Vespula germanica and V. vulgaris) are costly invertebrate pests in New Zealand, with large impacts on the local ecology and economy. Wasps eat honeybees (Apis mellifera), which has potentially devastating effects on hive health, as well as agricultural and horticultural industries. Vespex bait, which contains fipronil in a proteinaceous carrier, has recently been introduced for wasp control. In over a decade of reported trials, honeybees have never been observed foraging on Vespex, likely because the bait contains no sugars to serve as a bee food source. However, the potential for the control agent fipronil to enter beehives has not been tested. Therefore, here, we investigated this using a liquid chromatography–mass spectrometry assay of fipronil and two of its environmental breakdown and metabolic derivatives, fipronil desulfinyl and fipronil sulfone. We did not detect fipronil in any of the worker bee, bee larva, honey or pollen samples (n = 120 per product) collected from 30 hives over a 2-year period. Furthermore, although we detected fipronil desulfinyl in one honeybee sample, this is thought to have originated from a single individual, representing a rare occurrence of intoxication, and there was no evidence that Vespex was the toxicant source. There was also no evidence of trophallactic transfer of fipronil or its derivatives in any of the hives sampled. Previous studies have reported the impairment of individual bee performance at fipronil doses similar to the detection limit of our study. However, our results provide confidence that if undetectable intoxication was occurring, it would involve an acute exposure for those few individuals affected, with minimal impairment to colonies. Therefore, we conclude that the use of Vespex in the vicinity of honeybees does not result in significant hive uptake while effectively reducing wasp pressure on honeybee colonies.Introduced wasps ( Apis mellifera L. 1758) worldwide ). Sampling was carried out twice at BB and once at PB , giving exposure . In addiexposure –47. TherAll solvents (acetonitrile and hexane) that were used for sample preparation and analysis were of analytical grade and were purchased from Fisher Scientific . Water was purified with an Arium 611UV system . The QuEChERS kits, including ceramic homogenisers, extraction and purification salts, and primary secondary amine (PSA), were obtained from Agilent Technologies, Inc. . Fipronil (98% purity) was purchased from AK Scientific , while its two environmental and metabolic derivatives (fipronil sulfone and fipronil desulfinyl) and the internal standard , which were of 99.9% purity, were purchased from Sigma-Aldrich . Mixed standard solutions were prepared in acetonitrile.4, 0.2 g of NaOAc and 0.2 g of PSA) in deionised water (3 mL) and shaken vigorously for 2 minutes. The IS  was then added along with an additional 6 mL of acetonitrile and 2 mL of hexane. The sample dispersion was centrifuged at 4,000 × g for 5 minutes at 15°C, following which the supernatant (7 mL) was recovered, transferred to a new Falcon tube containing purification salts and a stationary phase  and vortexed for 2 minutes. This was then filtered through cotton wool into a test tube and the retentate was washed with acetonitrile (3 mL). The solvent was evaporated to dryness and then reconstituted in 3:2 acetonitrile/water (1 mL), which was filtered through a 0.45-μm PTFE disk into 2-mL LC-MS vials for analysis. To establish calibration curves (1–100 ng/mL), commercially available blank honey was analysed in which no fipronil or fipronil derivatives were detected, and this honey was then spiked with pesticide standard solutions and mixed with a vortex mixer to ensure homogenous distribution. All samples and calibration standards were analysed in triplicate, and all analyses were performed blind in the laboratory, with analysts unaware of the source of the samples.Sample analysis for the presence of fipronil and its derivatives followed the methods of Kasiotis et al.  and Parath sample.There were some minor differences in sample preparation between 2015 and 2016. In 2015, fipronil desulfinyl was not available as a standard and so was not tested for. In addition, no IS was used in 2015, with sample recoveries instead being monitored by using blank honey spiked with 1 mL of a 200 ng/mL fipronil and fipronil sulfone calibration solution for every 24m/z range, 100–1100; MS/MS acquisition rate, 2 spectra/s; MS/MS m/z range, 50–500; collision-induced dissociation (CID) gas, N2; CID gas pressure, 16 psi; isolation width, m/z 4; collision energies, 17 or 20 eV; total analysis time, 10 minutes. Any pesticide signals that were detected were normalised to the IS intensity. Agilent Technology’s MassHunter Workstation software, including Qualitative Analysis and Quantitative Analysis (version B.06.00), was used to process the MS and MS/MS data.Liquid chromatography analysis was carried out using an Agilent Technologies  6530 Q-TOF LC-MS system equipped with a Kinetex C18 column . The chromatographic gradient was optimised for separation of fipronil and its two derivatives along with the IS . ChromatThe LC-MS/MS method  was evalThe samples of 50 worker honeybees weighed 6.143 g, 7.436 g and 6.589 g , providing an estimate of 0.134 g per bee or 7 honeybees per gram.r2 values of >0.99 for the standards and >0.98 for the spiked samples  that were examined in 2015 tested positive for fipronil above the LOD. Three honey samples that were collected prior to bait deployment did return trace levels (between the LOD and LOQ) of fipronil sulfone, however, although all of these failed to register above the LOD for any of the compounds of interest when freshly prepared samples were retested.50 for an individual bee for both oral and dermal routes of exposure  at a level that was well above the LOQ and at the typical 24–48-hour LDsure see . However50 for a worker bee  was based on the published protocol of Kasiotis et al.  but contAs mentioned above, the finding that no other samples from the same hive or from 29 other hives in wasp treatment areas contained fipronil desulfinyl suggests that this result may have originated from a single honeybee. Assuming the complete photochemical degradation of fipronil to fipronil desulfinyl (see ), 6 ng o2 and often less than one tray per 10,000 m2. Second, residual wasp bait that is not taken by pest wasps becomes a dry solid within 3 days and is removed from the bait stations after 3–8 days without replacement. Consequently, the environmental exposure of bee colonies to the toxicant  and the period over which they could develop sub-lethal effects is shorter than indicated by published toxicity tests [The potential exposure of honeybees to fipronil or its derivatives during wasp control will differ from that experienced when fipronil is used as an active ingredient in insecticides for crop seed treatment, crop sprays or ground treatment for termites . In hortty tests , 52–53, Vespula wasps being less likely; however, neither scenario has been observed by the authors or reported by beekeepers who have used Vespex. It should also be noted that worker bees appear to have a negligible role as vehicles of fipronil associated with wasp baiting as we found no evidence for the trophallactic transfer of fipronil or its derivatives.The pathways by which fipronil can be transferred from wasp bait to the bee hive are also limited. Exposure could result from the direct contact of worker bees with the bait, with the entry of intoxicated 1/2 111–350 days; [Three honey samples that were obtained from both study areas before wasp baiting occurred returned trace amounts of fipronil sulfone but not fipronil. The retesting of each sample yielded negative results but we cannot exclude the possibility that trace levels of fipronil sulfone just below the LOD were present. Therefore, we consider that these results indicate the low-level presence of this toxicant. The source of these detections was not identified but several possible scenarios exist. First, cross contamination may have occurred in the laboratory. However, this seems extremely unlikely as mixed standards were used and only 3 out of 480 samples returned positive results for only one fipronil derivative. Long-term stability studies on the soil degradation of fipronil have shown that this toxicant can take almost a year to break down . TherefThese positive results, together with the low LOD/LOQs obtained, demonstrate that the method used was capable of detecting fipronil and its derivatives in bee and bee-product samples–a series of otherwise negative results could bring into question the validity of the chosen method.V. vulgaris and V. germanica on bees and bee services have been estimated to cost the New Zealand economy over NZ$70M annually [Vespula wasps globally [>90% reductions in the activity of wasps from the use of Vespex wasp bait is previously reported for similar sites . Since tannually , our finglobally , 14, incglobally , Argentiglobally –57 and tglobally –59, indiS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigValues are means ± standard errors.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


This study aims to experimentally determine the role of intermolecular forces on the contact angle of vegetable oil droplets. Contact angles were recorded using a microscope and measured using digital software. The results show that the surface tension of vegetable oils is influenced by the London force between the electron clouds of molecules. The process of cooling increases vegetable oil contact angles, due to the decreased kinetic energy of constituent molecules and increased London force on the molecules. A decrease in temperature causes the surrounding water vapor to condense, which adheres to the droplet surface (due to the hydrophilic properties of molecules). Hydrogen bonds develop after moisture adheres to the surface. Further, water molecules on the droplet surface reduce the surface tension, because of hydrogen bonds between the molecules on the droplet surface and moisture. Hydrogen bonds among the molecules force water molecules to accumulate on the droplet surface, which suppresses the droplet surface; therefore the contact angle decreases. Wettability, statistical physics, and fluid dynamics have always been associated with the wetting phenomena of liquids on solids –4. The aFouling is a problem in processes that use a membrane. Fouling causes a significant decrease in fluxes by several types of dopants (impurities) such as adsorption, pore-clogging, and the aggregation of solutes on membrane surfaces. Most research on fouling has been on large deposits, such as pore closing by a layer of dirt. However, research on fouling due to small deposits, such as vegetable oil droplets, has been scarce . Some reCertain forces must be overcome to enable the cleaning of impurities when fouling occurs: cohesive forces in impurity materials (deposits) and adhesion forces between the deposits and the surface. Researchers have suggested the need for improving cleanability as a solution for the problem of fouling .The measurement of contact angles between solid surfaces and droplets is a procedure performed by researchers to evaluate the cleanability of droplets. Moreover, the contact angles between solid surfaces and droplets are the result of adhesion forces between liquids and solids . (LDPE), polyethylene terephthalate (PET), stainless steel, and glass). These were then compared to some thermodynamic models, and the results showed a strong correlation between the thermodynamic model of Young–Dupré and the experimental results.In examining cleanability, several researchers tested the correlation of the thermodynamic model of adhesion forces between droplets and substrates, using experimentally measured adhesion forces . ResearcOther studies regarding cleanability have been conducted, using a statistical testing method. Olive oil was dropped onto various coated substrates: ceramic coating, quasicrystalline coatings, polytetrafluoroethylene coating, and silicone coating. The substrates were then heated, and the results showed a relationship between decreased contact angles and increased temperatures .Another experiment on cleanability proved that liquid metal dripped onto ceramics causes an increase in contact angles, with decreased temperatures. Surface roughness, porosity, and structural transformation of the substrate affected the increase in contact angles. Moreover, changes in droplet contact angles due to volume addition (or reduction) have also been investigated. Further, it has been proved that activation energy  affects contact angle changes .Several studies have discussed changes in contact angles. However, it is difficult to find an explanation about the roles of intermolecular forces in contact angle changes, especially in long-chain molecules. Therefore, this study used three kinds of vegetable oil droplets to clarify the role of intermolecular forces of long carbon-chain molecules, especially the force that emerges from polar and nonpolar molecules. The selected vegetable oils were coconut oil, jatropha curcas oil, and sunflower oil. These vegetable oils were chosen because they contain different molecules. Most of the content of coconut oil is saturated fatty acid molecules, which are polar. Jatropha curcas oil contains monounsaturated fatty acids, and sunflower oil contains polyunsaturated fatty acids, both of which are lower in polarity.Droplets of the selected vegetable oils were dripped onto a glass surface, as shown schematically in The oils tested in this experiment were coconut, jatropha curcas, and sunflower, and their fatty acid compositions are tabulated in Tables The results show that the cooling process affected the contact angles and the changes in contact angle occurred in all the samples. 18H34O2) is an unsaturated fatty acid with one double bond, linoleic acid (C18H32O2) is an unsaturated fatty acid with two double bonds, and lauric acid (C12H24O2) is a saturated fatty acid.Viscosity is influenced by the constituent fatty acids of vegetable oils. Jatropha curcas oil contains a large quantity of oleic acids and sunflower oil contains a large quantity of linoleic acids, while coconut oil contains a large quantity of lauric acids. Oleic acid ) were affected by intermolecular forces. The relationship between (-dα/dT) and temperatures (T) is shown in The changes of contact angles of vegetable oil droplets  (the highest of the tested oils), linoleic acid (33.55%), and some other fatty acids. Sunflower oil contains linoleic acid (66.19%), oleic acid (22.35%), and other fatty acids. Meanwhile, coconut oil has a fatty acid composition of 40.77% lauric acid, 13.36% tetradecanoic acid, and other fatty acids. The molecule structure geometries of oleic, linoleic, and lauric acid  are presented in α) at room temperature occurred in jatropha curcas oil (20.65°), followed by sunflower oil (18.92°) and coconut oil (9.06°). This is in accordance with Washburn's theory (cited by [The largest contact angle (cited by ) that hicited by , the vis18H34O2) belongs to the group of long-chain molecules. Linoleic acid (C18H32O2) is a little shorter than oleic acid, while lauric acid (C12H24O2) is much shorter than oleic acid, as shown in Oleic acid  decrease the surface tension see . A decreAfter the contact angles decreased, they appeared to increase again. The increased droplet contact angle of jatropha curcas oil was from 14.17° to 17.65°. Meanwhile, the droplet contact angle of sunflower oil increased from 14.02° to 16.69°, and that of coconut oil increased from 6.88° to 9.27°. This increase was due to the accumulation of moisture condensed on the droplet surface, and water molecules bind each other to hydrogen bonds see .Hydrogen bonding occurs due to the attraction (interaction) between dipoles of hydrogen atoms and oxygen. An attractive force occurs because oxygen has high electronegativity, and hydrogen is a nonmetallic element that has low electronegativity. When hydrogen binds to highly electronegative elements (such as oxygen), the combined electron couples are more attracted to oxygen, so that oxygen becomes more negative than hydrogen .In the condensation process on the droplet surface, the distance between water molecules is close enough for attraction, resulting in water accumulation . This exThis research found that the droplet contact angle of jatropha curcas oil decreased from 17.65° to 15.5°. Further, the droplet contact angle of sunflower oil decreased from 16.69° to 12.94°, while that of coconut oil reduced from 9.27° to 4.46°.This research has validated that changes in contact angles of vegetable oil droplets due to decreased temperatures are influenced by the intermolecular forces in their constituent molecules. Further, increased contact angles occur due to the decreased kinetic energy of molecules, and this decrease in kinetic energy is evidenced by changes in molecular geometry.One of these intermolecular forces is London force, and its interaction increases as the temperature decreases. This results in an increase in the viscosity of the liquid, which changes the contact angle.The hydrophilic properties of fatty acid molecule heads attract moisture to the droplet surface, and the water vapor adhered to the droplet surface forms hydrogen bonds.Hydrogen bonds contribute to contact angle changes, and they are formed by the high electronegativity in oxygen with the more positive hydrogen. Hydrogen bonds bind water to the droplet surface and this damages the surface tension, thus lowering the contact angle on the droplet.Moreover, hydrogen bonds bind water in greater amounts; therefore, water is accumulated on the droplet surface. Water accumulation causes the contact angle to seemingly increase. In large quantities, water accumulation will depress the droplet, thereby decreasing the contact angle.


One drawback of traditional forms of medical ocular dosage is drug dilution by tear; moreover, drugs are rapidly drained away from pre-corneal cavity by tear flow and lacrimo-nasal drainage. Prolonging contact time with different strategies and mucoadhesive vehicles will help to continuously deliver drugs to the eyes. For this study, we prepared and evaluated the effects of a nanostructure lipid carrier (NLC) on propranolol hydrochloride as a hydrophilic drug model for rabbit corneal permeation. Propranolol hydrochloride NLC was prepared using cold homogenization. The lipid was melted, then the drug and surfactant were dispersed and stirred into the melted lipid. This fused lipid phase was scattered in aqueous solution containing the cosurfactant at 4 °C and then homogenized. We evaluated particle size, drug loading, drug release, and NLC permeability through rabbit cornea as well as the formula’s effect on the cornea. Our results show that drug loading efficiency depended on the surfactant/lipid ratio (S/L) and the percentages of liquid lipid and Transcutol  (as solubilizer). Drug release data were evaluated with the Higuchi model and a significant correlation was shown between the S/L ratio and the amount of drug released after 4 and 48 h. NLC formulations improved propranolol hydrochloride permeation. We conclude that the effect of the NLC formulations was due to mucoadhesive and film forming properties. Topical drug delivery is a convenient mode of drug administration for ocular diseases. Yet, bioavailability through traditional ocular dosage forms, such as eye drops, is very poor. Several factors—such as pH, lachrymal secretion, blinking, tear flow, and epithelial barriers—influence ocular drug bioavailability . DrainagNanoparticle-based products demonstrate three capabilities, including enhanced drug permeation, sustained and controlled drug delivery, and targeted drug delivery. Encapsulation of drugs in these colloidal carriers improves therapeutic effectiveness in comparison with traditional ocular dosage forms (such as eye drops), as has been shown by studies of different nanostructured carriers ,7,8,9. TNanostructured lipid carriers (NLC) are prepared using solid and liquid lipids, surfactant, and water and range in size from 50 to 1000 nm . PreviouPropranolol hydrochloride was obtained as a gift from Daropakhesh Pharmaceutical Company . Oleic acid, Span 20, and Tween 80 were purchased from Merck . Cellulose acetate membrane (12 KDa) was obtained from Sigma Aldrich  . Male rabbits weighting 2.5–3 kg were purchased from Animal House . Compritol ATO 888 and Transcutol P were gifted by Gattefosse . All other chemicals were analytical grade.w/v) including Compritol ATO 888 (solid lipid) and oleic acid (liquid lipid) were melted, then 0.5% drug, 1.5–3% Tween 80+ Span 20 (1:1) as surfactants and 0–1% Transcutol as solubilizer were dispersed and stirred into the melted lipid, followed by sonication for 2 min. This fused lipid phase was cooled rapidly by placing in dry ice. Then, the drug-containing solid lipid was pulverized to microparticles by mortar milling. Microparticles were scattered in aqueous solution containing 2% propylene glycol as cosolvent at 4 °C . Then, the suspension was passed through a homogenizer for three cycles of 20 s each at 2000 bar.Propranolol hydrochloride NLC was prepared by cold homogenization according to the procedure described in Lidtke et al. . Lipids Mean particle size and the polydispersity index of propranolol-hydrochloride-loaded NLCs were calculated by laser light diffractometry using a Malvern Mastersizer SM 2000 K . Samples were prepared by dispersing NLCs with a sufficient amount of water, stirred, and followed by sonication for 2 min.The EE% and LC% determined by high pressure liquid chromatography (HPLC)  with 1 mL/min flow rate as mobile phase and ultra violet (UV) at 280 nm as detector). For this purpose, using an indirect method, 2 mL of each NLC formulation were centrifuged at 20,000 rpm for 20 min and the amount of free drug in the supernatant was measured. The amount of encapsulated drug was calculated by subtracting the amount of free drug from that of the initial drug . For the2).Drug release profiles were obtained by static diffusion cells with thermoregulated water jacket, with temperature maintained at 32 °C. A defined amount of the NLC formulation was centrifuged and the precipitate was collected and suspended in water. Five millilitres of NLC suspension was used as the donor phase and 33 mL of buffer phosphate (pH = 7) as receptor phase. Samples were collected in the determined intervals over 48 h . The dru2/5% CO2, bubbled across each compartment for 15 min. Thereafter, the donor phase was replaced with 2 mL of NLC containing 0.05 mg/mL propranolol hydrochloride, and 7 mL glutathione buffer was used to fill in receptor chambers [A) was 0.7 cm2 and Equations (3) and (4) describe Papp (apparent permeability coefficient) and Jss (flux or rate of permeation in steady state).Male rabbit corneas were used for the permeation studies. The experiments were performed and approved  in accordance with the guidelines for animal use of Ahvaz Jundishapur University of Medical Science (Iran), which were prepared by the National Academy of Sciences and published by the National Institutes of Health. Rabbits were sacrificed by intravenous ketamine injection and fresh corneas were excised, immediately weighed, and preserved in glutathione bicarbonate ringer (GBR) buffer. The corneal permeation studies were done using homemade Franz diffusion cells with horizontal design. In this experiment, the natural curvature of cornea was maintained. First, donor and receptor compartments were filled with GBR and tissue oxygenation performed using a mixture of 95% Ochambers . AqueousC0 is the drug concentration loaded in donor.In these equations, ΔQ/Δt is the steady state at the linear portion of the plot of amount of drug in the receptor chamber against time . A is thIncreased hydration of corneal tissue indicates cornea damage by drug dosage forms, evaluated by different methods such as differential scanning calorimetric (DSC) . StudiesResults are presented as the effect of NLC formulation on transition temperature and enthalpy of the transition phase.3 factorial design was used to investigate the combined effect of three independent variables on the physicochemical and corneal permeation properties of different formulations that were prepared using cold homogenization. The effect of variables on formulation properties and corneal permeation was evaluated by Student’s unpaired t-test or one-way analysis of variance (ANOVA)  at p < 0.05.A 2Different formulations’ properties are illustrated in Factorial and variance analysis were performed in order to evaluate the impact of independent variables on EE%. The results illustrate that all variables had a significant impact on EE%; however, an increase in variables led to elevated drug loading. Since propranolol hydrochloride is a naturally hydrophilic compound, while the nanoparticles are lipophilic, the maximum loading capacity of 61% (Formulation (1)) is acceptable. On the other hand, the values of LC% were between 1.1% and 2.31%, with maximum and minimum values provided by Formulations (2) and (4), respectively.As mentioned above, surfactant concentration impact on particle size was not significant, a finding that shows consistency with some previous experiments, though it differs from others. For example, for nitrofurazone, the particle size increased with elevation of co-surfactant concentration , while tThe experiment was carried out in phosphate buffer with a pH of 7 and drug release was followed for48 h. In order to determine the effect of independent variables on drug release, the percentages of drug released after 4 h (R4) and 48 h (R48) were measured. R4 values measure rapid release of the component, whereas R48 quantifies the slow release rate . AqueousAs can be observed, the S/L ratio and %L percentage had a significant impact on drug release after 48 h. The results illustrate that an increase in the mentioned variables leads to a decreased drug release rate. These two variables were the reasons for increasing drug loading. Therefore, an increase in oleic acid and surfactant contents results in increasing propranolol hydrochloride loading and simultaneously causes a decrease in the drug’s release rate. Minimum amounts of R48 correspond to Formulation (1), where all three variables were at the highest level. On the other hand, Formulation (4), in which two variables (S/L and %L) were at minimum levels, had the highest value of R48. It seems that increasing the surfactant content promotes drug solubility in lipid matrix; consequently, drug loading in nanoparticles increases, whereas release rate decreases.p < 0.05); an increase in S/L ratio promotes drug loading after 4 h. However, R48 showed similar behavior. In addition, although liquid lipid percentage significantly affected R48, an increase in liquid lipid content resulted in a meaningful decrease at R48.This impact was not significant for R4 (p = 0.22). A comparison between the impact of %L on R4 and R48 indicates that oleic acid (OA) had no short-term impact on drug release, while the longer time period induced elevated drug loading in nanoparticles followed by a decrease in drug release rate. Therefore, S/L ratio and surfactant content play an important role in adjustment of optimum drug loading and release.The drug release profile from NLC follows a two-step process: an initial rapid release with higher slope followed by a slow release with lower slope in the release profiles. Our results demonstrate that S/L ratio alone had a significant effect on R4  and the permeation rate, which can be obtained from the slope of drug amount against the time curve (Jss). Tlag was also determined by using the cumulative amount of permeated drug against time in a steady state. These parameters were measured on the basis of an infinite dose, considering sink condition. The results demonstrate that less than 10% of drug amount in the donor phase permeates through cornea, while the maximum concentration in the receiver phase was not more than 10% of drug saturation concentration in the receiver phase. Thus, sink condition and steady state were maintained. Different permeation parameters were determined, including amount of drug permeating the surface area after 5 h (QQ5 was 1.705 for Formulation (4), whereas the minimum content was 0.625 for Formulation (6). It should be mentioned that all formulations had significantly higher values of Q5 than the control (p = 0.001). It seems that only the S/L ratio had a significant but opposite effect on Q5. In other words, increased surfactant content led to an increase in loading and a significant decrease in Q5. We conclude that the effect of surfactant on Q5 was mainly due to drug loading. In addition, Transcutol impact on Q5 was not significant, perhaps due to its percentage in formulations. The effect of different concentrations of Transcutol as a permeation enhancer should be evaluated in another study. NLCs with a lipophilic nature and tendency toward cornea increased drug partitioning into cornea, causing promotion in Q5. The effect of independent variables on Jss was similar to Q5, with the lowest and highest amounts of Jss at 0.155 and 0.4 for Formulations (6) and (4), respectively. In addition, NLC formulations caused a significant p value increase in comparison to the control group (p = 0.001). According to our results, S/L ratio impact upon Jss was significant; S/L increase leads to Jss decrease.The maximum Jss is similar to Q5. Since Q5 and Jss parameters were strongly influenced by drug concentration in the donor phase, and because of different loading efficiency in formulations, a negligible variation in Jss and Q5 was observed due to different drug concentrations. The Papp in cornea was determined in order to normalize Jss regarding drug concentration in the donor phase. The highest permeation coefficient was 0.068 for Formulation (4), while the lowest was 0.012 for Formulation (1). Comparison between permeation coefficients illustrates that although drug amounts in the NLC formulations’ donor phase were much higher than the control’s, all formulations had significant impact on P parameter.T% and S/L ratio integration with In order to evaluate cornea heat behavior, thermograms were prepared by heating in temperatures ranging from −20 to 120 °C. Different thermograms were adjusted for cornea exposed to buffer, cornea in contact with NLC formulation, and NLC formulation, respectively .Several parameters, such as phase transition enthalpy ∆H and phase transition temperature, were calculated.The results illustrate that cornea has three transition phases at temperatures of 2.7, 74, and 85 °C, respectively. The transition phase was thoroughly eliminated for cornea in contact with NLC formulations at 2.7 °C. In addition, in other two-phase transitions showed negative shifts; phase transition temperatures were 12.5 and 19.5 °C, respectively. Phase transition enthalpy significantly decreased. Therefore, it seems that formulations absolutely affect cornea structure and alter phase transition. It should be mentioned that phase transition at 2.7 °C occurs due to melting free water existing in cornea .As a conclusion, NLC formulations absorb free water in cornea, and due to film forming and mucoadhesion properties they result in extended existence in the ocular system. For human cornea, one transition due to collagen denaturation in 56–74 °C was reported . The impNLC is the second generation of lipid nanoparticles with advantages of SLNs, while overcoming limitations such as low EE%, poor long-term stability, and possibility of drug expulsion. NLC shows great ability for ocular drug delivery due to better compatibility and modified drug release kinetics ,26. In tDrug loading and release profile of NLCs depend on several factors such as production parameters , lipid and drug properties, and surfactant concentration. Propranolol hydrochloride is a water-soluble compound which has no tendency toward the lipid phase; on the other hand, the preparation method for nanoparticles is cold homogenization. The drug release profile suggested the loading model followed by solid solution and drug-enriched shell patterns. Mostly solid solution patterns occurred during cold homogenization, where the drug substance was homogenized in solid lipid matrix. Shell patterning predominated during warm homogenization, especially for hydrophilic drug substances. However, during cooling, redistribution from the aqueous phase to lipid core occurs and drug remains in the outer matrix by film formation. As cold homogenization was applied in this study and the burst effect was negligible, the solid solution pattern dominated. Drug migration towards the shell occurred during cooling, as it has high hydrophilicity, so that drug content was much higher in the outer matrix than in the core, also resulting in a drug-enriched shell pattern. We conclude that the drug release profile follows both solid solution and drug-enriched patterns.The effect of interaction between drug and Compritol ATO 888 on the loading model and drug release through SLN has been reported for tetracaine and etomidate with low melting points and prednisolone with a high melting point ,28. The Jss was normalized by concentration adjustment in the donor phase, permeability through cornea was affected by S/L ratio.Additionally, in another study the impact of different factors  was studied for nitrofurazone-loaded SLN properties . NitrofuTlag; thus, practically prepared formulations in the present study did not show any obvious effect on shortening the time needed for approaching cornea equilibrium. In general, drug permeability through cornea includes two steps: first, partitioning or rapid distribution of drug from carrier to cornea, and second, permeation in different cornea layers. In order to determine the effects of different formulations on each step, the relation between nano-carrier properties and permeation parameters through cornea was recognized initially. The result indicates that there are no significant integrations between particle size, Jss and P (p = 0.244). Therefore, particle size was not an important factor for Jss and P, practically speaking. Since NLC formulations are not capable of Tlag alteration, we conclude that formulations did not change cornea structure and consequently had no impact on diffusion through different cornea layers.In terms of permeability, the results show that independent variables did not significantly relate to Papp coefficient was about 1.5 times higher than control. However, in a recent study Transcutol didn’t significantly impact ocular absorption, while NLCs increased P coefficient up to 20 for propranolol hydrochloride.On the other hand, since the formulations influenced P coefficient, they mainly affect drug partitioning into cornea. In other words, NLC formulations, due to their lipophilic nature, reach the inner parts of the eye. This property was also reported regarding tobramycin ocular delivery in rabbit eye, which showed that drug concentration was significantly higher in all ocular tissues after ocular and intravenous administration of tobramycin SLN formulation, with respect to reference formulations, and only tobramycin SLN allowed drug penetration into retina . In addiPapp parameter was much higher than that of lipophilic compounds such as ibuprofen. Mucoadhesion, increased corneal retention time, and enhanced permeation due to cellular uptake by corneal epithelial cells were reported as the main reasons for ocular delivery of topical lipid nanoparticles [In conclusion, due to the hydrophilic nature of propranolol hydrochloride, its impact on articles . Based oIn conclusion, NLC formulations improved propranolol hydrochloride as a hydrophilic compound permeation through rabbit cornea. On the other hand, this effect of nanoparticles may increase drug partitioning from carrier into cornea. Thermograms of cornea after pretreatment with NLCs demonstrated lose of free water due to surfactant. Structures responsible for transition phases were affected by NLCs. However, the effect of NLCs on permeability parameters demonstrated no structural change to cornea. It is suggested that structures belonging to transition phases are not responsible for the corneal barrier against drug permeability. Drug loading efficiency for hydrophilic compounds like propranolol hydrochloride was suitable. Drug release profile demonstrated low burst effect and a good sustained release property. Formulation (1), with the highest loading capacity, demonstrated the lowest amount of drug permeated through cornea. This finding suggests that formulation characteristics are the main factor for determination of drug permeability through rabbit cornea. Based on permeation data, Formulation (4) can be deemed the best NLC formulation that caused the highest drug permeability through cornea.


However, compared with scutellarin, it is very difficult to obtain scutellarein from Nature. Therefore, the present study focused on establishing an efficient route for the synthesis of scutellarein by hydrolyzing scutellarin. Neurological deficit score and cerebral infarction volume with the administration of scutellarein were then used to compare its neuroprotective effects on focal cerebral ischemia/reperfusion in rats induced by middle cerebral artery occlusion (MCAO) with those of scutellarin. The results showed that scutellarein had better protective effect on focal cerebral ischemia/reperfusion than scutellarin, which laid the foundation for further research and development of scutellarein as a promising candidate for ischemic cerebro-vascular disease.Scutellarein, the main metabolite of scutellarin  Erigeron breviscapus (vant.) Hand.-Mazz., has been clinically used in China since 1984 to treat acute cerebral infarction and paralysis induced by cerebrovascular diseases such as hypertension, cerebral thrombosis, cerebral haemorrhage −. The synthesis route of scutellarein was outlined in Scutellarin (0.5 g) was added to solutions of Het al. [The rats were randomly divided into six groups n = 10/group): sham operation group; MCAO group without pretreatment; MCAO group pretreated with scutellarin (100 mg/kg); MCAO groups pretreated with different dose of scutellarein (100, 50, 25 mg/kg [/group: set al. ,17.Neurological deficit was assessed 24 h after reperfusion according to a modified method established by Bederson  as follo0/A'A × 100%, where 0A was the weight of the infarction areas, was the weight of the whole brain [The cerebral infarct volume was measured using TTC staining . All thele brain ,21.2SO4 in 90% ethanol under a N2 atmosphere. The synthesis method solved the problem of the low yield of scutellarein, and provided a possible route for its industrialized production. The MCAO assay clearly demonstrated that scutellarein had better neuroprotection effects than scutellarin on focal cerebral occlusion/reperfusion by decreasing neurological deficit score and cerebral infarction volume, which suggested that scutellarein would be a more promising potent agent for the therapy of ischemic cerebrovascular disease.In summary, an efficient route for the synthesis of scutellarein by hydrolyzing scutellarin was reported. Essential to the synthesis was the use of H


Soil suppressive to the soybean cyst nematode (SCN), a major yield-limiting pathogen of soybean, plays an important role in biological control. Field and greenhouse experiments were conducted to evaluate the effects of tillage, crop sequence, and biocide application on SCN suppression in corn-soybean cropping systems in Minnesota. The experiment was a split-plot design with no-tillage and conventional tillage as main plots, and six crop-biocide treatments  as subplots with four replicates. Soil samples were taken from each plot at planting, midseason, and harvest each year for SCN egg counts, and soybean yield was determined. In addition, soil samples collected from each plot at midseason were assayed for suppressiveness to SCN. Tillage had minimal effect on SCN population density and soybean yield. Annual rotation with corn reduced SCN population density, but also reduced soil suppressiveness as SCN egg population density increased in the following SCN-susceptible soybean compared with soybean monoculture. Rotation with SCN-resistant soybean and corn was the most effective in reducing SCN population density. The bactericide streptomycin did not affect SCN populations but the fungicide captan increased SCN population density. The biocide formaldehyde was the most effective in reducing the level of suppressiveness to SCN. The greenhouse study confirmed that the soil was suppressive to SCN, but failed to detect effects of tillage, crop sequence, and biocide field treatments. This study demonstrated that the soil in the fields was suppressive to the SCN, and biological agents, especially fungal antagonists, were involved in nematode suppression. Heterodera glycines Ichinohe, is widely distributed throughout most soybean producing regions in the world (Soybean cyst nematode (SCN), he world . This nehe world . Crop rohe world .The beneficial effects of rotation of corn and soybean have been studied extensively, and in general, rotation increases the yields of both corn and soybean crops . HoweverSince the late 1980s, conservation tillage is used increasingly in the USA to limit soil erosion, preserve soil moisture during drought, improve water quality, increase organic matter, and reduce fuel costs . HoweverGenerally, agricultural soil has low levels of buffering against plant diseases. However, there are some soils which greatly suppress a specific pathogen and, most importantly, the suppressiveness can be transferred by small portions of soil . SpecifiBiocide treatments using fumigants, such as methyl bromide and formaldehyde, or fungicides, such as captafol, successfully reduce or eliminate the soil suppressiveness in fields . While cThis research was conducted at two field sites in Waseca County in Southern Minnesota for four years. Site 1 was located at the University of Minnesota Southern Research and Outreach Center (44°04′21″ N 93°31′21″ W) in Waseca, Minnesota, which had been planted to soybean continuously for 37 years and no-tillage had been practiced for the past 11 years before the experiment was established in 2009. Site 2 was in a commercial field (43°52′38″ N 93°43′14″ W) in Minnesota Lake, Minnesota, which had been in soybean monoculture for more than 20 years by 2005 when the field was planted to corn for four years prior to this experiment. The soil at Site 1 was a Nicollet clay loam , and the soil at Site 2 was Webster clay loam . The soils in both fields were demonstrated to be suppressive to SCN .The experiment was a split-plot design with no-tillage and conventional tillage as main plots, and the crop sequence-biocide treatments as subplots with four replicates . The croFor the formaldehyde treatment, 6.8 liter of 38% formaldehyde  in 220 liters water was applied by irrigation in the four central rows  3 wk before planting. Appropriate safety instructions were followed when applying formalin. For the streptomycin and captan treatments, 18 grams of streptomycin sulfate (Sigma S 5601) (7.75 kg a.i./ha) and 27 grams a.i. of captan (80% wettable powder) (11.6 kg a.i./ha) each in 220 liters of water were applied by a pump from a tank into the surface of four central rows 1 wk before planting and every 2 wk after planting for two months (five times per year).The conventional tillage treatment was fall chisel plowing after harvesting, and field cultivation followed by a finishing implement prior to planting. Fertilizer application was based on soil fertility test recommendations of the University of Minnesota Soil Test Laboratory. Fertilizer nitrogen in the form of urea with Agrotain nitrogen stabilizer was applied to corn plots only at the rate of 180 kg/ha in 2009, and 225 kg/ha in 2011. No other fertilizers were applied in soybean and corn during the 4yr. Corn and soybean were planted between late May and early June, and harvested between early October and mid-November depending on the soil and weather conditions each year. The SCN-resistant soybean cultivar was Latham EX547 RR N (PI 88788 source of resistance), SCN-susceptible soybean cultivar was Pioneer brand 92B13, and corn cultivar was DeKalb 46–61. All of the soybean and corn cultivars were resistant to glyphosate (2-phosphonomethylamino acetic acid), and glyphosate was used for both pre-emergence and post-emergence weed control. No insecticide or additional fungicide was used.3 of soil with a semiautomatic elutriator  was collected from each subplot in a systematic pattern across the two central rows at planting to assess initial (Pi), midseason (Pm) approximately 2 months after planting), and final population (Pf) at harvest. The soil was passed through a 5-mm aperture sieve and mixed thoroughly. The soil samples were stored in a cool room (4 °C) before being processed. Cysts were extracted from a subsample of 100 cmutriator  and sepautriator . Eggs weutriator  and collTo validate soil suppressiveness of each field treatment, soil samples from each plot were collected at midseason each year from 2009 to 2012 for a greenhouse assay. However, the results of 2011 were not reported because of insufficient SCN infection of soybean in the greenhouse. Approximately 4.5 kg soil was taken from 10 locations in each plot systematically to depth of 15 to 20 cm with a shovel. Each of the soil samples was passed through 5-mm aperture sieve, mixed thoroughly, and divided into three subsamples. Each subsample received one of three treatments: (i) 100% autoclaved field soil, (ii) 10% autoclaved field soil + 90% untreated field soil, and (iii) 100% field soil. The soil was autoclaved for 1 hr at 121°C.2 hatching solution  after 4 wk of planting to minimize the effects of autoclaving on soil fertility.Each subsample of soil was placed in a 15-cm-diameter pot. Seven soybean ‘Freeborn’ (PI 88788 source of resistance) seeds, that had been treated with 0.5% NaOCl for 3 min, were sown in each pot. Freeborn, which was susceptible to the population of HG 2.5.7 with a female index of 65% but resistant to the SCN populations (HG type 0) from the field plots, was used to minimize the effect of initial populations in the untreated soil. Pots were arranged in completely randomized blocks and maintained in the growth room with an average temperature of 28°C (range 20–30°C). After 1 wk, the plants were thinned to provide four plants, and 5,000 SCN J2 were added in six holes, 3 cm deep, around the soybean plants in each pot. The soil was supplied with a P-N-K fertilizer . After cutting soybean shoots at the soil surface, the soil ball was broken and thoroughly mixed. Nematode egg population densities were determined with the procedures described previously .P < 0.05 unless otherwise stated. In the field experiments, egg population density was transformed by x0.2 to x0.5 and yield data were not transformed. In contrast, egg population density was transformed by log(x), and shoot dry weight data were not transformed in the greenhouse experiment.The general linear model (GLM) procedure in Statistical Analysis System (SAS) Version 9.2  was used to perform the split-plot analysis of variance (ANOVA). Dependent variables were evaluated for normality and transformed as necessary before performing the ANOVA. Significant differences were reported at 3 soil at Site 1 and only 102 eggs/100 cm3 soil at Site 2. No effect of tillage on the egg population density was observed in most of the 12 sampling occasions over the 4 yr at both sites, except that no-tillage reduced midseason egg population density at both sites in 2010, but increased midseason egg population density at Site 1 in 2012 as compared with conventional tillage  only in the second year at Site 1, but decreased soybean yield (7.38%), the first year at Site 2. This agrees with previous studies reporting an inconsistent, yet positive, effect of conventional tillage on soybean yield . Tillage3 soil. This confirms that corn was relatively ineffective as a non-host in reducing SCN populations (Although the non-host corn reduced SCN population density, 1 yr of corn was not sufficient for SCN management, and when SCN-susceptible soybean was planted after the annual rotation of corn, SCN population density increased rapidly to the level similar to that in monoculture. This result was similar to a previous study , which dulations . Interesulations .Pasteuria nishizawae was responsible for an SCN-suppressive soil in Illinois (Hirsutella rhossiliensis and H. minnesotensis are frequently isolated from SCN J2 (Among the biocide treatments, the application of formaldehyde seemed to be the most effective for disrupting nematode suppressiveness. The increased SCN egg population density under formaldehyde treatment over the 4 yr supported the evidence of presence of antagonistic agents that attack nematodes in different stages – either egg, juvenile or adult – in the suppressive soil . While cIllinois . Hirsutem SCN J2 , and theThere was no effect of biocide treatment effect on soybean yield. This indicates that the increase of SCN population density in the formaldehyde and captan treatments at Site 1 was not due to better growth of soybean and greater food source, and rather that the increase can be attributed to the reduction of biocontrol agents in the soil. Conversely, the increased SCN population density in the biocide treatments did not reduce the soybean yield at Site 1. This result was similar to previous studies in which there was no increase of soybean yield of resistant cultivars as compared with susceptible cultivars in the field . The lacIn the greenhouse assay of soil suppressiveness to nematodes, transferring a small amount of soil to conducive soil is one of the most commonly used methods to detect specific suppression in soil . This teIn conclusion, tillage had little effect on the soil suppressiveness. Annual rotation with corn reduced SCN population density, but also reduced the level of soil suppressiveness and increased SCN population density in the following SCN-susceptible soybean as compared with the soybean in monoculture. Rotation with SCN-resistant and non-host corn was the most effective in lowering SCN population density. The biocide formaldehyde was the most effective in reducing the level of suppressiveness and increasing SCN population density through time, while captan also increased SCN population density in some sampling occasions at Site 1. This field study demonstrated that the soil in the fields was suppressive to the SCN, and biological agents were involved in nematode suppression.


Increasing evidence shows that antibiotic use in pregnancy may increase the risk of childhood asthma but epidemiologic studies are still limited and findings are inconsistent. Meanwhile, exclusive and prolonged breastfeeding may prevent children from allergic diseases. We aimed to assess the association between prenatal antibiotic use and the risk of childhood asthma, and explore whether breastfeeding modifies the risk.We conducted a case-control study in Shanghai, China, from June 2015 to January 2016. A total of 634 asthma cases and 864 controls aged 3–12 years were included. Multiple logistic regressions were used to estimate crude and adjusted odds ratios (aOR).The prevalence of antibiotic use in pregnancy in the cases and controls was 7.1 and 3.5%, respectively. A significant association between prenatal antibiotic use and childhood asthma was observed , particularly in boys  and children with family history of allergic disorders . However, this association existed only in children who were not breastfed exclusively in the first six months of life  but not in children who were exclusively breastfed . Likewise, exclusive breastfeeding also decreased the association between antibiotic use in pregnancy and asthma in boys and in children with family histories of allergic diseases.Antibiotic use in pregnancy was a risk factor for childhood asthma. However, this risk may be attenuated by exclusive breastfeeding in the first six months of life, especially among high-risk children.The online version of this article (10.1186/s12884-018-1936-5) contains supplementary material, which is available to authorized users. The prevalence of childhood asthma has been increasing over the past 30 years , 2. A nuSeveral other factors, such as male gender and family history of allergic disorders, have long been found to be risk factors for childhood asthma . While gInterestingly, several studies  suggest The purpose of the present study was to investigate the association between antibiotic use in pregnancy and the risk of childhood asthma, and the possible role of breastfeeding in modulating the risks using data from a case-control study on childhood asthma.https://ginasthma.org/]. Controls were non-asthma patients at the similar age from outpatient general pediatrics and pediatric surgery clinics. 697 cases of childhood asthma and 1099 potential controls were recruited. Mother-child pairs were excluded if they had missing information on maternal antibiotic use in pregnancy, delivery mode, feeding patterns within the first six months of life, child age and multiple births. Only three mothers smoked in pregnancy and, therefore, were excluded. Children with a history of wheezing were also excluded from the controls, leaving a total of 634 asthma cases and 864 non-asthma controls for analysis , feeding patterns within the first six months of life and baseline characteristic. Unfortunately, no information was collected on reasons for antibiotic use, types of antibiotics or dosage. Breastfeeding practice in the first 6 months postpartum was classified as exclusive breastfeeding, mixed feeding and bottle feeding. Exclusive breastfeeding was defined as infants only receiving breast milk without solid and liquid food (except water).We first compared the maternal and infant characteristics between cases and controls by Cochran-Mantel-Haenszel Chi-square and Student’s t test. We then examined the association between antibiotic exposure in pregnancy and childhood asthma. The modifiable effect of the exclusive breastfeeding within the first 6 months of life on this association was then explored. We specifically tested for the association of antibiotic use in pregnancy with childhood asthma stratified by child gender and family history of allergic disorders and examined the modifiable effect of exclusive breastfeeding within the first 6 months of life on the association in high-risk children. Also examined were interactions between antibiotic use during pregnancy and (1) feeding patterns, (2) child sex, (3) family history and (4) child age, respectively, on the asthma development. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated by multiple logistic regression models using SAS version 9.2 . The covariates adjusted in our models were: maternal age at delivery (years), maternal education level , child age (years), child gender , gestational age at birth (weeks), delivery modes , exclusive breastfeeding within the first 6 months of life , family history of allergic disorders . These covariates were selected according to prior knowledge , 21. WheP = 0.002). The prevalence of exclusive breastfeeding within the first 6 months of life, however, did not differ between the cases and controls  (Table Demographic characteristics were presented in Table 5) Table .Table 1CTable Table Table P-values were 0.06, 0.4, 0.1, 0.9, respectively).No significant interactions were found between antibiotic use during pregnancy and (1) feeding patterns, (2) child sex, (3) family history and (4) child age, respectively, on the asthma development  is relatively small, these bacteria play a very important role in the intestinal colonization [Animal studies showed that labeled bacteria placed in maternal gut could appear in breast milk. Although the number of maternal gut microbes that migrate into breast milk . However, this difference was mainly due to our exclusion of controls who had history of either asthma or wheezing. If these exclusions are considered legitimate, then exclusions due to other reasons were similar between the cases and controls


The composition of human milk varies widely and impacts the ability to meet nutrient requirements for preterm infants. The purpose of this study is to use a large dataset of milk composition from donors to a milk bank to: (1) describe the macronutrient variability in human milk and how it contributes to the ability to meet the protein and calorie targets for the preterm infant using fortification with commercially available multi-nutrient fortifiers; (2) assess how temporal versus subject effects explain macronutrient variability; (3) determine how macronutrient variability contributes to the nutrient distribution in pooled donor milk.This is a retrospective, observational study that analyzes the macronutrient data of 1,119 human milk samples from 443 individual donors to a milk bank. We test fortification strategies with potential basic, intermediate, and high protein and calorie commercial fortifiers. Additionally, we simulate the random pooling of multiple donors to model the impact of macronutrient variability on pooled donor milk.2 > = 0.50) than a time-effect (R2 < = 0.28). When pooling multiple donors, variability was reduced by increasing the number of donors randomly selected for a pool or targeted pooling based on macronutrient analysis of donor pools. Over 75% of mature milk samples fortified with a basic protein fortifier did not meet daily protein targets of 3.5 g/kg without exceeding volumes of 160 ml/kg/day.Fat was the most variable nutrient and accounted for 80% of the difference in calories. A subject-effect predicted more of the variability after 4 weeks postpartum in all macronutrients (RThere is a strong individual signature to human milk that impacts the pooling of donor milk, and the ability to meet protein and energy requirements for the preterm infant with basic and intermediate protein and calorie fortifiers. Growth velocity is frequently monitored in the neonatal setting due to evidence that adequate growth in preterm infants is associated with improved neurocognitive development . Higher The underlying variability in human milk must be considered when trying to meet the nutrient needs of the preterm infant. Several trends in the macronutrient composition of human milk are well described in the literature: a decline in protein and an increase in fat content during the early days following parturition ,13; greaDespite improvements in growth velocities in Very Low Birth Weight (VLBW) infants (less than 1500 grams), a study of over 360,000 VLBW infants between 2000 and 2013 found that 50% were classified as growth restricted at hospital discharge, defined as falling below the 10th percentile of weight for postmenstrual age on the Fenton growth chart . The purThis is a retrospective, observational study of the macronutrient composition of human milk provided by donors to the Mothers’ Milk Bank of North Texas  and received between November 2015 and July 2016. MMBNT follows the guidelines of the Human Milk Banking Association of North America (HMBANA) [Approved donors collect and freeze expressed breast milk from individual pumping session in containers , typically in 1–4 ounce volumes, until they are ready to send the aggregate collection of milk to MMBNT for processing. When MMBNT receives a donor shipment, the frozen containers from an individual donor are sorted by pump date, grouped into one or more Deposits, and stored, frozen, until processing. MMBNT records the estimated volume, expiration date, and donor ID for each Deposit. The expiration date is set to the earliest pump date in the Deposit plus one year. During daily milk processing at MMBNT, for each donor selected, one or more frozen Deposits are thawed and aggregated into a Donor Pool. That is, Donor Pools can consist of the milk from single or multiple Deposits, representing a range of pump dates, all from the same donor.Once each donor’s thawed milk is mixed into a Donor Pool, a sample of it is analyzed for fat, protein, and lactose using a MilkOScan FT 120 . The MilkOScan uses Fourier transformed infrared spectroscopy (FT-IR) to measure macronutrients based on unique vibration patterns of chemical functional groups in each class of macromolecule. Precision and accuracy for measuring protein and fat in human milk using IR technology and the MilkOScan have been reported by others, though variability with lactose measurement was observed, likely due to interference from human milk oligosaccharides ,22. MMBNMacronutrient data for each Donor Pool is logged in MMBNT’s Timeless  database and a spreadsheet. Other information available in MMBNT’s dataset includes: Donor ID, Baby Date of Birth (DOB), Baby Term , Deposit Receipt Date, Deposit Expiration Date, and Deposit Volume.Several values were calculated. For Donor Pools that contained a single Deposit, Pump Date was computed using Deposit Expiration Date less one year. For Donor Pools with multiple Deposits, Pump Date was weighted based on the expiration date and volume of each Deposit within the Donor Pool. Lactation Stage was computed based on Pump Date less Baby DOB and was reported in weeks postpartum. Milk type was assigned based on analysis of the dataset to determine when protein concentrations stabilized in the first 7 weeks postpartum. Based on this analysis, Transition Milk was defined as Lactation Stage less than or equal to four weeks and Mature Milk was defined as Lactation Stage between 4 and 52 weeks. A final category, Extended Milk, was classified as Donor Pools with a Lactation Stage greater than 52 weeks postpartum based on evidence that macronutrient composition changes in the second year postpartum . CalorieTo analyze how fortification would affect the protein and calorie content of the Donor Pools, commercial fortifiers were identified and hypothetical post-fortification protein and energy content was calculated based on the manufacturer’s mixing instructions. Using the manufacturer’s mixing instructions and assuming milk composition of 1 g/dL protein, 7 g/dL lactose, 3.5 g/dL fat, and 19 kcal/ounce, Similac Human Milk Fortifier Concentrated Liquid  mixed to 24 kcal would increase protein by 1.0 g/dL and was considered a basic protein fortifier. Similac Human Milk Fortifier Hydrolyzed Protein Concentrated Liquid and Prolacta +6  would increase protein by 1.5 g/dL and were considered intermediate protein fortifiers. Prolacta +10  would increase protein by 2.5 g/dL and was considered a high protein fortifier ,24. BothData were excluded for multiple reasons. For example, for some Donor Pools, the calculated Pump Date was earlier than the Infant DOB, likely due to data entry errors . For the purpose of maintaining conservative expiration dates, when milk is donated without a pump date on the label, MMBNT assigns the pump date as the infant DOB. For this reason, Donor Pools with a Pump Date equal to the Infant DOB were excluded from the analysis . Donor Pools from unique donors that had identical macronutrient values is suggestive of final pooling values versus individual donor pool values and were therefor excluded . To avoid masking effects associated with Stage of Lactation, Donor Pools that contained multiple Deposits for which the difference between earliest and latest Pump Dates was greater than 7 days were also excluded from the analysis .2) were calculated to determine the impact of Lactation Stage (a time-effect) and Donor ID (a subject-effect). The full dataset, which reflects the actual population of Donor Pools available for creating pooled donor human milk, was used to simulate random pooling of 1, 2, 3, 4, and 5 donors per pool. Briefly, the simulation involved confirming independence between deposit volume, protein, and fat and using historical data; generating values for these characteristics of each deposit independently using the empirical distributions; combining deposits into random pools using a variety of numbers of donors per pool; and calculating volume-weighted averages. Two-thousand random pools were generated for each number of donors in a pool (1 through 5). Simulated results were compared to historical data from MMBNT of pools created between November 2015 and July 2016 to target 19+ kcal/ounce using macronutrient profiles from individual Donor Pools. This research received an exempt status from the University of Pittsburgh Institutional Review Board (protocol #PR017070192).Statistical analysis was conducted in Minitab , Microsoft Excel  and SAS Enterprise Edition 9.4 . The full dataset was used to describe prevalence of Donor Pools by Baby Term  and by Lactation Stage , with differences in distribution evaluated using a Fisher’s Exact test. Descriptive statistics were computed on a subset of the data containing a single Donor Pool per donor in order not to violate statistical rules of independence between observations in a dataset. For donors with multiple Donor Pools, the first donation chronologically was used. Within this data subset, differences in Lactation Stage were evaluated using one-way ANOVA analysis with a Tukey test for multiple comparisons, and theoretical effects of fortification were explored. To explore subject versus time effects, a subset of the data containing donors with multiple Donor Pools was used. Coefficients of determination  and 84.4%  of the Donor Pools were from donors who had given birth at term. Donors of preterm infants represented 13.1% of donors (58/443) and 13.8%  of Donor Pools while bereaved donors of deceased infants represented 2.3% of donors (10/443) and 1.9%  of Donor Pools. There was a significant difference (P < 0.001) in the distribution of Donor Pools by Donor Term and Lactation Stage, with Term and Preterm donors providing mostly Mature Milk , while bereaved donors provided the highest percentage of Transition Milk (47.6% of Donor Pools). Forty-eight percent of the donors (214/443) had multiple Donor Pools in the dataset. The mean and median number of Donor Pools per donor were 2.5 and 2, respectively, while the maximum number of Donor Pools in the dataset from a single donor was 29.In a cross-sectional analysis of the initial Donor Pool from each unique donor (N = 443), 33.4% (148/443) were Transition Milk, 61.6% (273/443) were Mature Milk, and 5.0% (22/443) were Extended Milk. Protein concentration was different by Lactation Stage (P < 0.001), with Transition Milk having the highest protein (1.3 ± 0.2 g/dL) followed by Extended Milk (1.2 ± 0.2 g/dL) and Mature Milk (1.0 ± 0.2 g/dL). Fat and calories were significantly higher (P < 0.05) in Extended Milk  compared to Mature Milk . A summary of macronutrient composition by Lactation Stage is presented in The distribution of macronutrients in the initial Donor Pools with a Lactation Stage greater than 4 weeks from 295 unique donors is presented in histogram format , along wIn unfortified milk, the median feeding volumes to reach protein targets of 3.5 to 5.0 g/kg ranged from 269 to 490 ml/kg of body weight, with lower volumes required for Transition Milk compared to Mature Milk due to higher protein content. Median daily feeding requirements ranged from 156 to 248 ml/kg with basic protein fortification, 129 to 199 ml/kg with intermediate protein fortification, and 96 to 143 ml/kg with high protein fortification. Box and whisker plots illustrate the distribution of feeding volumes required to reach various protein and calorie targets using fortified Transition and Mature milk .2 values ranging from 0.511 to 0.828) than the time-effect . When excluding Transition Milk where significant temporal changes are expected, the difference between the subject-effect  and the time effect  were even more pronounced. Results are summarized in Only donors with multiple Donor Pools were included in a bivariate analysis to explore the relationship between macronutrients and Lactation Stage (a time-effect) versus Donor ID (a subject-effect). When including all Lactation Stages, the subject-effect explained more of the macronutrients  of the production; high calorie milk  represented 10.2% (172/1686) of the production; and outpatient/skim/dairy-free milk represented 34.1% (575/1686) of the production. A distribution of the fat and protein content of MMBNT’s 1,111 pools intended for the NICU  is presented in Temporal changes in human milk composition are well-defined in the literature, with rapid changes observed in the first few weeks postpartum, as milk moves from colostrum to mature milk ,16,28,29The ability of Stage of Lactation (a time-effect) to predict the macronutrient composition of milk was small compared to the usefulness of Donor ID (a subject-effect) in predicting nutrient composition for all macronutrients in Transition and Mature Milk. This suggests that average values often assumed for human milk composition may not be representative of an individual’s milk composition. Others have reported a high inter-individual variability in the macronutrient composition in human milk ,18,32–35Understanding how the natural variability in human milk composition contributes to the ability to meet nutrient needs in the NICU is an important area of research. Koo et al. examined the impact of multiple commercial fortifiers in meeting preterm infant requirements and reported that macronutrient recommendations were typically met; however, they used “average” human milk values cited by the manufacture and did not consider the impact of individual variation . In our The impact of human milk macronutrient variability on pooled donor milk is not well understood. Smith et al. reported creamatocrit values ranged from 1.7 to 7.3% in 22 samples of pooled donor milk from 3 to 4 donors . A studyThe dataset represents primarily milk from women who delivered at term. In the NICU environment when feeding infants with mother’s own milk, we would expect higher protein content in the milk of mothers delivering preterm. This would translate into lower feeding volumes to reach protein targets. A systematic review of preterm and term milk composition found that the average protein difference was only 0.2 g/dL by the second week postpartum , which wThe macronutrient profile of human milk varies significantly by subject, with differences in fat composition explaining up to 80% of the difference in calories. Individual variability can impact nutrients in pooled donor milk. This variability can be reduced by increasing the number of donors in a pool or by target pooling. While protein is frequently considered a rate limiting nutrient for preterm infant growth, providing adequate total calories is also an important consideration in the NICU. Basic protein fortification of human milk typically requires feeding volumes above 160 ml/kg to reach protein targets of 3.5 to 4.0 g/kg. There is an urgent need for research into how the nutrient variability in human milk impacts preterm infant growth, body composition, and short-term and long-term health outcomes.S1 File(XLSX)Click here for additional data file.


Masters endurance runners can epitomize healthy aging; being reflective of the physiological processes of aging without the compounded effects of inactivity. The primary aim of the present study was to determine, using cross-sectional data, whether individuals taking up training after the age of 50 years can achieve the same level of athletic performance and musculoskeletal characteristics in their older age as those who trained all of their adult lives. A total of 150 master endurance runners  were divided into early starters  and late starters (started training after age 50 years). A comparative non-athletic group of 59 healthy older adults  were additionally included for analysis. Training intensity, age-graded performance (AGP) and musculoskeletal assessments were performed. Results showed that there was no difference between athlete groups for training intensity or age-graded performance, despite the 30-year difference in training history. Body fat percentage and leg lean mass did not differ between athlete groups, but were 17% lower and 12% greater, respectively, in athlete groups compared with controls. Power normalized to body mass did not differ between any groups. Spine BMD was lower in late starters than controls, while early starters did not differ from late starters or controls. Hip BMD did not differ between any of the groups. These findings show that the Masters athletes we studied that started intense endurance running after the age of 50 years had lower body fat and higher leg lean mass compared to non-athletes. Body composition and athletic performance of the late starters was very similar to those who trained all of their adult lives. Written informed consent was obtained from all participants. Masters runners were recruited as part of the United Kingdom-based “VIBE” study . Male anData for control participants was taken from the MYOAGE study, a European multi-center study of healthy aging and the methodology has been described previously . These pTherefore, the total number of complete datasets available for analysis was 209 and all participants completed measurements at the same laboratory and using the same equipment. Of those, 140 were male and 69 were female. For this study, the athletes were divided into early starter (ES) and late starter (LS) athletes. These groups were based on answers from a questionnaire asking each athlete to self-report the number of hours spent training  during different stages of their adult life . Early starter athletes were defined as those that had taken part in competitive endurance running throughout their adult lives, reporting intense training and competition at ages 18–29, 30–49, and 50 years and over. Late starter athletes were defined as those that had taken up intense training and competition after the age of 50 years, with no previous competitive training history. See Participants provided demographic, general health, lifestyle, and physical activity information by questionnaire [described previously ]. Self-rY-axis accelerations dataset was cleaned to remove any movement artifacts and any periods of nil data collection, most likely due to the participant not wearing the accelerometer. Activity data were normalized based on seven valid days of 14 h with ≥10 h recording time. Y-axis peaks were calculated based on accelerations that were higher than the previous and subsequent reading and recorded within 14 pre-specified g bands. These were condensed to three impact bands; low (≥0.5 to <1.0 g), medium (≥1.0 to <1.5 g) and higher (≥1.5 g) impact. All g values represent g over and above 1 g from earth’s gravitational force  was taken from hip and spine scans. All measurements were recorded after manual adjustment of the regions of interest. Repeat total body and hip scans were performed in eight participants within 1 month of the first scan. Using these repeat scans, the short-term error for our laboratory was 2.0% for hip BMD, 0.9% for spine BMD and 0.01% for whole body lean mass.Standing height was measured to the nearest millimeter and body mass was measured to the nearest 0.1 kg. Whole body, total hip and lumbar spine dual energy X-ray absorptiometry  scans were performed while the participant lay supine wearing a light cotton t-shirt to reduce measurement errors due to clothing absorption. Body composition (fat mass and lean mass) was taken from results of total body scans and regional analysis of legs and arms. Bone mineral density  as previously described . The widA Leonardo Jump Mechanography Platform  was used to assess lower limb muscle power during a countermovement vertical jump, as described previously . Resultsp > 0.05) showed no group × sex interactions for any of the variables so for this reason, together with the relatively small numbers of females in the ES group, data from both sexes were included within the same analysis. Univariate ANOVA (ANCOVA) analysis was used to identify differences between the three groups  with age and sex added as covariates, to account for the different mean ages and proportions of males and females between groups. Where significant differences were found, Fisher’s Least Significant Difference pairwise comparisons were performed. Differences between groups were considered statistically significant at p < 0.05.Statistical analysis was performed using SPSSv21 . Normality of distribution was assessed using the Shapiro–Wilk test, which showed that all data was normally distributed (presented as mean ± standard deviation) except for accelerometry data (presented as median (25th/75th) quartiles). Two-way ANOVAs , as recommended by 2p = 0.01, a medium effect size was set at η2p = 0.06 and a large effect size was set at η2p = 0.14. The values provided in the Results section show that the study was sufficiently powered to detect even small differences between groups for the main outcome variables of body fat percentage, lean mass, spine BMD, and vertical jump power.As participants included in our study were initially recruited to address other primary research questions, sample sizes for our analyses were fixed by the data already available. Our power to determine small, medium and large effect sizes can be ascertained from the observed effect sizes for the different outcome variables based on the partial eta squared (ηp < 0.0005). Pairwise comparison showed there was no significant difference between ES and LS for AGP (p = 0.294). Accelerometry collected over 7 days showed similar impacts in the low, medium, and high g bands between ES and LS groups . Pairwise comparisons showed that C were older than ES and LS, with no difference between ES and LS groups. Body mass and BMI were similar for the ES and LS athletes, but significantly greater for C compared with ES and LS groups (both p < 0.0005). The main reason for greater body mass in C was the higher body fat percentage  due to approximately 17% lower values for both athlete groups than C . Appendicular lean mass was also different between groups  and although values were similar between athlete groups, the LS had a significantly greater lean mass than C (p = 0.001). Total body lean mass did not differ significantly between groups , BMC , and Area  were significantly different between groups. There were no differences for these measurements between athlete groups, but LS had lower BMD , BMC , and Area  compared to C. There was no significant difference in Hip BMD (η2p = 0.024), BMC (η2p = 0.020) or Area (η2p = 0.005) between any of the groups (Spine BMD (e groups .2p = 0.020) and vertical jump power relative to body mass (η2p = 0.005) did not differ significantly between groups ; values were similar between athlete groups, but controls exhibited a greater power than LS (p = 0.006).Maximal grip strength  when comparing the results of the Masters athletes with those of the controls, where data were collected approximately 5 years apart. However, care was taken to follow exactly the same protocols as far as possible and the assessments were made in the same laboratory using the same equipment. The values for body composition and lean mass of the controls are within the ranges previously published for non-athletic but otherwise healthy older adults from larger, multi-center studies . NeverthThe Masters athletes within our sample taking up intense endurance running after the age of 50 years had lower body fat and higher leg lean mass than non-athletes by the age of 70 years and the values for body composition and athletic performance of the late starters were very similar to those of people whom had trained all of their adult lives.The study was conducted in accordance with the Declaration of Helsinki and approved by the University Research Ethics Committee and the National Research Ethics Committee Northwest (14/NW0275). All participants provided written informed consent.All authors contributed to the data collection, analyses, and wrote the manuscript. JP led the write up and directed the analysis of data.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.


PIK3CG single nucleotide polymorphisms  and patient responsiveness to clopidogrel to evaluate the risks of ischemia in patients with coronary heart disease (CHD).This study explores the associations between PIK3CG polymorphism. Haplotype and linkage disequilibrium analyses were performed. Prognosis analysis was performed using the Kaplan-Meier curve.The study consisted of 513 CHD patients who received clopidogrel as part of antiplatelet therapy, after percutaneous coronary intervention. According to the patient responsiveness to clopidogrel, the subjects were assigned to either clopidogrel-resistant (CR) or clopidogrel-sensitive (CS) groups. CR group was determined by patients’ platelet aggregation rate of ≥70% and poor responsiveness to clopidogrel, and CS group by patients’ platelet aggregation rates of <70% and good responsiveness to clopidogrel. Polymerase chain reaction using TaqMan probe was employed to detect Significant difference was found in genotype and rs1129293 and rs17398575 allele frequency between the CR and CS groups. Haplotype analysis indicated that the frequency of TG allele was higher in the CR group compared with the CS group, and the frequency of CA allele was lower in the CR group compared with the CS group. Patients with rs1129293 CT + TT genotype and T allele, rs1129293 AG + GG genotype and G allele exhibited an increased CR risk. Logistic regression analysis determined hypertension history as an independent risk factor for CR. The Kaplan-Meier curve suggests that distribution curve of cumulative probability nonischemic events was different between patients with rs1129293 and rs17398575 alleles. Stable CHD patients with TT genotype of rs1129293 allele and GG genotype of rs17398575 allele showed poorer prognosis compared to those with other genotypes and patients with acute coronary syndromes.PIK3CG SNPs (rs1129293 and rs17398575) and patients with poor responsiveness to clopidogrel. These findings show that this factor may contribute to an increased risk of ischemia in patients suffering from CHD.A positive correlation may exist between  Thus far, the exact mechanism of the interindividual variability has not yet been fully elucidated. In addition to clinical factors such as age, renal failure, obesity, diabetes, high plasma fibrinogen, and lack of adherence,–8 accumulating evidence suggest that genetic polymorphism is closely associated with patient's response to clopidogrel.,10Coronary heart disease (CHD) is a term used to describe a group of diseases that is characterized by a narrowing of the blood vessels that supply blood and oxygen to the heart. A lot of factors that have been correlated and associated with the onset of CHD include high blood pressure, smoking, diabetes, lack of exercise, obesity, poor diet, high blood cholesterol, and high blood sugar. Dual antiplatelet therapy (DAT) with aspirin and clopidogrel is a commonly recommended treatment used to prevent adverse cardiovascular events in patients suffering from CHD after stent implantation.PIK3CG), is a gene responsible for encoding the protein called PI3Kγ or p110γ which is located at the chromosome 7q223. P110γ is part of the PI3K family of enzymes that is involved in cellular functions such as cell proliferation, differentiation, and intracellular trafficking. Studies have suggested that PI3Kγ plays an important role in the development of cardiovascular diseases such as atherosclerosis, myocardial infarction, and thrombosis.,13 Its expression has also been detected in several cardiac cells including cardiomyocytes, vascular endothelial cells and smooth muscle cells, and platelets, thus establishing a link between its expression and various diseases.,15 P110γ expression is typically restricted and predominantly detected in leukocytes.Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit gamma  signaling pathway, PI3Kγ is a downstream isoform activated by GPCRs through the β/γ complex, which binds to heterotrimetric G protein subunitβ/γ and GPCRs.PIK3CG polymorphism in interindividual responses to clopidogrel. The study will be conducted by determining the different platelet aggregation rate in patients with 2 PIK3CG singly nucleotide polymorphisms (rs1129293 and rs17398575) and the occurrence of ischemic events by the means of an 18-month follow-up.In the present study, we aim to investigate the role of 22.1 Informed consents were obtained from all the subjects and their family members. This investigation was approved by the local institutional review board.This research was conducted according to the protocols regulations established by the Linyi People's Hospital. All the experiments conducted in this study were in accordance with the Declaration of Helsinki.2.2 and were currently receiving antiplatelet therapy with clopidogrel following a percutaneous coronary intervention (PCI) procedure. The exclusion criteria for this study were as follows: patients treated with cilostazol and IIb/IIIa receptor antagonists simultaneously, patients treated with calcium-ion antagonists, patients allergic to clopidogrel or showing contraindications to clopidogrel, patients suffering from hematological system diseases and deemed high-risk for bleeding and hemorrhage, patients with malignant tumors, patients who were pregnant, and patients suffering from severe hepatic and renal dysfunction. According to responsiveness to clopidogrel and their platelet aggregation rates, patients were assigned to 2 study groups: clopidogrel-resistant (CR) (platelet aggregation rates ≥70% with poor responsiveness to clopidogrel) and clopidogrel-sensitive (CS) (platelet aggregation rates <70% with favorable responsiveness to clopidogrel).A total of 513 diagnosed CHD patients were hospitalized in the Linyi People's Hospital between December 2012 and November 2014. All patients were diagnosed with coronary atherosclerotic heart disease2.3A questionnaire survey was conducted to collect data from all patients participating in this study. The survey included demographic data such as name, gender, age, height, and contact information. Patient's medical history , medicines  used and personal habits (smoking and drinking history) were also included.2.4 The operation was deemed successful if it fulfilled the following criteria: postoperative stenosis was <20%; thrombolysis in myocardial infarction (TIMI) was reestablished to normal flow which completely fills the distal coronary bed; and no serious implications were observed. The operation was seen as a clinical success when patients’ symptoms and signs of myocardial ischemia disappeared. Antiplatelet therapy was given to the patients according to the following regimen: Patients were given 300 mg of clopidogrel 24 hours before the surgery and 75 mg after the operation.All patients participating in this study have previously received conventional coronary angiograms before undergoing conventional PCI. Their results were determined in accordance with the American College of Cardiology/American Heart Association guidelines.2.5Blood samples, 5 mL each, were collected from the median cubital vein of patients who underwent antiplatelet therapy for at least 7 days (5 mL/person). Collected blood samples were transferred to an anticoagulative tube containing 3.8% sodium citrate , and thoroughly mixed and detected within a 2-hour duration to avoid damage to the blood contents. Turbidimetric platelet aggregometry (TPA) was applied to measure the platelet aggregation rate. Autologous platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared by centrifugation. According to the adenosine diphosphate (ADP) concentration difference (2 μmol/L) between PRP and PPP, platelet aggregation rate was determined using Optical principles. For post-clopidogrel therapy, the 2 μmol/L ADP-platelet aggregation rate was categorized as ≥70%. Patients who had aggregation rates of >70% were considered as CR. In contrast, patients who had platelet aggregation rates of <70% were categorized as CS.2.62, dye, reaction buffer, PCR enhancer, optimizers, and stabilizer), 11.25 μL of DNA stoste, and double-distilled water. The PCRs were set according to the following conditions: 95°C predegeneration for 3 min, 95°C degeneration for 15 min, 60°C annealing for 1 min; the PCR was set to repeat for a total of 45 cycles. TaqMan probe was applied to detect the polymorphism in rs1129293 and rs17398575 SNPs. According to the fluorescence curve produced beforehand, real-time fluorescence quantification PCR machine was produced that reflected the  rs1129293 SNPs including CC, CT, and TT, and rs17398575 SNPs including AA, AG, and GG.Genomic DNA was extracted from anticoagulated whole blood sample in accordance with the instructions of the DNA extraction kit provided by QIAGEN GmbH, Hilden, Germany. Ultraviolet spectrometry set at 260/280 nm was used to detect optical density and for the measurement of DNA concentration. Two SNPs, rs1129293 and rs17398575, were selected. The primers of polymerase chain reaction (PCR)  over the upper limit of 95% reference range (0.03 ng/mL) along with at least 1 of the following phenomena: clinical symptoms of myocardial ischemia, new changes in myocardial ischemia shown by electrocardiogram, pathological Q waves detected in electrocardiogram, and newly emerged loss in myocardial viability and regional wall motion abnormalities on image maps.2.8t and chi-square test (χ2). Genotype distribution was detected according to the Hardy-Weinberg equilibrium, and χ2 test was employed to help determine SNPs genotype distribution and allele frequency. SHEsis software was used for haplotype analysis. The association between genotype and prognosis of CHD patients was analyzed using a Kaplan-Meier curve. The risk of clopidogrel-resistance in patients was evaluated with a logistics regression analysis. P <.05 was considered as statistically significant.Statistical analysis was performed using SPSS 21.0 software for data analysis . Comparisons between groups of general information were conducted by 33.1t Test showed that the differences in the treatment of patients with hypertension using ACEi, ARB, and proton pump inhibitors between CR and CS groups were significant . The 2 groups showed no significant differences in their age, gender proportion, body mass index (BMI), history of smoking, drinking, stroke, hyperlipidemia, diabetes, and usage of statins and β receptor blockers  (Table Patients were divided into CR (n = 101) and CS n = 412) groups, depending on how well they responded to clopidogrel.  groups, 3.2rs112929 gene was marked by 6-carboxyfluorescein (FAM), and G allele in rs112929 was marked by green fluoresent protein of Victoria (VIC). After PCR amplification, 3 different results  were presented. G allele in the rs112929 gene was marked by FAM, and A allele in rs112929 was marked by VIC. After PCR amplification, 3 different results  were presented , suggesting that selected patients were representative of the population . Patients with rs1129293 CT + TT genotype were 2.45 times more susceptible to CR than the patients with rs1129293 CC genotype . Additionally, patients with rs1129293 T allele were 2.334 times more susceptible to CR than the patients with rs1129293 C allele . Patients with rs17398575 AG + GG genotype were more susceptible to CR with a 6.069 times higher risk of CR than the patients with rs17398575 AA genotype . Moreover, patients with rs17398575 G allele were more susceptible to CR with a 2.270 times higher risk of CR than the patients with rs17398575 A allele   was performed using the SHEsis software. Haplotyping results revealed that the TG frequency was higher in the CR group compared with the CS group, whereas CA frequency was lower in the CR group compared with the CS group  .Logistic regression analysis was conducted using CR as the dependent variable, and hypertension history, usage of ACEI or ARB, proton pump inhibitor, and genotype frequency of rs1129293 and rs17398575 as independent variables. Table 3.6P < .05) between the ischemic event group and the ischemic event-free group   Fig. .P <.05)  (Table P <.05)  between CR and CS groups. This suggests that the rs1129293 and rs17398575 SNPs may act as useful biomarkers for the prediction of patient responsiveness to clopidogrel. Furthermore, patients with rs1129293 CT + TT and rs17398575 AG + GG genotypes showed a higher risk of CR than the patients with rs1129293 CC and rs17398575 AA genotype. We could thus interpret that both rs1129293 CT + TT and rs17398575 AG + GG allelic variations could be risk factors for CR. On basis of our results, we suggest that genotyping of the PIK3CG gene should be conducted in patients before the administration of clopidogrel.Our findings show there was a significant difference in the genotype and allelic frequencies of 2Y12, thereby allowing the drug to exert its inhibitive role in platelet aggregation.,29 Studies have proven that PI3Kγ is activated by Gi-coupled P2Y12 ADP receptor and PI3Kγ-deficient platelets. Though PI3K responds to thrombin and thromboxane, it showed a reduction in both platelet aggregation and fibrinogen after stimulation with ADP.,30,31 We hypothesized that there could be a relationship between PIK3CG polymorphisms and interindividual responsiveness to clopidogrel. Based on this hypothesis, we could determin the significance of the PIK3CG SNPs (rs1129293 and rs17398575) before drug administration. By conducting a 6-month follow-up, results suggested and found that the occurrence of ischemic events varies with the different genotypes of rs1129293 and rs17398575. We believe that different rs1129293 and rs17398575 genotypes may relate to different PI3Kγ expression, and therefore, lead to different degrees of platelet aggregation, which is determinant factor for ischemic events.–34 However, we did not have the opportunity to investigate the possible links and mechanisms between the association of genotypes rs1129293 and rs17398575 and how they relate to PI3Kγ expression. The aforementioned aspect should be studied in future.Upon entering the human body, clopidogrel produces an active role through enzymatic oxidation by cytochrome P450 2C19. This is followed by an inevitable blockade of the combination between ADP and its platelet receptor P Previous studies have identified pathophysiological links between PI3Kγ and blood pressure regulation.,37 Carnevale et al suggest that PI3Kγ in hypertension may be a novel therapeutic target that controls vascular myogenic tone and target organ damage. However, in contrast to our study, a previous study also demonstrates that statin usage, smoking, overweight, diabetes, age, and female sex are all associated with the efficacy and good responsiveness to clopidogrel. A possible explanation for this relationship could be attributed to the fact that the platelet responsiveness value was not calculated from the difference between pre- and post-clopidogrel therapy. Therefore, the associations between clinic date and platelet responsiveness may be undercalculated. Our limitation in this study is due to our lack of direct evidence to support the association between PIK3CG polymorphism and the antiplatelet responsiveness of clopidogrel to use for the measurement of plasma clopidogrel concentrations. In conclusion, our study revealed that patients with TT genotype of SNP rs1129293 and GG genotype of SNP rs17398575 showed a poorer prognosis compared with patients with other genotypes. The SNP rs1129293 is proven to be associated with decreased high-density-lipoproptein cholesterol. Additionally, a large number of studies showed high-density-lipoproptein cholesterol to be a significant, strong, and independent inverse predictor of cardiovascular risk, and is correlated with the occurrence, development, and prognosis of CHD.–42In addition to gene polymorphism, we found that patients’ history of hypertension also affects their responsiveness to clopidogrel. Hypertension is widely proven to be a potential cause for a reduced antiplatelet effect of clopidogrel and aspirin.PIK3CG SNPs (rs1129293 and rs17398575) may be associated with the interindividual variability of responsiveness to clopidogrel, whereas rs1129293 T and rs17398575 G alleles are considered to be risk factors for the onset of CHD. Moreover, PIK3CG SNPs (rs1129293 and rs17398575) may be also related to the incidence of ischemic events within 6 months. With current development of many drugs used to treat CHD, recent evidence indicates that the ticagrelor provides a faster, greater, and more consistent platelet inhibition activity in comparison with clopidogrel. Thus, a possible direction to be headed in following investigations is the comparison between the drug responsiveness of clopidogrel and ticagrelor.In summary, our study has highlighted that Clopidogrel remains common and widely used to treat patients who suffer from CHD, especially for those who are financially challenged. Current alternative forms of treatment are either too expensive for the regimen to be maintained or have other consequences, such as ticagrelor. We believe that our study shows a favorable clinical net benefit for the treatment of patients suffering from CHD, and hope that the screening process of genotypes will be useful for future clinical implications. With our clinical data, we think that more experiments need to be conducted to further validate our results, and confirm the aforementioned associations.We would like to give our sincere appreciation to the reviewers for their helpful comments on this article.