a molecular assembler can be used to produce any desired sequence of amino acids in high precision.Although the field of molecular nanotechnology started to make experimental progress in the 1990’s, the basic concepts were introduced by Richard Feynman in his iconic there’s plenty of room at the bottom lectureOne possible interpretation of Drexler’s design of an assembler in terms of chemistry, is shown in Fig. 27, we discovered a system that we realized shares many features of the previously envisaged molecular assembler, that system is described here. Early in the system’s lifecycle, the reaction between two (phase-separated) reagents produces surfactants that self-assemble into functional supramolecular aggregates; the assembler. These aggregates self-replicate , resulting in an exponential increase in their concentration. However, instead of self-replicating until all available starting materials are consumed, at some concentration the assembler reaches a steady state. During this steady state, the assembler produces a new product (in this case polymers). After the available starting materials have been depleted, the assembler consumes itself, resulting in self-destruction of the aggregates. Whereas this system in no way guides the polymerization reaction by positioning reactive molecules with atomic precision, as envisioned by Drexler, it functions by one of the reactants creating an encapsulated self-assembled space. Like Drexlers vision of an assembler, this confined space brings the reactants together, allowing for a reaction to take place. This system provides opportunities to design transient supramolecular systems capable of performing functions such as controlled uptake of a systems component and synthesis as well as polymerization.During the course of our work on self-replicating and out-of-equilibrium supramolecular systemsExperiments with the assembler were performed for various temperatures, pH, reactant concentrations, and stirring rates. In all cases, the assembler’s lifespan is characterized by the same three distinct phases; formation, function, and self-destruction. The transition between these three phases is fluid, showing no abrupt changes.1 in buffered water and hydrophobic dodecane-1,12-dithiol (compound 2). The reaction between the two reagents results in the formation of (bola)amphiphilic molecule 3. These amphiphiles self-assemble into supramolecular aggregates29, which constitutes the molecular assembler  is increased. This in turn, results in exponential growth of the concentration of 3, which is characteristic for autocatalytic reactions  trimer of disulfide. This process results in destruction of the amphiphilic building block. As this disulfide trimer, produced in the destruction step, still contains two thiol moieties, it can react with additional amphiphile to result in further chain growth. The driving force of the overall process is the conversion of high-energy disulfide 1 and thiol 2 to the thermodynamically favored disulfide polymer and thiol 4. In previous examples of autocatalytic reactions on monofunctional reactants, the formation of the bisalkyl-disulfide as the thermodynamic product marks the end of reactivity. In the case of compound 2, the presence of functional thiol groups on both ends of the alkyl chain that remain intact upon disulfide formation, constitutes a continuous driving force for further polymer growth. The combined processes during this phase can be regarded as a metabolic-cycle-like system, in which the assembler acts as the catalyst  is sufficient.In contrast to most autocatalytic reactions where product concentration increases until the starting materials are consumedyst Fig. . It prod1 is used, implying that the lifetime of the assembler is solely limited by the availability of 2. As the supply of compound 2 becomes smaller, the rate of formation of the amphiphile building block is reduced. As amphiphile destruction does not necessarily require 2, but can also use previously formed polydisulfides (which still contain thiol moieties), this process is hampered to a lesser extent -destruction of the assembler, making it an example of a transient species.In our experiments, an excess of compound ime Fig. . When ev37. This property has been exploited by others in the use of cell-penetrating poly(disulfide)s (CPD)38, which can be used to deliver drugs, proteins, and other compounds across cell-barriers into cells40. As the final destination of CPDs is highly dependent on polymer length, it is vital to exert control over polymer length40.During these experiments, the growing disulfide polymers precipitate from the solution and can be easily collected by filtration. Polydisulfides are a useful class of compounds because the disulfide bond is easily cleaved to cause polymer destruction when desired5) can be added to induce polymer capping41. Once 5 has been incorporated on both sides of a polymer molecule, no more thiols are present, stopping further polymerization. The ratio of monothiol (5) to dithiol (2) thus determines the final average chainlength  resulted in much larger variations, involving significant changes in the concentration of the assembler’s building blocks ([3]) throughout the experiment  is independent of [1], resulting in an overall increase in [3]. In addition the effect of pH on [3] was investigated. By increasing the pH, more negatively charged alkylthiol becomes present, potentially resulting in both faster formation and destruction of 3. The lifetime of the system is significantly shorter when pH is increased  on the polymerization process. Although the corresponding surfactant aggregated in the form of vesicles instead of micelles, these vesicles played no role in the (very slow) polymerization process (see SI).Although tuning the reaction time and/or using polymer capping provide adequate control over the average polymer size, the course of the experiments proved sensitive to several reaction parameters. Variation of the reaction temperature simply resulted in changes in polymerization speed and lifetime of the assembler (see SI). Tuning the reactant concentration  reaction, resulting in the production of the intended disulfide polymers. Although the assembler exists as a transient species, it can maintain its concentration at a constant level for extended periods of time. The consumption of bis-sulfide fuel keeps the system in this out-of-equilibrium state so long as reactant is available. It thereby appears able to maintain its concentration through a primitive form of dynamic kinetic stability. The sensitivity of the system to changes in [1] and pH, point toward the system being controlled by kinetic rather than thermodynamic factors. After one of the reagents, required for the assembler’s building block formation, gets depleted the assembler will continue to produce polymers, resulting in self-destruction of the system. Overall, this study provides an alternative direction for the design of functional systems based on balancing the incorporation, and consumption of the assembler’s building blocks in a series of steps, at least one of which must be irreversible (under the operating conditions) to provide the sense of directionality or asymmetry necessary to allow the assembler to operate in time. Although this system in no way allows the production of products by positioning reactive molecules with atomic precision, it relies on an encapsulated self-assembled space to direct the reaction between otherwise (phase)-separated reactants. This solves the common challenge in the design of molecular assemblers of realizing nanoproduction line-like processes, where overcoming diffusion and Brownian motion is difficult44. The concept of using encapsulation to create confined reaction environments is by itself not new. Confinement can result in altered chemical properties, which in turn can change reaction rates and selectivity45. Encapsulation has been extensively used on enzymes, using surfaces46 as well as three-dimensional compartments such as (giant) vesicles, protein cages or (reverse) micelles47. This approach is not limited to a single enzyme, but can involve a series of catalytically active species, creating a cascade of chemical reactions48. In contrast to our current work, the supramolecular structures providing the confinement in these examples are generally composed of a secondary compound, and not the  active species itself. Although our present system relies on supramolecular structures that spontaneously emerge and are controlled by reaction kinetics, other strategies which rely on external stimuli  can be envisaged that would provide spatiotemporal control over catalytically active aggregates.Although the exact molecular assembler envisioned by Eric Drexler will remain elusive to chemists, man-made analogs that approximate the concept have been recently devisedIn summary, a spontaneously emerging molecular assembler, able to produce disulfide polymers was discovered. Two phase-separated components react to form amphiphiles that self-aggregate into the assembler, in the form of supramolecular micelles. An initial period of exponential growth owing to self-replication is followed by a period of constant amphiphile concentration, which is sensitive toward experimental conditions, and heavily dependent on pH and reactant concentration. During this period, the assembler’s building blocks are both produced and consumed, whereas polymers are produced. By adjusting temperature and reaction time, or by adding a reagent that causes polymer capping, control over polymer size can be achieved. Once the starting material has been depleted, the assembler continues to use its own building blocks to allow further polymerization, resulting in self-destruction of the system. Throughout this process, the assembler uses its self-encapsulated space to bring the reactants for the polymerization process into contact. Although the reactants are not placed together with atomic precision (as for Drexler’s assembler), the confined space created by the self-assembly of one of the reactants drastically increases their contact. This effectively overcomes the necessity of diffusion and Brownian motion that would otherwise limit the feasibility of molecular assembly-line processes. This work thereby provides alternative directions for the development of functional supramolecular systems.1, which was obtained from Fluorochem) and were used without further purification. Synthesis of compounds 2 and 3 is described in the Supplemental information. Flash column chromatography was performed using silica gel . Thin layer chromatography analyses were performed on Merck Kiesegel 60 F254 0.25 mm precoated silica plates. All 1H NMR and 13C NMR spectra were recorded using a Bruker AVIII HD Nanobay 400 MHz spectrometer. Chemical shifts are given in ppm with respect to tetramethylsilane as an internal standard , 4 mL, 0.5 M TRIS buffer (pH 8.00) and a small stirring magnet (0.5 cm) were added to a 7 mL flat vial. The solution was stirred at 200 rpm and the vial was heated to 40 °C. After compound 1 had completely dissolved, 27 µl compound 2  was carefully added on top of the water layer as an oil (by heating it to 40 °C), and stirring was continued. Samples were taking regularly, by carefully extracting 20 µL of the aqeous layer. The extracted solution was immediately added to 1 mL of a 62 mM aqeous solution of maleimide  to quench all remaining thiols. Samples were analyzed as described in the ultra performance liquid chromatography (UPLC) section. To determine the growth of the disulfide polymers (present as a light yellow solid that floated on top of the water layer), some solid was carefully extracted using a spatula and subsequently dried using paper tissue. The average chainlength was determined based on the thiol to disulfide ratio, that could be accurately determined using 1NMR spectroscopy and GPC analysis , weight average molar mass (Mw), and dispersity (Ɖ) were calculated using Shimadzu LabSolutions GPC analysis program. Pure compound 2 (monomer) and three samples obtained at consecutive time points in the polymerization experiment were analyzed using both 1H NMR and GPC. In addition, a polymer sample of the final time point was depolymerized  and subsequently analyzed using NMR and GPC to show conversion back to the monomer. Comparison between the two methods, as well as the unmodified GPC data, are given in Supplementary Table GPC analysis of polydisulfides was performed on a Shimadzu LC-20AD instrument, equipped with a refractive index (RI) detector and two PSS SDV 5 μm linear M columns. High-performance liquid chromatography grade tetrahydrofuran was used as the eluent at 1.0 mL/min at 30 °C. Samples were passed through 0.2 μm olytetrafluoroethylene filters prior to analysis. Monodisperse polystyrene standards were used for calibration. Number average molar mass  was used. A mixture of water: MeCN: 5% TFA in H2O with a gradient of 93:2:5 changing to 0:95:5 over 5 min was used as the mobile phase. All peaks were analyzed at a wavelength of 330 nm  and were filtered using a microfilter (poresize 0.22 µm) before measurements. Samples were placed in a 1 mL plastic disposable cuvette and were heated to 60 °C using an equilibrated heating probe. Measurements on solution containing compound 3 gave a maximum intensity for a particle size of ~7 nm, regardless of the concentration, which sizes were further confirmed by iSCAT measurements .Aggregate sizes were analyzed with DLS measurements, using a Malvern Zetasizer Nano ZEN5600 apparatus. All samples were prepared by dissolving 3. Samples with varying concentrations of 3 in a 0.5 M TRIS buffer (pH 8.00) were prepared and their surface tension was determined at 25 °C (in triplicate) using a Kruss K10ST tensiometer. The CMC (0.011 mM) is the point at which an increase in the concentration of 3 no longer results in a decrease in the surface tension .Ring tensiometry measurements were used to determine the critical micelle concentration (CMC) of compound Supplementary Information


Electrospinning, as a promising platform in multidisciplinary engineering over the past two decades, has overcome major challenges and has achieved remarkable breakthroughs in a wide variety of fields such as energy, environmental, and pharmaceutics. However, as a facile and cost-effective approach, its capability of creating nanofibers is still strongly limited by the numbers of treatable fluids. Most recently, more and more efforts have been spent on the treatments of liquids without electrospinnability using multifluid working processes. These unspinnable liquids, although have no electrospinnability themselves, can be converted into nanofibers when they are electrospun with an electrospinnable fluid. Among all sorts of multifluid electrospinning methods, coaxial electrospinning is the most fundamental one. In this review, the principle of modified coaxial electrospinning, in which unspinnable liquids are explored as the sheath working fluids, is introduced. Meanwhile, several typical examples are summarized, in which electrospun nanofibers aimed for the environment remediation were prepared using the modified coaxial electrospinning. Based on the exploration of unspinnable liquids, the present review opens a way for generating complex functional nanostructures from other kinds of multifluid electrospinning methods. Electrospinning, a simple and straightforward method for preparing nanofibers ,2,3,4, iShown in However, among all the complex nanostructures, the core-shell should be the most fundamental and the most important one . It is nIn 2002, the first publication about core-shell structure was published based on electrospraying, or electrohydrodynamic atomization, a sister technique of electrospinning . Later, Shown in Apparently, the involvement of unspinnable sheath fluid can greatly promote the capability of creating complex nanostructures for functional applications. This is because the spinnable filament-forming polymers are very limited, slightly over 100 kinds by estimation , but theThe advantages of modified coaxial electrospinning over the traditional one lies on not only the great treatment capability about unspinnable fluids prepared from numerous raw materials, but also in creating different kinds of nanostructures. Shown in In the traditional blending electrospinning, the working process is regulated by a series of variables, including the properties of the solution, experimental parameters and surrounding conditions ,37,38,39No matter how many fluids are simultaneously electrospun using the multiple-fluid electrospinning processes, the above-mentioned parameters will similarly influence the working processes and the final products’ quality. The additional key parameter for the modified coaxial electrospinning should be the flow rates of sheath/core working fluids or the sheath-to-core fluid flow rate ratio. This is particularly vital for the modified coaxial electrospinning, in which solvent is exploited as a sheath fluid. Shown in R can be expressed as s/FcR = F, where Fs and cF are flow rates of sheath and core working fluids, respectively. When R is manipulated in a suitable range, the sheath solvent will make positive influences on the whole coaxial working process and benefit the quality of final solid nanofibers. However, when R value is beyond the limitation, i.e., the sheath solvent flow rate is too large, the beads-on-a-string or spindles-on-a-string may be generated. This is because the sheath solvent can’t be completely exhausted in time and in turn influence the solidification of the core fluid [The sheath-to-core fluid flow rate ratio re fluid .Based on coaxial electrospinning, the applications of electrospun nanofibers from unspinnable fluids for pharmaceutical applications have been broadly reported, which can provide different kinds of drug-controlled release profiles such as immediate release, sustained release and multiple-phase release ,44,45,46+-protein interactions for avoiding the frequent clogging, and thus ensure a continuous and robust preparation process. On the other hand, during the applications, this interaction is exploited to absorb lead ions from the polluted water for environmental remediation.It is well-known that biomacromolecules have the potential to become fine adsorbents for the removal of heavy metals ,48. Partv/v) ethanol/water mixture was surrounded by a transparent and unspinnable sheath fluid composed of 0.5% (w/v) sodium lauryl sulfate in 75%/25% (v/v) ethanol/water. An enlarged image shown in The difference between the traditional single-fluid blending process and the multiple-fluid process mainly lie in how to guide the fluids in an organized manner. Shown in 2+-polluted water. Adsorption results indicated that the adsorption process can be described using the pseudo-second-order model. Isotherm data fitted well to the Langmuir isotherm model, with a maximum adsorption of 89.37 mg/g for the nanoribbons prepared from the modified coaxial process. Desorption results showed that the adsorption capacity can remain up to 82.3% even after 5 cycles of re-use. These positive results apparently demonstrated that the unspinnable working fluids utilized in the modified coaxial electrospinning were able to stabilize the preparation process, to further downsize the nanoproducts’ width, and to improve their functional performances in environmental remediation.Cross-sections of different zein nanoribbons were prepared, which are compared in For the traditional single-fluid electrospinning, the filament-forming polymeric matrices must have enough molecular weight to support enough physical entanglements for forming electrospun fibers within their electrospinnable windows . Few repHowever, in a recent investigation, an oligomer Poly(2-aminothiazole) (PAT) was synthesized, and then this functional oligomer solution (0.1 g of PAT was dissolved in a mixed solvent of 2 mL DMAC and 2 mL ethanol) was mixed with a cellulose acetate  solution to create composite nanofibers. The sheath fluid was pure solvent acetone, which was pumped quantitatively through another booster to another inlet of a concentric spinneret for a smooth working process.The Hg(II) adsorption properties of the PAT/CA composite nanofiber mat from polluted water was tested. The adsorption capability was highly dependent on the pH value of solution, with a maximum at pH 6.5. The isotherm data fitted well to the Langmuir isothermal model. The adsorption kinetics data fitted well to the pseudo-second-order model. At a temperature of 298 K, the maximum adsorption capacity of 177 mg/g for PAT/CA fiber membrane with a very low PAT percentage (6.5 wt.%) was calculated. Through conversion, the adsorption capacity of the PAT in the composite fiber membrane is much larger than that of the nanoparticle-type PAT. The desorption experiments revealed that the desorption percentage remained at 80.2% after being used for three times. Here the unspinnable sheath solvent smoothed the electrospinning processes to prevent the spinneret clogging of CA, resulted in solid phase separation to lead to a surface distribution of functional ingredient, i.e., PAT particles, and thus provided an improved functional performance of removing Hg(II) from water. Certainly, the PAT-loaded nanofibers, as a non-woven mat, were more convenient than the PAT powders themselves for environmental remediations and less possibility of secondary pollutions due to nanoparticles themselves.Through the modified coaxial electrospinning, it is possible to directly distribute the inorganic particles on the surface of polymeric nanofibers, in which nanosuspension can be explored as the sheath working fluid. Unfortunately, the inorganic nanoparticles are very easy to precipitate during the working processes, which may stop the continuous preparation. However, a combined strategy based on the modified coaxial electrospinning using salt solution as a sheath working fluid can overcome this issue. w/v) polyacrylonitrile (PAN) in N, N-dimethylacetamide (DMAc) as the electrospinnable core working fluid, a 10% (w/v) AgNO3 DMAc solution was employed as a sheath fluid, a modified coaxial process was successfully carried out to create the salt coating PAN nanofibers. The electrospun nanofibers were then exposed to 254 nm ultraviolet light for 24 h to reduce the Ag+ to Ag nanoparticles (NPs). The prepared products are shown in With a solution of 15% (3 solution of 0.1 was not able to follow the core PAN fluid jet during the fast bending and whipping process. This results in a separation of the sheath AgNO3 solution and a non-uniform deposition of AgNO3 on the surface of nanofibers (3 on the surface of nanofibers. When the Ag+ on the surface of PAN nanofibers was reduced to Ag NPs, the nanofibers from a larger sheath fluid flow rate ratio had a more uniform but smaller size, and a tighter distribution than those from a smaller ratio (+ to Ag NP on the surface of nanofibers should be beneficial to long-term stability of the system and prevent Ag leaching from the fibers. These Ag NPs-loaded nanofibers showed fine antibacterial performances and should be candidates for friendly ecological environmental applications.The influence of the key parameter, i.e., the sheath-to-core fluid flow rate ratio was investigated. The results demonstrated that the smaller flow rate of AgNOnofibers c,d. Thuser ratio c,e. It iThe popularity of electrospun nanofibers has a close relationship with their small diameters, huge surface area and large porosity ,53,54. HAs the fast development of electrospinning, modified coaxial electrospinning is upgraded from the traditional one and shows strong vitality. Its usefulness lies in both stabilizing working process for creating high quality monolithic nanofibers, but also in generating solid core-shell nanostructure and nanocoating. What is more, almost any kinds of functional raw materials, regardless of filament-forming property, can be treated into nanofibers to exert their functional performances by taking advantages of the unique properties of electrospun non-woven mats. The above-mentioned several examples demonstrated that the modified coaxial processes can be utilized to manipulate the nano products’ shape and size facilely, to incorporate oligomer into the nanofibers, and to coat inorganic salt on the surface of polymeric nanofibers. These preparations are impossible using the traditional processes. Based on the combination of modified coaxial electrospinning and the tremendous active ingredients for treating pollutants in our environment, a new platform can be built to develop more and more functional nanostructures for environmental remediation. The new nanomaterials and the corresponding applications resulted from the modified coaxial electrospinning using unspinnable liquid as a sheath working fluid is still at its infant time period. It is anticipated there are more and more possibilities in future, an unspinnable fluid can be treated with one or more spinnable fluids in many ways such as as tri-axial ,63 and sJust as the traditional electrospinning, one of the most important challenges for the modified coaxial electrospinning is to create nanofibers on a large scale ,72,73. A


Context: It has been reported that isoprostanes (IPs) have a role in the pathophysiology of ductus arteriosus during the fetal and neonatal period. Our aim in this study was to assess if urinary IPs (uIPs) levels correlate with the risk of developing a hemodynamically significant patent ductus arteriosus (hsPDA) in preterm infants.Materials and methods: Infants with 23 + 0 – 33 + 6 weeks of gestational age and respiratory distress syndrome (RDS) were consecutively enrolled. Urine samples were collected on the 2nd and 10th day of life (DOL) for uIPs measurement. Echocardiography for hsPDA diagnosis was performed between 24 and 48 h of life. Regression analysis was performed to assess the correlation between uIPs and hsPDA. Receiver operating characteristic (ROC) curve analysis was used to evaluate the accuracy of the uIPs in predicting the occurrence of hsPDA.Results: Sixty patients were studied: 33 (55%) developed a hsPDA, 27 (45%) had ibuprofen hsPDA closure, and six (10%) required surgical closure. uIPs levels decreased from the 2nd to the 10th DOL. Adjusted regression analysis demonstrated that uIPs on the 2nd DOL were associated (p = 0.02) with the risk of developing a hsPDA. A cut-off level of 1627 ng/mg of creatinine of uIPs predicted the development of a hsPDA with a sensitivity of 82% and a specificity of 73%.Conclusion: Early measurement of uIPs on the 2nd DOL is a reliable biomarker of hsPDA development and, alone or combined with other markers, might represent a non-invasive tool useful for planning the management of PDA in preterm infants. Patent ductus arteriosus (PDA) is a frequent complication in preterm infants with respiratory distress syndrome (RDS) and 60–70% of preterm infants of <28 weeks' gestation receives medical and/or surgical treatment for PDA . The proCurrently, functional echocardiography is the best tool to diagnose PDA in preterm infants, to monitor its progress, and to determine its treatment . In factRecently, it was reported that isoprostanes (IsoPs) have a role in DA closure. IsoPs derive from the free radical-mediated peroxidation of phospholipid-bound arachidonate and, although they are mainly known as a biomarker of oxidative stress, they might induce physiological effects during the fetal and neonatal period, such as DA vascular constriction mediated by stimulation of thromboxane receptors . Van derOn the basis of these considerations we hypothesized that uIPs level might predict the development of a hemodynamically significant PDA (hsPDA). Thus, our aim was to assess uIPs levels in a cohort of very preterm infants and correlate them with the risk of hsPDA development.A prospective center-based study was carried out from June 2014 to December 2014 at the neonatal intensive care unit of Careggi University Hospital of Florence. The study was approved by the local ethics committee. Infants with gestational age from 23 + 0 to 33 + 6 weeks with respiratory distress syndrome (RDS) were consecutively enrolled after written parental informed consent. Exclusion criteria were major congenital malformations, chromosomal disorders, inborn errors of metabolism, and acute renal failure.Urine samples of 2 ml were collected from each infant on the 2nd and 10th day of life (DOL) by placing a gauze in the infants' diaper. The samples were frozen and maintained at −80°C until analysis, which was performed at the Laboratory of Oxidative Stress of the University of Siena, Italy. Urinary isoprostanes (uIPs) were measured using tandem mass spectrometry (GC-MS) according to the methodology described by Casetta et al. . UrinaryAll enrolled infants underwent echocardiography between 24 and 48 h of life. The diagnosis of hsPDA requiring pharmacological treatment was made by echocardiographic demonstration of a ductal left-to-right shunt, with a left atrium to aortic root ratio >1.3 or a ductal size >1.5 mm , excludi2) on 2nd and 10th DOL, occurrence and duration of non-invasive respiratory support , and mechanical ventilation , sepsis, BPD, necrotizing enterocolitis (NEC), intraventricular hemorrhage (IVH), periventricular leukomalacia (PVL), retinopathy of prematurity, and stay-in-hospital duration. Sepsis was defined as positive blood culture. BPD was defined as oxygen requirement at 36 weeks of PMA , rate and percentage, or median and interquartile range (IQR). The Student's 2 at samples collection. In the final multivariable regression model, relevant perinatal and neonatal factors that showed an association with uIPs with alpha level <0.05 were included. Data analysis was performed using IBM SPSS Statistics version 20 .Univariate regression analyses were performed to assess the correlation between uIPs and PDA. The following variables were entered into the model: GA, BPD, NEC, IVH, mechanical ventilation, and mean FiOTo analyze the impact of the ‘uIPs level on the 2nd DOL on the occurrence of hsPDA, we used ROC (receiver operating characteristic) curve analysis. The test's ability to classify patients as those who will develop hsPDA or not is represented by the area under the ROC curve (AUC). The AUC can be in the range of 0.5–1.0, wherein an AUC of 0.5 indicates that the classification model is of no value, and a value of 1.0 indicates perfect diagnostic accuracy. In turn, the cut-off point of the ROC curve indicates the uIPs level that gives the most true and the least false indications of hsPDA development, and therefore has the best predictive power.Post hoc analyses demonstrated that the population size of our study allows an 85% statistical power to detect as significant (p < 0.05) the difference between uIPs levels measured on the 2nd and the 10th DOL.n = 60) are summarized in p < 0.01)  . Signifi < 0.01) .n = 33) of patients developed a hsPDA, 45% (n = 27) had ibuprofen hsPDA closure, and 10% (n = 6) failed pharmacological treatment and underwent surgical closure. Eleven patients (18%) received two courses of ibuprofen. Infants with hsPDA had lower GA and Apgar score at 5 min, higher incidence of IVH, PVL, CLD, ROP, NEC, sepsis, mortality, and lengthier stay in NICU compared to infants without PDA (Fifty-five percentage (hout PDA .p < 0.01) (p < 0.05) with a lower GA and a higher occurrence of hsPDA. After adjusting for GA, BPD, IVH, NEC, and mean FiO2 the association between uIPs and hsPDA remained significant .Infants without PDA showed a significantly lower uIPs in comparison with infants with ibuprofen-treated hsPDA and who needed surgical closure (after failure of ibuprofen therapy) ( < 0.01) . Univarip < 0.0001), showing the best prognostic cutoff point at uIPs = 1627 ng/mg of creatinine, with a sensitivity of 82% and a specificity of 73%  had hsPDA on the 10th DOL. Urinary IPs were similar in infants with or without hsPDA . UnivariThis is the first study to investigate the possible predictive role of uIPs levels on the development of hsPDA. We found that uIPs on the 2nd DOL were higher in infants who developed hsPDA than in infants who did not, and were higher in infants who had hsPDA refractory to drug therapy requiring surgical closure than in infants who had pharmacological closure of hsPDA. It is noteworthy that the correlation between uIPs levels on the 2nd DOL and the development of hsPDA remained statistically significant after adjusting for the main potential confounding factors. Thus, uIPs levels might be a valuable biomarker of hsPDA, as confirmed by ROC analysis which demonstrated that a uIPs cutoff level of 1627 ng/mg of creatinine on the 2nd DOL predicts a hsPDA with a sensitivity of 82% and a specificity of 73%.Isoprostanes are a product of free radical-induced injury by peroxidation of lipids and their synthesis can easily occur in preterm newborns due to their limited antioxidant defenses and large production of free-radicals according to several factors, such as hyperoxia, inflammation, infections, free iron release, and activation of arachidonic acid cascade . The rolOn the 10th DOL uIPs decreased in comparison with the 2nd DOL levels, and differences between infants without hsPDA, with hsPDA responding to pharmacological therapy, or requiring surgical closure disappeared. This reduction might be due to the maturation of infants who achieved a more favorable balance between anti-oxidant and pro-oxidant factors and overcame the acute phase of respiratory distress syndrome, in agreement with previously reported inverse correlation between IPs synthesis and gestational age . MoreoveOur results confirmed the findings of Longini et al. who studied 43 infants with gestational age <33 weeks and hsPDA, and found that uIPs decreased after PDA closure with ibuprofen . MoreoveLimitations of our study include the small number infants with hsPDA which precludes the possibility of using regression analysis to evaluate the possible correlation between uIPs levels and the risk of undergoing surgical closure of hsPDA. Moreover, our population has a wide range of gestational ages which might affect the range of uIPs levels. However, we are confident that the high quality of data analyses could contribute to minimize the effects of this limit on our results.In conclusion, we found that uIPs levels measured on the 2nd DOL are significantly correlated with the risk of developing a hsPDA. We demonstrated that a uIPs cutoff level of 1627 ng/mg of creatinine can predict a hsPDA with a sensitivity of 82% and a specificity of 73%. Early measurement of uIPs, alone or combined with other markers, might represent a reliable, non-invasive biomarker useful for planning the management of PDA in preterm infants.We found that early and painless measurement of uIPs levels predicts the development of a hsPDA with a sensitivity of 82% and a specificity of 73%. Since the proper management of PDA is continuously debated between neonatologists, the discovery of reliable biomarkers of hsPDA might be useful in the clinical practice in driving its management together with the findings of echocardiography. Thus, if a rapid method for their dosing might be developed, uIPs could be one of these promising biomarkers.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Pediatric ethics committee of Tuscany region. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.CC, MT, IC, VL, ML, FB, GB, and CD have made substantial contributions to conception and design of the study, critically revised the article, and have given their final approval of the version to be published. CC and CD performed the data statistical analyses and wrote the manuscript. CC, MT, IC, and VL collected data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antimicrobial resistance (AMR) is a critical global health threat with a disproportionate impact on low-income and middle-income countries (LMICs) due to their higher burden of infections, reduced laboratory surveillance infrastructure and fewer regulations governing antimicrobial use among humans or animals. While there have been increasing descriptions of AMR within many LMICs in WHO’s Western Pacific and South East Asian regions, there remains a paucity of data from Pacific Island countries and territories (PICTs). The PICTs represent 22 predominantly middle-income countries and territories with a combined population of 12 million people and 20 official languages, spread over hundreds of separate islands spanning an area corresponding to more than 15% of the earth’s surface. Our paper outlines the present state of the evidence regarding AMR in PICTs—discussing the present estimates of AMR and their accompanying limitations, important drivers of AMR, as well as outlining key priorities and potential solutions for tackling AMR in this region. Significant areas for action include developing National Action Plans, strengthening laboratory surveillance systems and educational activities targeted at both healthcare workers and the wider community. Ensuring adequate funding for AMR activities in PICTs is challenging given competing health and environmental priorities, in this context global or regional funding initiatives such as the Fleming Fund can play a key role. There is a paucity of antimicrobial resistance (AMR) data from Pacific Island countries and territories (PICTs), especially for pathogens deemed ‘critical’ by WHO.Important potential drivers of AMR in PICTs include antimicrobial selection pressure in humans and animals, healthcare transmission of AMR pathogens, poor community understanding of AMR and increasing population movement and travel.excess, without jeopardising antimicrobial access for those who need them.In addressing AMR, a challenge for PICTs is trying to curtail antimicrobial Key priorities for PICTs responding to AMR should include developing National Action Plans, strengthening laboratory surveillance systems and educational activities targeted at both healthcare workers and the wider community.Providing funding for AMR can be difficult for PICTs in the setting of multiple competing health and environmental priorities; global or regional funding initiatives can play an important role.The emergence of antimicrobial resistance (AMR) represents a growing health threat that could undo decades of medical progress. Previously innocuous infectious or routine surgical procedures, such as Caesarean sections, may become life-threatening events in a ‘postantibiotic era.’3The Pacific Island countries and territories (PICTs) consist of 22 members of the Secretariat of the Pacific Community, with the omissions of Australia, New Zealand, USA and France . All are6The impact of AMR has been well described in other countries from the WHO Western Pacific Region10Escherichia coli resistant to third-generation cephalosporins was 0% in Kiribati (n=72), 12% in Fiji (n=2895), 24% in  and 77% in Micronesia (n=158).10The existing knowledge of AMR in PICTs was summarised in a recent systematic scoping review identifying 65 relevant studies published since 1950.Klebsiella pneumoniae surveillance isolates from a Fiji hospital were resistant to carbapenems.To date, carbapenem resistance—one of the most challenging and concerning examples of AMR—appears to be uncommon. For example, less than 1% of 2175 Accurately defining the magnitude of the problem of AMR in PICTs using existing literature has many challenges . First, Box 1Limited volume of dataSmall number of published studies.Small number of tested isolates.Acinetobacter baumannii).Paucity of surveillance data especially notable for some ‘critical’ bug/drug combinations  or subgroups .access to antimicrobial susceptibility testing.Under-representation of populations with limited or absent Limited quality of dataMany PICT laboratories lack rigorous quality assurance processes.Many PICT laboratories face shortages of necessary equipment or trained staff.Subsequent potential for AMR to be missed or misclassified.Long-term successful AMR surveillance in PICTs is possible, however, requires significant resources, infrastructure, capacity building and support. One example is the WHO Gonococcal Antimicrobial Surveillance Programme, active in certain PICTs for over 25 ye ars.17From the global literature, we have extrapolated some of the main drivers that are likely to influence the development and spread of AMR in the Pacific. Under the headings below we have provided context and highlighted the existing state of knowledge in PICTs. A schematic representation of drivers of AMR and potential solutions is presented in Antimicrobial use can exert selective pressure on micro-organisms, promoting the development and persistence of AMR isolates. Human antimicrobial consumption continues to rise globally, driven mostly by increases among LMICs.23Campylobacter carriage among 97% of chicken neck-skins at the point of slaughter. Of concern 47% of isolates were resistant to ciprofloxacin, an important antimicrobial regularly used in humans.26Use of antimicrobials in animals can also contribute to AMR. Animals may be given antimicrobials not only to treat existing infections, but also for prophylaxis, metaphylaxis (treating an entire group after a subset develop symptoms or disease) or growth promotion. There is little data on rates of antimicrobial use in animals in PICTs. Only five PICTs are member countries of the World Organisation for Animal Health (OIE), and the OIE’s annual report on regional antimicrobial use in animals provides aggregated data only, not country-level data.AMR acquisition can occur not only due to antimicrobial selective pressure, but also the direct transfer of multidrug-resistant organisms (MDROs) between humans, animals or the environment. This is a particular concern in healthcare settings, where high volumes of antimicrobials are used, infections are more likely to be due to MDROs and organisms can easily spread from patient to patient either directly or via healthcare workers and hospital equipment. Healthcare-associated infections are at least twice as common in LMICs than in high-income settings.Poor community knowledge about the purpose of antimicrobials, and the risks of AMR, can drive inappropriate demand for these products. Similarly, unnecessary prescribing of antimicrobials—for instance, for syndromes that are predominantly viral in aetiology—also contributes to excessive antimicrobial use. A survey of 112 Samoans living in New Zealand demonstrated common misconceptions around the role of antimicrobials: over 80% believed they could treat common colds, and very few (8%) were aware of the concept of AMR.32Traditionally, PICTs experienced low volumes of airline traffic due to their geographical dispersion, small populations and high costs of fuel and parts. However, the recent emergence of low-cost carriers has led to greater capacity on existing airline routes and the introduction of new routes.Another potential transmission source of AMR for PICTs is medical tourism.AMR is an important global health threat to which PICTs are particularly vulnerable. Globally, the danger of AMR has been recognised and efforts made to mobilise a response. The WHO’s ‘Global Action Plan on AMR’ is the internationally agreed framework for action.The Global Action Plan calls on countries to develop National Action Plans (NAPs) that are aligned with the global strategy but take into account local resources, national priorities and local governance arrangements.Regular and reliable surveillance should be the cornerstone of any AMR response. It provides an accurate understanding of AMR prevalence and changes over time, permitting the development of targeted AMR policies informed by local data. However, as highlighted earlier, AMR surveillance in PICTs can be variable, hindered by a lack of trained staff or consistent access to the necessary equipment and materials. Currently data are often recorded in hard copy, and cannot rapidly be linked to a centralised system or other facilities. PICTs should aim to develop their national laboratory infrastructure so that AST can be reliably performed on WHO Priority PathogensCorrect selection of antimicrobials by healthcare providers is also important to reduce AMR. Such stewardship is assisted by targeted education of healthcare workers, and developing standard treatment guidelines.Antimicrobial prescribing can also be optimised by rapidly differentiating between bacterial infections and other febrile illnesses. Studies from LMICs in South East Asia have demonstrated modest reductions in antimicrobial use with point-of-care testing for C-reactive proteinAwareness campaigns can help reduce unnecessary demand for antimicrobials, especially in the outpatient setting.excess should not concurrently deny antimicrobial access to those patients who require them.Lastly, when considering stewardship initiatives in PICTs, it is essential to recognise that more deaths currently occur in LMICs due to inadequate access to antimicrobials, rather than AMR.Allocating adequate funding towards AMR in PICTs is challenging. Competing national budget priorities—for instance, the rapid increase in non-communicable diseases,Globally, there has been a significant increase in funding for AMR research and initiatives in LMICs, most prominently through the Fleming Fund.47The small size and relative isolation of PICTs can be advantageous when monitoring antimicrobial consumption: there is little parallel importation of medicines, and national coverage of dispensaries is easier to achieve. Presently, many PICTs monitor their stockpiles and movements of medicines using software such as mSupply,49There is scope for this existing digital data to be analysed and presented as a proxy for antimicrobial consumption: providing accurate, real-time data down to the level of individual regions or clinics. In addition to the ability to contribute data to WHO’s Global Programme on Surveillance of Antimicrobial ConsumptionRates of both AMR and antimicrobial consumption are rising globally, especially within LMICs. Such increases are likely being replicated within PICTs, which face similar challenges including high rates of infectious diseases, low community knowledge around antimicrobials and AMR, and limited laboratory surveillance capacity. However, precise estimates of AMR prevalence within humans, and especially within animals, are hindered by a scarcity of data.WHO has taken a leading role in coordinating a global response to AMR, most notably through its Global Action Plan. Within PICTs, we suggest that addressing AMR should initially focus on: (1) optimising governance and cross-sectoral collaboration through the establishment of NAPs, (2) optimising surveillance through strengthening laboratory capacity and (3) optimising antimicrobial awareness through community education activities and provision of standard treatment guidelines for clinicians. Additionally, we believe PICTs have the potential to rapidly develop advanced surveillance systems for antimicrobial consumption in humans by building on existing IT infrastructure and improving laboratory systems. While providing reliable funding for AMR activities may be challenging for PICTs in the setting of multiple competing health and environmental priorities, global or regional funding initiatives can play an important role. Technical and financial support for sustainable collaborations and international partnerships with organisations with established AMR surveillance systems can help reduce the barriers faced by PICTs.


A kinetic investigation found that the orders of polymerization with respect to EBiB, Pd NP and MMA were 0.23, 0.50, and 0.58, respectively. Proton nuclear magnetic resonance (1H NMR) combined with matrix-assisted laser desorption ionization time of flight mass spectroscopy (MALDI-TOF) and gel permeation chromatography (GPC) were used to prove that the macromolecular chain had an end-group of EBiB residue. The electron spin resonance (ESR), transmission electron microscope (TEM), and X-ray photoelectron spectroscopy (XPS) results reveal that the reaction of EBiB with Pd NPs caused a bromo atom (Br) transfer from EBiB to Pd NPs and resulted in the generation of EBiB residue radical to initiate the polymerization of MMA and the formation of PdIIBr2 on the surface of Pd nanoparticles.A facile and versatile approach for the synthesis of ultrahigh molecular weight poly(methyl methacrylate) (PMMA) at mild conditions was developed. Certain organic halides combined with a catalytical amount of palladium nanoparticles (Pd NPs) were found to be very effective in initiating polymerizations of methyl methacrylate (MMA), methyl acrylate, vinyl acetate and other vinyl monomers. An ultrahigh molecular weight PMMA with a number-average molecular weight of 4.65 × 10 It is reasonable for the polymerization to proceed faster at higher temperatures according to the Arrhenius equation, but the chain transfer and chain termination rate constants also increase with the increase in temperature, resulting in a decrease in PMMA molecular weight. These results indicate that the combination of EBiB with catalytical amount  of Pd NPs provided a facile and novel approach for the synthesis of ultrahigh molecular weight PMMA at mild conditions. The combination was also found to polymerize MA, BA, and VAc , ethyl 2-bromopropionate (EBP), 2-bromoisobutyric acid ethyl ester (EBiB), and carbon tetrachloride  was prepared at [MMA]/[EBiB]/[Pd NP] = 60:17:1 at 70 °C and then analyzed by 1H NMR (3), methylene (–CH2–), and methoxyl groups (–OCH3), respectively, in the repeating unit of PMMA (2CH3). The molecular weight of PMMA was therefore calculated to be 2530 Da from the integration of peak g and peak f, which is very close to the molecular weight of 2620 Da measured by GPC, indicating that the PMMA macromolecular chain was end capped with EBiB residue. According to the literature /[initiator]/[copper catalyst] = 1.72 × 103:1:2.5, and a EBiB/Pd NP initiated polymerization at [MMA]/[EBiB]/[Pd NP] = 1.72 × 103:1:0.1, respectively. Obviously, the conventional radical polymerization presented strong typical signal of carbon-centered radicals trapped by PBN from the propagating PMMA chain /[EBiB]/[Pd NP] = 1.05 × 105:76:1 at 70 °C. The orders of polymerization with respect to EBiB, Pd NP and MMA were found to be 0.23, 0.50, and 0.58, respectively. It was found that the polymerization of MMA was initiated by the radicals produced from the reaction of EBiB with Pd NPs. The reaction involved a bromo atom (Br) transfer reaction from EBiB to Pd NPs, which caused the generation of EBiB residue radical to initiate the polymerization and the formation of PdIIBr2 on the surface of Pd NPs.In this paper, a facile and versatile approach for the synthesis of ultrahigh molecular weight PMMA at mild conditions has been developed by the polymerization of MMA using EBiB as an initiator in the presence of a catalytical amount (10.1 ppm) of Pd NPs. An ultrahigh molecular weight PMMA with a number-average molecular weight of 4.65 × 10


Accurately calculating the entropy of liquids is an important goal, given that many processes take place in the liquid phase. Of almost equal importance is understanding the values obtained. However, there are few methods that can calculate the entropy of such systems, and fewer still to make sense of the values obtained. We present our multiscale cell correlation (MCC) method to calculate the entropy of liquids from molecular dynamics simulations. The method uses forces and torques at the molecule and united-atom levels and probability distributions of molecular coordinations and conformations. The main differences with previous work are the consistent treatment of the mean-field cell approximation to the approriate degrees of freedom, the separation of the force and torque covariance matrices, and the inclusion of conformation correlation for molecules with multiple dihedrals. MCC is applied to a broader set of 56 important industrial liquids modeled using the Generalized AMBER Force Field (GAFF) and Optimized Potentials for Liquid Simulations (OPLS) force fields with 1.14*CM1A charges. Unsigned errors versus experimental entropies are 8.7 J K Molecular liquids are present in numerous systems in chemistry and biology. However, methods to calculate their entropy are scarce or limited in scope. Entropy quantifies the probability distribution of quantum states of a system and, together with energy, determines a system’s stability. The most common route used to determine entropy is indirect, being as a difference with respect to a reference state, typically the ideal gas or a non-interacting set of atoms. The entropy difference may be extracted from integrated heat capacity changes or from the Gibbs energy difference, either as its temperature derivative or as a difference with enthalpy . While tThe probability distributions to evaluate entropy are typically over the coordinates of the system, which may be Cartesian coordinates, bonds-angles-dihedrals, or interatomic distances. Histogram-based methods, because of their arbitrary bin-widths, can only give the entropy difference relative to a reference, which is typically the uniform distribution. Even then, the entropy difference may be unrealistic for strongly interacting systems such as those with covalent bonds because of the omission of quantum effects which necessarily keep the entropy non-negative. For this reason, histogram methods are often restricted to softer degrees of freedom such as dihedrals or atomic distances. The simplest approach ignores coordinate correlations by considering each coordinate separately, for example, in dihedral angles . Higher-Significant simplification of the theory, greater speed of convergence and a route to the direct calculation of entropy is provided by assuming a multivariate Gaussian probability distribution . EntropyHere we extend MCC to calculate the entropy of 56 important industrial liquids. These represent a class of system which no other method has been capable of calculating entropy except for the 2PT method, which has been tested on a smaller subset of 14 liquids , argon , water , carbon The entropy of molecular liquids is well captured at two different length scales : the molIn the cell approximation the potential energy surface is partitioned into energy wells, and in the multiscale approximation this partitioning is done at the molecule and united-atom levels. This brings about two kinds of entropy term: vibrational relating to the average size of the energy wells, termed a cell, and topographical relating to the probability of the energy wells. The vibrational term at each level is further partitioned according to the translational (transvib) and rotational (rovib) degrees of freedom. The translational component of the topographical entropy at the molecular level is zero for a pure liquid because exchanging identical molecules leads to no change. The rotational topographical entropy (topo) at the molecule level is termed the orientational entropy. At the united-atom level the translational topographical entropy is the conformational entropy, while the rotational component, corresponding to hydrogen-bond arrangements, is negligible for the liquids studied here. The total entropy per molecule for a liquid is therefore taken as the sum of six termsh is Planck’s constant, T is temperature and i and j ranging over the three axes m is the molecule’s mass, and All four vibrational entropy terms are calculated in the harmonic approximation using the equation for a collection of ous work , translaed using ,403)νiximation ,35 whereillatorsSvib=kB∑iνiximatioN is the number of united atoms and the six vibrations removed correspond to the six largest eigenvalues which are already accounted for as molecular translation and rotation. The procedure at the united-atom level to evaluate Equation  is similEquation  for unitEquation  for unitThe molecular topographical entropy Equation  only hasNc using (4)SMtoprotation , the equMolecules with a single united atom may still have orientational entropy at the atom-level if their hydrogens sufficiently break symmetry, so as to form distinct energy wells. Ammonia is included in this category, as water had been earlier , but met) method ,55 accort), gauche− ..SUAtopo,elementsρij=pijriy matrix , the totequals 1 , the tradihedral , its eigThe entropy was calculated for a series of 56 liquids using molecular dynamics simulations. All simulations were carried out with the sander module of the AMBER 14 simulation package . Each syFor equilibration, each system was minimized with 500 steps of steepest descent minimization, thermalized in a 100 ps molecular dynamics simulation at constant volume and temperature using a Langevin thermostat with a collision frequency of 5 psd values , in whicThe experimental entropies for most liquids were taken from the NIST Chemistry Webbook . If more ammonia , chlorof ammonia , methane ammonia , hydroge ammonia , hydroge ammonia  and carb ammonia . Values  ammonia ,70   8) into We have presented the multiscale cell correlation method to calculate the entropy of 56 molecular liquids from molecular dynamics simulations. The entropies are in excellent agreement with experiment for the OPLS and GAFF force field, with GAFF performing slightly better. Agreement is better than that of the 2PT method, which can also calculate the entropy of molecular liquids. The components of entropy give an insightful and intuitive understanding of the values obtained. With suitably chosen levels of hierarchy, the method is readily scalable to larger and more complex systems.


Delayed parkinsonism and dystonia are recognized phenomena in osmotic demyelinating syndrome (ODS). Dopamine receptor agonists and levodopa have been reported to benefit select patients.We report a patient with ODS with severe pseudobulbar deficits, parkinsonism and dystonia, poorly responsive to levodopa, who experienced a remarkable improvement with pramipexole.A marked response to pramipexole with lack of response to levodopa suggests a pre-synaptic source for his deficits coupled with injuries to non-nigral compensatory structures.This case highlights a dramatic response of osmotic demyelination-induced parkinsonism/dystonia to pramipexole. A lack of response to levodopa suggests deficits in the pre-synaptic nigral as well as non-nigral compensatory structures. Osmotic demyelinating syndrome (ODS) occurs as a result of overly rapid correction of hyponatremia . DependiWhile magnetic resonance imaging (MRI) is helpful in evaluating suspected ODS, the findings may take several weeks to become apparent . Typical2This case report presents a patient presenting with pseudobulbar deficits, parkinsonism and dystonia from ODS, whose symptoms were resistant to levodopa but markedly responsive to pramipexole.A 42-year-old man with history of von Willebrand disease type 1 was prescribed desmopressin after a recent nasal septoplasty in the setting of epistaxis post-surgery. Within days of discharge, patient presented to the hospital with a seizure. His sodium at the time of presentation was 122 mEq/L which was corrected to 132 mEq/L within a period of 24 hours. After transfer to our institution, ODS was confirmed on MRI which showed T2 hyperintense lesion at the central pons as well as T1 hypointensities at the bilateral lentiform nuclei . He showed gradual response on this regimen with improvement in his pseudobulbar palsy and facial masking. When the dose of pramipexole was increased to 1 mg three times daily there was a dramatic improvement in speech and movements. He was able to speak clearly in long sentences though the volume of speech was observed to be reduced. Facial masking and voluntary movements were markedly improved  was added without significant benefit. Due to the lack of efficacy with prior regimen, pramipexole was re-introduced and he responded again to modest dose (1.5 mg daily) Video .The pathophysiology of ODS is complex. Briefly, the cerebral adaptation to serum hyponatremia occurs via multiple mechanisms. The serum hypotonicity from the initial hyponatremia causes water to flow across the blood-brain barrier, increasing the brain water content as well as the intracellular water content . AstrocyTreatment of ODS remains largely symptomatic and supportive. Treatment with thyrotropin releasing hormone, plasma exchange, methylprednisolone and intravenous immunoglobulin has not shown clear evidence of efficacy . SymptomLevodopa supplementation allows the presynaptic neurons to release more dopamine into the synapse, resulting in greater stimulation of the dopamine receptors. In contrast, dopamine receptor agonists directly stimulate the dopamine receptors, bypassing the process of presynaptic storage and release of the neurotransmitter 67.6Review of multiple cases of movement disorder associated with ODS reveals a variable response to levodopa Table . It shouOur patient responded minimally to levodopa but showed a marked dose-dependent improvement with pramipexole. The worsening of his symptoms upon discontinuation of pramipexole and subsequent modest improvement upon re-initiation support its therapeutic effect. Another case report has also demonstrated the efficacy of pramipexole in extrapontine myelinolysis that presented in the setting of adrenal failure . In thisThe etiology of the dichotomous response in our case warrants further discussion. One explanation may be that the patient had such significant destruction of the presynaptic neurons from ODS that the amount of converted dopamine in the remaining few neurons could not result in clinically significant activation of postsynaptic dopamine receptors. Alternatively, because the postsynaptic dopamine receptors are intact, direct postsynaptic dopamine receptor agonists, such as pramipexole, could still achieve good clinical response as it bypasses the aforementioned presynaptic problem. In addition, direct dopamine agonists, with their longer half-lives than levodopa, may maintain a sustained activation of the postsynaptic dopamine receptors and result in a clinically significant response . LevodopThe lack of response to rotigotine compared to pramipexole should be discussed as both are nonergot dopamine agonists. While the mechanism is not completely understood, these dopamine agonists have different selectivity for different dopamine receptors . Rotigot


Jacob sheep are a rare ancient breed of sheep believed to have originated from the Mediterranean area but which are now kept throughout the world. These sheep have recently attracted medical interest due to the observation of a genetic disorder in the breed that can be used as an animal model of Tay–Sachs disease (TSD). This study aims to detect mutations in the Hexosaminidase A gene in Jacob sheep based on sequence analysis of the 284-bp fragment situated between exon 11 and intron 11 of the gene, a target sequence for site-specific mutation. This is the first study that has investigated Jacob sheep in Bulgaria for gene-specific mutations.A total of 20 blood samples were collected from Jacob sheep from the Rhodope Mountains. DNA was isolated from these samples, and a specific 284-bp fragment was amplified. The amplified products were purified using a polymerase chain reaction purification kit and sequenced in both directions.Target sequences were successfully amplified from all 20 investigated sheep. Sequence analysis did not show the homozygous, recessive, missense (G-to-C transition) mutation at nucleotide position 1330 (G1330→C) in exon 11, demonstrating that all of these sheep were a normal genotype (wild-type).Jacob sheep are considered a potentially useful animal model in advancing the understanding of pathogenesis and developing potential therapies for orphan diseases, such as those characterized by mutant GM2 gangliosides. The clinical and biochemical features of the Jacob sheep model of TSD represent well the human classical late-infantile form of this disorder, indicating that the model can serve as a possible new research tool for further study of the pathogenesis and treatment of TSD. Jacob sheep, also known as Spanish sheep, many-horned sheep, and piebald sheep, is a unique British breed of domestic sheep that has been bred in England for at least 350 years. Both males and females are horned, sporting two, four, or occasionally six horns. The origin of this breed is under debate, but according to one study that used endogenous retrovirus markers to investigate the history of sheep domestication, it is more closely related to old African and Southwest Asian breeds than to other British breeds . About 3Many diseases affecting Jacob sheep are of crucial importance to other animals as well, including an unusual form of asymmetric occipital condylar dysplasia  and Tay-Hex A gene, which encodes the α-subunit of lysosomal enzyme β-Hexosaminidase α [One of the most common diseases observed in Jacob sheep is TSD  , an autonidase α ,13. It inidase α -16. Althnidase α . β-N-Acenidase α . β-N-Acenidase α . The prinidase α .Hex A gene is situated on chromosome 7 (Hex A gene in Jacob sheep is few. This study aimed to detect mutations in the Hex A gene in Jacob sheep from Bulgaria.The Jacob sheep mosome 7  and has The experiments were carried out under permissions and the guidelines of the Bulgarian Academy of Sciences and the Bulgarian Ministry of Environment and Waters (no. 627/30.03.2015).In Bulgaria, Jacob sheep are represented by a single flock  located in the Rhodope Mountains. Specimens from this flock have been introduced to the Midalidare Estate winery , mainly to attract visitors. Some sheep from Midalidare Estate have been further transferred to the Agricultural Institute of Stara Zagora for breeding. For the purposes of the present study, unrelated animals from the winery and the Agricultural Institute were selected for analysis.The investigation, which took place in 2019, included twenty 4-5-month-old animals – nine males and 11 females . They we®, Terumo, USA) with K2EDTA as an anticoagulant. The samples were obtained from purebred lambs of the Jacob sheep breed provided by the Agricultural Institute of Stara Zagora, Bulgaria.A total of 20 blood samples (5 mL) from the jugular vein were collected using blood collection tubes , according to the manufacturer’s instructions. DNA concentration was spectrophotometrically determined. The quality of the isolated DNA was verified by 1% agarose gel electrophoresis and then staining with SimpliSafe  under UV light. The isolated DNA was stored at −20°C before analysis.Hex A gene was amplified using the following primers: forward 5′-CTTGCATGAGACTGTGTCCCGGG-3′; reverse 5′-GGGGACCAGGTTTGTGCTGTCC-3′. The primer set covered a partial region of intron 11 (53 bp), the entire exon 11 (186 bp), and a partial sequence of intron 12 (44 bp). All PCR reactions were performed with 10 ng/μl DNA in a final volume of 50 μl . PCR conditions were as follows: Initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, primer hybridization at 50°C for 30 s, elongation at 72°C for 1 min, and final elongation at 72°C for 10 min. In addition, a negative control was also included for all PCR reactions.The amplified fragments were separated and visualized by 1% agarose gel electrophoresis, followed by staining with SimpliSafe . Fragment size was determined using the Gene-Ruler™ 100 bp Ladder Plus . Amplified products were purified with a PCR purification kit  to remove excess primers and nucleotides and were then sequenced in both directions using a PlateSeq Kit  per the manufacturer’s instructions.Based on the sheep genomic reference sequence EU 579459.1 , a 284-bHex A gene) in the amplified products were analyzed using DNAStar software  [All 20 obtained DNA sequences were manually edited and aligned with the MEGA7 software program  using thWI, USA) . The obtJacob sheep were originally called piebald, spotted, or Spanish sheep, and it has been suggested that Jacob sheep were present in England as early as the 1500s . In 2011Hex A gene did not demonstrate a homozygous, recessive, and missense (G-to-C transition) mutation at nucleotide position 1330 (G1330→C) in exon 11. A BLAST search in the GenBank database found only two additional identical sequences of mRNA, both coding for the Ovis aries hexosaminidase subunit alpha protein XM_027971423 and EU579459.1. These sequences covered exon 11 of our sequence entries. Our results from the partial sequence analysis of the This mutation is uncommon in Jacob sheep. Using a PCR-restriction fragment length polymorphism analysis, investigation of 104 genomic DNA samples from Jacob sheep in three geographically distinct flocks showed the overall incidence of carriers to be only 14.4%  . The smaMoreover, the sequence analysis from this study did not even find carrier Jacob sheep, that is, animals heterozygous for the wild-type and recessive mutant allele of the gene. Carrier identification is important, because carriers appear normal and live a normal life; however, breeding a mutation-carrying Jacob sheep to a normal Jacob sheep results in a 50% chance of producing another carrier lamb and a 25% chance of a TSD-afflicted Jacob sheep.Hex A sequence similarity with humans. Thus, in terms of research, the Jacob sheep are one of the most useful animal models of spontaneously developing TSD. Because of this genetic similarity, the clinical and biochemical features of the Jacob sheep model are likely to mirror well those of the human infantile form of TSD. Furthermore, the level of residual enzyme activity is also similar [One important feature of TSD is that its clinical manifestations in Jacob sheep are the closest to the pathological effects observed in humans . Most im similar . This meHex A did not show the specific G1330→C mutation at the 3’-end of exon 11 in any of the investigated lambs; therefore, the studied animals were all normal, possessing the wild-type allele.The sequence analysis of the fragment of This study, the first report examining the genotype of Jacob sheep in Bulgaria, may be used as a starting point for genotyping additional flocks. This will allow the prevention of TSD in sheep through identification of afflicted individuals and will help avoid the breeding of carriers in Jacob flocks.PH and JK conceived and designed the research. BN and TA carried out all experiments and performed the data analysis. NP and SL drafted the manuscript. All authors read and approved the final manuscript.