Inexpensive high-resolution silicon (Si) X-ray detectors are required for on-site surveys of traces of hazardous elements in food and soil by measuring the energies and counts of X-ray fluorescence photons radially emitted from these elements. Gated silicon drift detectors (GSDDs) are much cheaper to fabricate than commercial silicon drift detectors (SDDs). However, previous GSDDs were fabricated from 10-kΩ·cm Si wafers, which are more expensive than 2-kΩ·cm Si wafers used in commercial SDDs. To fabricate cheaper portable X-ray fluorescence instruments, we investigate GSDDs formed from 2-kΩ·cm Si wafers. The thicknesses of commercial SDDs are up to 0.5 mm, which can detect photons with energies up to 27 keV, whereas we describe GSDDs that can detect photons with energies of up to 35 keV. We simulate the electric potential distributions in GSDDs with Si thicknesses of 0.5 and 1 mm at a single high reverse bias. GSDDs with one gate pattern using any resistivity Si wafer can work well for changing the reverse bias that is inversely proportional to the resistivity of the Si wafer. In X-ray fluorescence spectroscopy, the capacitance of a pin detector increases with the active area of the detector because the anode (n-type layer) and the cathode (p-type layer) have equal areas. The increase in the capacitance degrades its performance. However, SDDs have a much smaller capacitance than pin detectors . This ispt large . The ano− Si substrate with a resistivity (ρSi) of 10 kΩ·cm, an energy resolution of 145 eV at 5.9 keV was obtained from a 55Fe source at −38 °C [2 by irradiating X-ray photons through a 0.1-mm-diameter pinhole [To fabricate low-cost X-ray detectors, we have designed several simple-structure SDDs without MOSFETs or implanted resistors –43, one t −38 °C . The eff pinhole .The 10-kΩ·cm Si wafers are more expensive than the 2-kΩ·cm Si wafers used in commercial SDDs. In the present study, to fabricate much cheaper X-ray detectors, we used a device simulation to design adequate gate patterns for GSDDs formed from 2-kΩ·cm Si wafers.2.GSDDs have a cathode and only one p-ring, and to which the same reverse bias can be applied. 2) are formed, and the anode, p-ring, ground rings, and cathode are metallized. During metallization, the innermost p-ring is also metallized in SDDs, whereas gates are formed in GSDDs.In SDDs and GSDDs, n-type layers (anode and ground rings) and p-type layers  are fabricated by the same processes. In SDDs, multiple inner p-rings located between the anode and the p-ring are formed. Compared with GSDDs, the extra fabrication processes in SDDs are for creating the built-in MOSFETs or implanted resistors to couple the p-rings together electrically, which lowers the yield rate of detectors. The passivating oxide layers . All the simulations were performed by solving Poisson's equation and the carrier continuity equations. This provides a complete description of the system in terms of electrical quantities, such as electric potential and electric field distributions, carrier densities, and current densities.− Si substrate (dSi) were 0.5 and 1 mm, and the values of ρSi were 2 and 10 k Ω·cm. The radius of the anode (Ra) at the center of the cylindrical GSDD was fixed as 0.055 mm, which kept the capacitance of all GSDDs small. The widths of the p-ring (Wp), p-type floating rings (Wf), and n-type ground rings (Wg) were 0.545, 0.03 and 0.39 mm, respectively. The gap between the p-ring and the floating ring (Gpf) and the gap between the floating and ground rings (Gfg) were all 0.04 mm. The thickness of SiO2 on the cathode (dc) was 0.75 μm. The thickness of SiO2 on the other side (dg) was changed to constrain the electric field in the SiO2 between the gates and the Si substrate at ≤ 2.5 MV/cm, which is less than the SiO2 breakdown electric field of 10 MV/cm [2 near the SiO2/Si interface (QF) was fixed as 3 × 1010 cm−2, which has been reported for the present fabrication process [18 cm−3, and the donor densities of the anode and ground rings were 1 × 1019 cm−3. The depths of the cathode, p-ring, anode, ground rings, and floating rings were all 1 μm.The thicknesses of the n10 MV/cm . The sheGa1 was the gap between the anode and the innermost gate, and G12, G23, G34, G45, G56 and G67 were the gaps between the gates, from the innermost to outermost. G7p was the gap between the outermost gate and the p-ring. W1, W2, W3, W4, W5, W6 and W7 were the widths of the seven gates, from the innermost to outermost, respectively. The radii of the cathode (Rc = 3 mm) and the GSDD chip (Rchip = 3.5 mm) were fixed. As a result, the area inside the inner edge of the p-ring (Sarea) was 18.9 mm2, which is nearly equal to that of commercial small-area SDDs. The same reverse bias voltage(VR) was applied to the cathode, the p-ring, and all the gates.Seven gates were considered in this study. 4.4.1.dSi and ρSi of the n− Si substrate were 0.5 mm and 10 kΩ·cm, respectively, and dg was 0.75 μm. In Gate A, the values of W1, W2, W3, W4, W5, W6, and W7 were 0.1, 0.1, 0.19, 0.29, 0.39, 0.47 and 0.51 mm, respectively. Ga1 was 0.04 mm and G12 and G23 were both 0.03 mm. G34, G45, G56, G67 and G7p were all 0.05 mm.The VR of −60 V for Gate A. The voltage midway between the p-ring and the cathode was −37 V, and the electric field along the electric potential valley was strong enough to make all the electrons produced by an X-ray photon flow smoothly to the anode. Therefore, the electrons produced within the radius of the inner edge of the p-ring can be directed to the anode, indicating that the effective active area is approximately 18 mm2.55Fe source at −38 °C [2 by irradiating X-ray photons through a pinhole with diameter 0.1 mm [We fabricated GSDDs using the design of Gate A. In the GSDD, an energy resolution of 145 eV at 5.9 keV was obtained from a t −38 °C . The effr 0.1 mm , which i4.2.Si was decreased from 10 kΩ·cm to 2 kΩ·cm. VR of −60 V. Because the voltage drops at G67 and G7p were too large, the electric potential was almost zero between the anode and the outermost gate, and also over approximately 60% of the n− Si substrate, where the electrons produced by an X-ray photon are recombined with the holes produced by the X-ray photon.The value of ρ− Si substrate,VR was increased from −60 to −300 V, following the relationTo deplete the whole nVR of −300 V. It is clear from VR follows VR of −300 V was twice that of a commercial 0.5-mm-thick SDD using a 2-kΩ·cm Si wafer. Therefore, a gate pattern that can reduce VR was investigated. Because in G67 and G7p is too large, G67 and G7p in Gate B were decreased from 0.05 to 0.02 mm. The G34, G45 and G56 values were also decreased from 0.05 to 0.02 mm, and G23 was decreased from 0.03 to 0.02 mm. The value of Ga1 was increased from 0.04 to 0.07 mm, so that the potential at the innermost gate could be increased and the potential around the anode would not be zero. To keep Rc in Gate B the same as Rc in Gate A, the values of W3, W4, W5, W6 and W7 were changed to 0.21, 0.31, 0.41, 0.51 and 0.54 mm, respectively.VR of −200 V. Because the voltage midway between the p-ring and the cathode was −92 V, the electric field along the electric potential valley was strong enough to make all the electrons produced by the X-ray photons flow smoothly to the anode.VR from 200 to 150 V, which is VR of commercial 0.5-mm-thick SDDs using 2-kΩ·cm Si wafers, in Gate C the values of Ga1, G12, G23, G34, G45, G56, G67 and G7p were changed to 0.11, 0.02, 0.01, 0.01, 0.01, 0.01, 0.005 and 0.005 mm, respectively. To keep Rc in Gate C the same as Rc in Gate A, the values of W1, W2, W3, W4, W5, W6 and W7 were 0.01, 0.05, 0.24, 0.34, 0.44, 0.54 and 0.60 mm, respectively.To reduce VR of −150 V. In the electric potential distribution, the voltage midway between the p-ring and the cathode was approximately −78 V, and consequently the electric field along the electric potential valley strong enough to make all the electrons produced by the X-ray photons flow smoothly to the anode.4.3.To detect traces of hazardous or radioactive elements in food, soil, and the human body effectively, the absorption of X-ray fluorescence photons of these elements, such as Cd (23.1 keV) and Cs (30.8 keV), by GSDDs must be increased. However, the thickness of the Si substrates in commercial SDDs is approximately 0.5 mm; thus, the absorbed fractions of Cd and Cs X-ray fluorescence photons are 29.1% and 14.4%, respectively. In contrast, for a 1-mm-thick Si substrate, the absorbed fractions increase to 49.7% and 26.8%, respectively. In other words, the commercial SSDs up to 0.5 mm thick can detect photons with energies up to 27 keV for X-ray absorbance higher than 20%, whereas our gate pattern for the GSDD can detect photons with energies up to 35 keV. Here, we simulate the electric potential distribution in the GSDD with a Si thickness of 1 mm.dg was changed from 0.75 to 3 µm to avoid SiO2 breakdown caused by the high electric field. In Gate D, the values of Ga1, G12, G23, G34, G45, G56, G67 and G7p were changed to 0.33, 0.06, 0.02, 0.02, 0.02, 0.02, 0.01 and 0.01 mm, respectively. To keep Rc in Gate D the same as Rc in Gate A, the values of W1, W2, W3, W4, W5, W6 and W7 were changed to 0.02, 0.07, 0.18, 0.28, 0.38, 0.47 and 0.51 mm, respectively.In the 1-mm-thick GSDDs, − Si substrate, the value of VR was increased from 150 to 600 V, following the relationTo deplete the whole nVR of −600 V. The voltage at the saddleback was approximately −175 V. Because the average electric field toward the anode along the electric potential valley was approximately 950 V/cm, the average electron drift velocity was higher than 1 × 106 cm/s at the operating temperature (≤0 °C). This was caused by the electron mobility of 1450 cm2- V−1·s−1 in the Si substrate at room temperature [perature . This indSi of 1 mm and ρSi of 2 kΩ·cm, a reverse bias of approximately −1500 V is required to deplete the whole Si layer. However, for the GSDD, a reverse bias of only −600 V was required, which is an advantage of GSDDs.For a Si pin diode with 5.GSDDs are inexpensive Si X-ray detectors because of their simple structure. Although we have investigated GSDDs with 10-kΩ·cm Si because we have designed thicker GSDDs to detect X-ray photons with high energies, 10-kΩ·cm Si wafers are much more expensive than 2-kΩ·cm Si wafers, from which commercial SDDs are fabricated. Therefore, GSDDs with 2-kΩ·cm Si were investigated to develop low-cost X-ray detectors that can accurately detect photon counts and energies of X-ray fluorescence photons with energies of up to 35 keV. Device simulations of GSDDs with 0.5- and 1-mm-thick, 2-kΩ·cm Si substrates indicated that the X-ray detectors should work well when they are produced by using current fabrication processes. GSDDs with one gate pattern can work well for any resistivity Si substrate if the reverse bias is inversely proportional to the resistivity of the Si substrate. These findings indicate that the cost of portable X-ray fluorescence instruments can be reduced considerably.


Idiomarina woesei strain W11T was isolated from the Andaman Sea. Heterotrophic growth was observed at 30 to 37°C and pH 7 to 9. It grows in the presence of 0.5 to 12% (wt/vol) NaCl, and optimum growth occurred at 5 to 6% NaCl. The estimated genome is 2.4 Mb and encodes 2,305 proteins. Idiomarina woesei strain W11T is a Gram-negative, short rod aerobe that belongs to the class Gammaproteobacteria  (https://github.com/lh3/wgsim). Illumina reads were assembled with simulated read pairs using Allpaths-LG (version r46652) .The genome was annotated using the JGI Microbial Genome Annotation Pipeline . Genes wIdiomarina woesei contained 17 contigs in 16 scaffolds, with a total genome size of 2.4 Mb and N50 contig size of 317.6 kb. The largest contig was 842.6 kb, with a G+C content of 48.0%. The draft genome sequence has 2,305 candidate protein-coding genes (CDSs), 47 tRNAs, and 8 rRNAs.The draft genome assembly of LIPW00000000. The version described in this paper is the first version, LIPW01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Yarrowia lipolytica is an emerging scope that has not been fully scrutinized, especially for what concerns cultivation conditions of newly generated engineered strains. In this study, by combining flux balance analysis (FBA) and Plackett-Burman design, we screened chemicals for lycopene production from a metabolically engineered strain of Y. lipolytica. Lycopene concentrations of 126 and 242 mg/L were achieved correspondingly from the FBA-independent and the FBA-assisted designed media in fed-batch cultivation mode. Transcriptional studies revealed upregulations of heterologous genes in media designed according to FBA, thus implying the efficiency of model predictions. Our study will potentially support upgraded lycopene and other terpenoids production from existing or prospect bioengineered strains of Y. lipolytica and/or closely related yeast species.Genome-scale metabolic models embody a significant advantage of systems biology since their applications as metabolic flux simulation models enable predictions for the production of industrially-interesting metabolites. The biotechnological production of lycopene from  Yarrowia lipolytica, categorized as GRAS , has been established as a promising fermentation platform. Recently, a complete procedure for engineering Y. lipolytica for lycopene production was developed. Nonetheless, apart from the culture medium and genetic manipulation procedure provided [Y. lipolytica under lipid accumulation conditions [Y. lipolytica. One of the most efficient tools for yielding high titers of carotenoids in engineered strains is based on knock-out or overexpression of specific genes coupled with computational approaches [Carotenoids represent a subcategory of tetraterpenoid pigmented lipid compounds made by plants and various fungi and bacteria . Majorlyprovided , no deepprovided . Furthernditions . Therefoproaches ,16,17. Mproaches ,19.In silico models are valuable for comprehending the biochemical aptitudes of an organism and to foresee the influence of genetic and/or medium perturbations on growth and metabolic fluxes [in silico channel for scrutinizing biological networks in a quantitative approach. Contrary to mechanistic simulations that rely on precise kinetic information, FBA is based on the stoichiometric matrix and a biologically important objective function for identifying optimal reaction flux distributions which are subsequently used to unravel the metabolic capabilities of the studied system [c fluxes ,21. Fluxd system .Y. lipolytica for lipid production and predicted a minimal culture medium for lipids biosynthesis. Afterward, we have updated this model for generating model information for terpenoids production. The updated model is apt to provide reliable information when farnesyl diphosphate (FPP), one of the key precursors of terpenoids backbone in Y. lipolytica, is resolved as the objective metabolite. We hypothesized that such a metabolic network model can support the prediction of culture media for ameliorating the yield of lycopene in biotechnological applications.The present-day FBA is in a wide way to guide designs of experiments. In a previous study, we constructed a genome scale metabolic model of in silico data rarely match with the in vivo metabolic behavior. However, by combining GSMN (FBA) results and statistical methods such as Plackett-Burman design, it is possible to develop an efficient culture medium for the production of valuable objective products.Statistical design methods, especially the Plackett-Burman design, are commonly employed for optimization of fermentation media. The Plackett-Burman designs are usually resolution III, highly efficient two level fractional factorial screening designs for studying N-1 variables using N runs, where N is a multiple of 4. They use the minimum number of runs to quickly identify the factors with a significant effect on the response. However, the choice of the initial set of chemical compounds is laborious since these compounds are generally randomly selected. In addition, though GSMN can predict the nutritional requirements for a given biological system, the accuracy of the amount of a single predicted compound to be added is difficult to determine because Y. lipolytica harboring only crtE, crtB, and crtI genes. As model predictions do not correctly reflect the in vivo situation, we combined modelling data with Plackett-Burman factorial design of experiments for optimizing cultivation media. Furthermore, we compared lycopene production in modeling suggested media with other media designed based on compounds usually reported in the literature through fermentation scale up experiments.Therefore, in this paper, we run FBA by means of COBRA Toolbox 2.0  to foresY. lipolytica has been realized. In order to establish a reference medium for evaluation of the FBA efficiency, we first selected a set of 23 factors in the light of preliminary experiments and literature review for the designing of FBA-independent cultivation medium. Bioprocess improvement experiments were carried out using the engineered strain Po1f-1312E-1269IB. In a first step, we screened a set of chemicals without considering the flux balance analysis (FBA). The matrix of the Plackett-Burman design used in regard of this achievement is reported in 10 or the root square (Sqrt) for data transformation. Subsequently, the Pareto chart was used for analyzing the effects of different components and selecting those to be included in the model. There are two different t limits plotted on the graph based on the Bonferroni or family-wise corrected t and a standard t for individual effects tested. Effects above the Bonferroni Limit were added to the model as certainly significant factors. Effects above the t-value Limit were possibly significant and added if they make sense. Effects below the t-value limit were likely insignificant. Pareto charts of all experimental data are reported in Up to date, no deep investigation for development of fermentation media for lycopene production from 3, MgSO4·7H2O, FeCl3·6H2O, FeSO4·7H2O, yeast extract, fructose, BaCl2·2H2O, NiCl2·6H2O, KH2PO4 and vitamin B1 in this decreasing order. The effect of MnCl2·4H2O, Na2MoO4·2H2O and KI were also positively significant  while the positive effect of K2HPO4 was not significant. The set of factors including CuCl2·2H2O, NiSO4·6H2O, CoCl2·6H2O, H3BO3, ZnCl2, MnSO4·7H20, NaCl, Glucose, (NH4)2SO4 were found as inhibitors of biomass production. The highest biomass concentration obtained was 16.14 g/L with max/min value of 25.22. The final equation in term of coded factors was: Sqrt (Biomass) = 2.24 − 0.38 × A − 0.18 × B − 0.043 × C + 0.29 × D + 0.15 × E − 0.13 × G − 0.095 × H + 0.072 × J + 0.089 × K + 0.28 × L + 0.082 × M + 0.29 × N + 0.37 × O − 0.095 × Q +0.22 × R + 0.25 × S − 0.20 × T + 0.14 × U + 0.020 × V + 0.12 × W + 0.19 × X.The results showed that biomass production was significantly (effects above the Bonferroni Limit) improved by the effects of KNO4·7H2O, FeCl3·6H2O, fructose, KNO3, NiCl2·6H2O, vitamin B1 and KH2PO4 had significant positive effects. The maximum lycopene concentration obtained in flasks was 9.18 mg/L. The final equation in terms of coded factors was: Sqrt (Lycopene)= 0.75 − 0.52 × A − 0.12 × B − 0.2 × C + 0.32 × D + 0.18 × E + 0.059 × G + 0.074 × H + 0.081 × J + 0.052 × K + 0.049 × L + 0.049 × M + 0.3 × N + 0.26 × O − 0.064 × P − 0.071 × Q + 0.28 × R − 0.15 × T + 0.11 × U − 0.17 × V + 0.14 × W + 0.072 × X.For lycopene production, MgSO4·7H2O, NiCl2·6H2O, ZnCl2, MnSO4·7H20, KI, MnCl2·4H2O, FeCl3·6H2O, KNO3, fructose, KH2PO4, vitamin B1, BaCl2·2H2O. Other components had negative effects.The maximal lycopene content obtained from the 24 runs was 1.11 mg/g dry cell weight (DCW). Lycopene content was positively promoted by the effects of MgSO4·7H2O (14.74), NiCl2·6H2O (0.097), H3BO3 (0.099), ZnCl2 (0.15), MnSO4·7H20 (0.4), KI (0.012), MnCl2·4H2O (0.2), Na2MoO4·2H2O (0.1), FeCl3·6H2O (2.87), KNO3 (0.05), Glucose (19.9), fructose (20), yeast extract (0.96), (NH4)2SO4 (0.51), KH2PO4 (10) and vitamin B1 (1.00E-02).The post-optimization experiments conducted to the optimized medium (PM medium) with the following composition (g/L): MgSOY. lipolytica (yli v1.7) that was updated from model iYL619_PCP by correcting topological network and parameters of model (model yli v1.7 is available at http://www.echaoceshi.com/ECLOSB/CN/Default.aspx). In model yli v1.7, R0763 is the only reaction, in which farnesyl diphosphate (FPP) is produced; R2000 is the reaction of growth (biomass formation reaction). Besides, the maximum flux of R2000 (specific growth rate) is 0.0350 h−1 in the glucose minimal media. The model yli v1.7 and iYL619_PCP predicted the equal glucose minimal media. The composition of minimal media in the experiment is as follows: glucose (20 g/L), NH4SO4 (3 g/L), KH2PO4 (2 g/L), in which the maximum specific growth rate is 0.0352 h−1. This indicates that model yli v1.7 predicted the maximum specific growth rate more accurately  was implemented to determine metabolic flux distribution with the genome-scale metabolic network model of curately .l-isoleucine, l-leucine, l-valine, l-asparagine, l-histidine, l-methionine, l-tryptophan, guanine, and l-lysine. The additional exchange reactions were those of O2, thiamine diphosphate, succinate, phosphate, ammonia, 4-aminobutanoate, ergosterol, ethanolamine, d-fructose, urea, d-glucose, hypoxanthine, and (R)-pantothenate. The number of negative exchange reactions and fluxes varied widely with the nature of the carbon source opted for. Putting aside the fact that FBA only gives one possible random solution instead of looking at all the possibilities , it seems that the only “non-zero” values in this table are those corresponding to O2 exchange, d-fructose exchange and d-glucose exchange. All other values from −8.05e-35 to −1.46e-13 are most probably rounding errors of the LP solver, and so all those values could be considered as zeros. However, in order to predict the potential benefit of supplementing the minimal media with of above amino acids or several cofactors in terms of FPP production based on FBA metabolic flux distributions, we relaxed the exchange reactions of model yli v1.7. We first tested effects of 18 amino acids (only 18 amino acid can be utilized in model yli v1.7) on FPP production in a definite specific growth rate (90% of the maximum specific growth rate), and found that all the 18 amino acids facilitated FPP production in different degrees  were found as amino acids that could be supplemented to the minimal medium owing to the guarantee of C/N ratio more than 10. Additionally, we also tested effects of 10 factors on FPP production in a definite specific growth rate  with average biomass production of 14.23 and 16.53 g/L leading to lycopene content of 2.90 and 2.45 mg/g. Approximately equal quantities of lipids (1.55 and 1.53 g/L) were yielded on both media and corresponded to lipid contents of 10.90% and 9.23%.Taking into account the results of the above simulations, we applied the Plackett-Burman design for experimental validation of FBA-predicted factors. The design matrix and responses obtained for biomass, lycopene and lipid production in Plackett-Burman design screening of FBA-predicted factors are correspondingly reported in 2PO4, ethanolamine, guanine, l-methionine, l-tryptophan, l-valine, l-asparagine, d-glucose, and d-fructose had positive effects on biomass production. Fructose was the most significant terms (effect above the Bonferroni Limit) followed by KH2PO4, l-valine, ethanolamine, nicotinate, l-asparagine, guanine, l-tryptophan, and glucose. The list of compounds composed of pyridoxine hydrochloride, succinate, 4-aminobutyric acid, Na2SO3, hypoxanthine, l-histidine, l-isoleucine, and l-leucine had negative effects on biomass production. The negative effects of 4-aminobutyric acid, l-histidine, and l-leucine were not significant. The effects of thiamine pyrophosphate and l-methionine were not significant for biomass production. The final equation of biomass production obtained after ANOVA analysis in terms of coded factors was:The analysis of the effects of FBA-predicted factors on the biomass production showed that thiamine pyrophosphate, nicotinate, KHBiomass = 1761.61 +1.00 × A − 1.16 × B + 1158.95 × C − 1.25 × D − 0.77 × E +2.70 × F − 1.70 × J + 1.57 × L − 1.18 × M + 1.51 × N − 0.71 × O + 0.63 × P + 1.41 × R + 1.75 × S + 1.52 × T − 1.85 × U − 0.66 × V + 1.14 × W + 3.28 × X.l-tryptophan, l-isoleucine, ergosterol, and thiamine pyrophosphate (effect above the Bonferroni Limit) followed by ethanolamine and (NH4)2SO4 . Although l-asparagine had a positive effect, this effect was not significant. All other compounds had an inhibitory effect on lycopene production. The final equation in terms of coded factors was:Six factors were found to have significant positive effects on lycopene production. The most significant factors were 10 (Lycopene) = 1.10 + 0.055 × A − 0.049 × B − 0.14 × D − 0.056 × E − 0.046 × F + 0.026 × G − 0.15 × J + 0.080 × K + 0.049 × L − 0.11 × M − 0.010 × N − 0.085 × P + 5.397e-004 × Q + 0.14 × R − 0.027 × S + 8.320e-003 × T + 0.095 × U − 0.14 × V − 0.053 × W − 0.077 × X.Logl-isoleucine was found as the most significant factor having positive effect on lycopene content, followed by l-tryptophan, ergosterol, thiamine pyrophosphate, and l-histidine. Ethanolamine and (NH4)2SO4 had negligible positive effects on lycopene content. The large number of factors negatively influenced the lycopene content.The final equation for lycopene content in terms of coded factors was:10 (Lycopene content) = −42.56 + 0.033 × A − 0.025 × B − 28.22 × C − 0.12 × D − 0.035 × E − 0.10 × F + 0.012 × G − 0.12 × J + 0.067 × K + 0.015 × L − 0.073 × M − 0.041 × N + 0.020 × O − 0.10 × P + 0.10 × R − 0.065 × S − 0.017 × T + 0.14 × U − 0.12 × V − 0.072 × W − 0.13 × X.Log4)2SO4, and l-lysine had significant positive effects. The effects of KH2PO4, glucose, and thiamine pyrophosphate were also moderately significantly positive. l-methionine, urea, l-histidine, nicotinate, and hypoxanthine significantly inhibited lipid production. The effects of other components were not significant. The final equation for lycopene content in terms of coded factors was: For lipid production, ergosterol, 2SO4, and l-leucine. l-histidine had no significant positive effect. The rest of predicted components strongly repressed lipid accumulation.Lipid content was positively affected by 2PO4, 0.5 g/L (NH4)2SO4, 0.1 g/L ergosterol, 0.3 g/L ethanolamine, 5 g/L lysine, 5 g/L tryptophan, 10 g/L isoleucine, 20 g/L fructose, and 10 g/L glucose.Based on the above observations and multiple post-optimization experiments, the optimization process gave MP medium constituted of 1 g/L asparagine, 6 g/L KHl-isoleucine, ergosterol and (NH4)2SO4. This suggested the positive correlation between lipid accumulation and lycopene biosynthesis. This relationship between both pathways may be influenced by the composition of the culture medium since we found diverse discrete distribution of the concentration of these compounds in the different culture media. In addition, high lipid content did not necessarily imply high lycopene content.The correlation analysis was carried out using the Pearson correlation analysis method with a two-tailed test of significance. The correlation analysis was applied to the data obtained from the Plackett-Burman design responses obtained with the FBA-predicted factors. We obtained a Pearson correlation of 0.49 with a significance of 0.01. The correlation between both variable was significant at the 0.05 level. Furthermore, we found commonness between both variables when positive factors were taken into account. Specifically, lycopene content and lipid content were both significantly stimulated by l-tryptophan, (NH4)2SO4, KH2PO4, thiamine, and l-isoleucine in this decreasing order. The rest of the components including glucose, l-asparagine, and ethanolamine had moderate negative effects on biomass production. The highest biomass production of 33.58 g/L was obtained with run 10. The elimination of factors with negative effects in the first PBD driven on the FBA predicted components did not significantly affect biomass production, showing that their elimination was not due to errors. Similarly, KH2PO4, fructose, ergosterol, (NH4)2SO4, and l-tryptophan promoted lycopene accumulation. A lycopene production of 4.35 mg/g DCW (76.62 mg/L) was achieved under the coordinated effect of these compounds in shake flasks. Only KH2PO4 had positive effect on lycopene content.Following the first Plackett-Burman design (PBD), we conducted another PBD  in orderThis second tour PBD suggested that the selected components were further optimizable for biomass and lycopene concentration but did not really impact on lycopene content. Furthermore, some of components with positive effects in the first round of screening were found inhibiting lycopene production. This could be explained by the fact that the scale of choice of these components was reduced.YALI0B16126g which is annotated as protein farnesyltransferase/geranylgeranyltransferase type-1 subunit alpha. On the contrary, significant effects were recorded for YALI0C18755g, the gene encoding for -farnesyl pyrophosphate (FPP) specific hexaprenyl-diphosphate synthase. This gene was significantly stimulated by the effects of l-tryptophan, l-lysine, l-isoleucine, l-asparagine, KH2PO4, and fructose. The high number of positive factorial effects corroborated with the fact that FPP was set as the target metabolite in the FBA simulation. Only ergosterol, thiamine, and glucose had inhibitory effects on this gene. In addition, the ditrans, polycis-polyprenyl diphosphate synthase -farnesyl diphosphate) gene YALI0C18799g was activated by l-tryptophan, ergosterol, l-asparagine, and to some extent KH2PO4 while other components displayed negative effects. YALI0D17556g, another gene responsible for -FPP specific ditrans, polycis-polyprenyl diphosphate synthase, was significantly stimulated by ethanolamine but repressed by l-tryptophan. The expression of the gene in charge of protein farnesyltransferase subunit beta, YALI0D14762g, was similarly triggered by l-lysine, l-asparagine, fructose, KH2PO4, and l-tryptophan and to a minor extent by glucose. Only l-tryptophan had a positive effect on YALI0D17050g (geranylgeranyl diphosphate synthase). The rate-limiting gene of the mevalonate pathway governing the expression of hydroxymethylglutaryl-CoA reductase (HMGCR), YALI0E04807g, was significantly activated by factors including l-lysine, l-asparagine, l-tryptophan, KH2PO4, and fructose but significantly inhibited by ergosterol and thiamine. The activation of this gene was necessary for converting HMG-CoA into mevalonic acid and subsequent metabolites of the terpenoids biosynthesis backbone. YALI0E05753g -farnesyl diphosphate synthase) was significantly stimulated by l-tryptophan, KH2PO4 and to a minor extent by L-asparagine. However, the lycopene biosynthesis genes introduced into the engineered strains, although highly expressed, were activated by numerous positive factors. However, the number of positive effects was not as significant as expected. This could be explained by the fact that the FBA was targeted towards FPP synthesis. Specifically, the expression of the crtE, crtB, and crtI genes were all promoted by ethanolamine (significant effect) and l-lysine, l-isoleucine, glucose, (NH4)2SO4, ergosterol, and glucose to a trivial degree.In order to get insights into the effect of selected factors on genes of the terpenoids backbone, we realized quantitative RT-PCR experiments to explore expression levels of these genes. No significant effect was found for zwf and crt genes. ACT1 while all heterologous genes were upregulated. The upregulation of heterologous genes implied that the carbon metabolism was efficiently directed to FPP biosynthesis and subsequently permitted lycopene production. Nevertheless, different amounts of biomass and lycopene yielded on different runs of culture media could be explained by divergences in expression patterns as depicted in Overall, the results showed that FBA-predicted and PBD screened factors differentially affected genes of the terpenoids backbone, Indeed, though some heterologous genes were highly expressed in certain media, the level of produced lycopene did not match with expression levels. We found that some media components were inhibiting genes of the terpenoids backbone at different levels but the inhibition did not necessarily imply low production of lycopene. Each gene of the terpenoids backbone was activated by specific selected factors whose coordinated actions allowed high lycopene production.Designed media were initially used for lycopene production in a 1.5 L fermenter with a working volume of 1 L in batch. The fed-batch operation started at 24 h and consisted in the supplementation of a feeding solution containing 200 g/L glucose and 200 g/L of fructose. The feeding (50 mL) was executed every 24 h. The pH was initially controlled at 5.5 and then controlled at 3 after 48 h when the color began to appear. The results of biomass and lycopene production on different media over the fermentation course are depicted in After 10 days of fermentation, we found a maximum biomass production of 40.2 g/L with a lycopene concentration of 126 mg/L from the medium designed without taking into account the FBA predictions (PM) a. LycopeY. lipolytica, as an oleaginous yeast, is able to accumulate significant quantities of lipid bodies under certain growth conditions. This property provides a reliable storehouse for carotenoids biosynthesis and, consequently, the heterologous production of carotenoids using Y. lipolytica may constitute a promising asset for large-scale operations. The heterologous synthesis of lycopene in Y. lipolytica requires the introduction of the biosynthetic genes  even if the expression of crtE is not necessary in various heterologous hosts including Y. lipolytica, and bioengineering approaches for integrating these genes in Y. lipolytica have been described previously [CrtE is a geranylgeranyl diphosphate synthase that catalyzes the conversion of FPP and IPP to GGPP and CrtB is a phytoene synthase that can condense two molecules of the C20 precursor, GGPP, to yield the first C40 hydrocarbon, the phytoene. This is followed by a series of sequential desaturations of phytoene to produce ζ-carotene, neurosporene, and, finally, lycopene [CrtI). The most advanced lycopene production using Y. lipolytica is the recent publication of Matthäus, Ketelhot, Gatter, and Barth [crtB and crtI of Pantoea ananatis were expressed in Y. lipolytica under the control of the TEF1 promoter of Y. lipolytica. Moreover, these authors undertook the modifications of native metabolic pathways in Y. lipolytica via the overexpression of bottleneck genes in the isoprenoid pathway (GGS1 and HMG1) and the knock-out of POX1 to POX6 as well as GUT2 genes and successfully enhanced lycopene production (16 mg/g DCW). In a previous study, taking into account the fact that the use of expression vectors pINA1269 and pINA1312, carotenoid genes crtE, crtB, and crtI can be successively integrated into the yeast chromosome with improved strain stability, we synchronically introduced combinations of these plasmids carrying lycopene biosynthesis genes and spawned lycopene producing strains. Further characterization of these engineered strains showed discrepancies in terms of lycopene and biomass production among engineered strains and suggested the impact of engineering tactics on lycopene biosynthesis in engineered strains.Relative to other yeasts, eviously . CrtE islycopene . This prnd Barth  in whichY. lipolytica and other yeast species for lycopene and other terpenoids molecules. Additionally, our study gave insights into the comprehension of the effects of some external factors on genes of the terpenoids pathway. Correct monitoring of these factors for media designing, coupled with adequate metabolic engineering tactics, will contribute to the fundamental research and the efficient production of lycopene even at the industrial level.In the present study, we aimed to establish an effective fermentation platform for lycopene production from the most robust engineered strain (Po1f-1312E + 1269IB) using statistical and GSMN modeling tools for screening chemical compounds and designing culture media. Efficient media were employed for fed-batch cultivation. The improvement of cultivation conditions  led to lycopene production of 242 mg/L of lycopene using the medium designed based on the variables screened from the FBA-predicted factors. This concentration of lycopene was approximately the double of that obtained in the medium designed based on the literature. The above results demonstrated the usefulness of FBA and modelling tools for improved production of lycopene in consistency with previous investigations showing that FBA simulations can be efficiently applied for experimental validation and improvement of the production of objectives production or biological processes ,28. The Y. lipolytica in order to obtain a dependable lycopene producing strain. The bioengineering work has been published in a local Chinese journal. The strain designated as Po1f-1312E + 1269IB, representing the recombinant strain obtained from the introduction of plasmid pINA1312 (carrying gene crtE) and pINA1269 (carrying genes crtI and crtB) into the host strain Y. lipolytica Po1f, was used in this study given that it displayed noteworthy growth and lycopene production behavior.Prior to cultivation improvement experiments, we first proceeded to the bioengineering of in silico Y. lipolytica terpenoids biosynthesis genome-scale metabolic model (GSM) yli v1.7 updated from our previous publication [Flux balance analysis (FBA) is the paramount method for estimating flux distributions and assumes that organisms utilize substrates adequately to maximize a fixed objective of model (biomass or a particular reaction of production). The FBA method can be used to reproduce environmental conditions necessary for achieving the maximization of the objective function of the model and is an integrated element of the COBRA 2.0 Toolbox in Matlab. The FBA was carried out using Matlab R2011a  and the COBRA 2.0 toolbox. We used an lication . The updn+1) runs, wherein n represents the number of modules to be screened. Each component was assigned two levels of concentrations (high concentration and low concentration). Firstly, a set of 23 factors selected in light of the preliminary experiments and literature review (data not shown), were combined in 24 trials for the designing of FBA-independent cultivation media. Following this, the FBA-driven predicted factors (n = 23) were used for conducting a Plackett-Burman design of 24 runs. Numerous post-optimization experiments were equally carried out on the basis of Plackett-Burman design.To identify the vital factors that affect biomass and lycopene production in the engineered strain and make breakthrough improvements, a saturated screening design based on the Plackett-Burman structures incorporated into Design expert software (version 8) was adopted. The total number of trials to be implemented according to Plackett-Burman was  with a working volume of 1 L at 30 °C. The aeration rate was fixed at 1 vvm (volume air per volume per minute) and the agitation rate was set to automatically respond to the dissolved oxygen (DO) concentration so as to maintain it at 50%. Sterile air filters with 0.2 µm pores were used for air transfer into bioreactor. The DO concentration in the culture broth was measured using a pO2 electrode. Silicone was periodically added as an antifoam agent. The fed-batch operation started at 24 h and consisted in the supplementation of a feeding solution containing 200 g/L glucose and 200 g/L of fructose. The feeding (50 mL) was executed every 24 h. The pH was initially controlled at 5.5 and then controlled at 3 after 48 h following the appearance of color.The optimized designed media were directly used for aerobic fermentation of the transgenic After cultivation, 5 mL of cell broth was harvested, centrifuged at 12,000 rpm for 5 min, washed twice with distilled water and dried at 105 °C for 48 h. The dry cell weight (DCW) was determined by gravimetrically measuring the weight of the dry cell pellets. Glucose measurement was monitored by a spectrophotometric procedure by means of a Glucose Reagent Kit  following the vendor’s guidelines. Lipid extraction protocol was as described in our previous publication .Following the cultivation end time, precise volumes of homogenous culture broth were harvested, centrifuged at 12,000 rpm for 10 min at 4 °C and washed twice with deionized water. The supernatant was discarded and the pellet was used for lycopene extraction. Prior to lycopene extraction, pellets were dissolved in 500 μL of dimethyl sulfoxide (DMSO), pre-warmed at 55 °C for 30 min, shook strongly for 30 s, and then maintained for an extra 30 min without shaking. After that, 500 μL of acetone was added to the DMSO extract and homogenized by vortexing for 30 s, followed by incubation at 55 °C for 15 min with intermittent shaking. The mixture was subsequently centrifuged at 12,000 rpm for 10 min, and the colored mixed supernatants were transferred into a new tube. The extraction step was repeated until all visible pigments in the residual cell pellet and supernatant were extracted. The obtained colored supernatants were pooled together and used for lycopene quantification via high-performance liquid chromatography (HPLC) analysis. All operations occurred on ice under dim light to prevent photodegradation, isomerizations, and structural changes of the carotenoids.The lycopene concentration of each extract was quantified by HPLC analysis which was performed on a Shimadzu LC-20A HPLC instrument equipped with a LC solution software program, LC-20AD pump, and a detector with a UV-Vis lamp. Specifically, it was carried out by injecting 10 μL of the crude carotenoid extract into a Zorbax SB-Aq C18-reverse-phase column . The extract was eluted under isocratic condition with a mobile phase containing 80% acetonitrile, 15% methanol and 5% isopropanol at a flow rate of 1 mL/min at 30 °C. Quantitative analysis was accomplished using a calibration curve obtained with a lycopene standard and final lycopene concentrations were obtained by normalization to the total initial volumes used during extraction.The cells were pelleted and total RNA was extracted as previously described . To keepACT1 was utilized as a housekeeping gene. The results were expressed as fractions of gene expression between the target gene and the housekeeping gene, ACT1 [To prevent the variation of starting materials amounts, ne, ACT1 .Y. lipolytica. The findings of the present study could have implications for improved production of lycopene using Y. lipolytica as a fermentation platform and promote terpenoids production at the industrial scale. The FBA and Plackett–Burman screening investigational design empowered the bringing about of significant variables for lycopene production from


Pre-cooling aims to decrease body core temperature prior to  and dece2max, 10sec of passive recovery) [Eight healthy subjects (22 (1.7) yrs) performed, in 31 °C, two experimental conditions: mid-cooling (Mid) and (Con), a 46-min intermittent exercise protocol consisting of multiple 2-min bouts  than in Con (0.61 (0.11) °C; 153 (1.20) beats.min-1), whereas ΔTskin did not differ between condition.No differences were observed between conditions in the initial 46 min of exercise. Τhe rate of rectal temperature drop (ΔTre) during the break was higher in Mid (-0.15 (0.02) °C) than in Con (-0.05 (0.02) °C) (p = 0.03), whereas the rate of change in skin temperature (ΔTskin) and heart rate (HR) were similar in the two conditions. Upon completion of the second exercise period, the rise of Tre was less profound (p = 0.05) and HR tended to be lower (p = 0.12) in Mid (0.42 (0.07) °C; 139 (1.57) beats.minImplementation of a "Mid" (wet bathrobe), cooling maneuver during the break between two intermittent exercise periods elicited a three times greater Tre fall. This effect was carried on to the second exercise period where the rate of Tre rise was lower, thus preventing excessive hyperthermia .These results suggest an effective and practical mid-competition cooling maneuver.


We determined the complete genome sequence of a coxsackievirus A16 strain (CVA16/SZ29/CHN/2014) from a fatal case in Shenzhen, southern China, in 2014. The strain was assigned to subgenotype B1b based on phylogenetic analysis of the VP1 gene. Picornaviridae, is a common causative agent of hand, foot, and mouth disease (HFMD) in children and infants (Coxsackievirus A16 (CVA16), a member of the enterovirus A species of the family  infants . CVA16 c infants . Worldwi infants , 5.On 13 May 2014, a 2.7-year-old boy manifested symptoms of hand, foot, and mouth disease accompanied by severe vomiting, lethargy, muscle twitching, and aseptic encephalitis. The boy died in a few days after ineffective treatment. The fecal specimen from this case was detected positive for CVA16 by real-time reverse transcription (RT)-PCR and propagated in rhabdomyosarcoma (RD) cell lines at the Department of Microbiology, Shenzhen Center for Disease Control and Prevention, Shenzhen, China. This is the first case of death caused by CVA16 in Shenzhen since sentinel surveillance for HFMD began in 2008.We designed a pair of universal primers EVA-F30 (5′-TTAAAACAGCCTGTGGGTTGTACCCACCCA-3′) and EVA-R36 (5′-GCTATTCTGGTTATAACAAATTTACCCCCACCAGTC-3′) to amplify the whole genome of CVA16 strain using a PrimeScript One Step RT-PCR kit version 2 . The amplification mixture was run under the following conditions: reverse transcription for 30 min at 50°C, initial denaturation for 2 min at 94°C, 45 cycles of 30 s at 94°C, 30 s at 55°C, 8 min at 72°C, and 10 min at 72°C. The amplified DNA product was sequenced by a commercial corporation  using a primer-walking method. Contigs were assembled using Sequencer version 4.9. The raw genome sequence was edited using BioEdit version 7.2.5 before submission to GenBank. Nucleic acid and amino acid sequence alignments were done with BioEdit version 7.2.5. Phylogenetic analysis was performed using MEGA version 5 , excluding the poly(A) tail. The 5′ UTR was found to be 746 nt, followed by an open reading frame (ORF) encoding the structural protein P1 , the nonstructural proteins P2  and P3 , and the 3′ UTR (82 nt). The contents of A, C, G, and U were 27.73%, 23.29%, 23.82%, and 25.16%, respectively, with G+C contents of 47.11%. The genome sequence of CVA16/SZ29/CHN/2014 was found to be closely related to the Chinese CVA16 strain BJ08/07 (GenBank accession no. JX068833) with 98.06% nucleotide identity by a BLAST research. The strain CVA16/SZ29/CHN/2014 was assigned to subgenotype B1b based on phylogenetic analysis of the VP1 gene.KM215267.The full-length sequence of CVA16/SZ29/CHN/2014 has been deposited in GenBank under the accession number


Poor systemic concentrations of lopinavir (LPV) following oral administration occur due to high cellular efflux by P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs) and extensive metabolism by CYP3A4 enzymes. In this study, amino acid prodrugs of LPV were designed and investigated for their potential to circumvent efflux processes and first pass effects. Three amino acid prodrugs were synthesized by conjugating isoleucine, tryptophan and methionine to LPV. Prodrug formation was confirmed by the LCMS/MS and NMR technique. Interaction of LPV prodrugs with efflux proteins were carried out in P-gp (MDCK-MDR1) and MRP2 (MDCK-MRP2) transfected cells. Aqueous solubility studies demonstrated that prodrugs generate higher solubility relative to LPV. Prodrugs displayed higher stability under acidic conditions and degraded significantly with rise in pH. Uptake and transport data suggested that prodrugs carry significantly lower affinity towards P-gp and MRP2 relative to LPV. Moreover, prodrugs exhibited higher liver microsomal stability relative to LPV. Hence, amino acid prodrug modification might be a viable approach for enhancing LPV absorption across intestinal epithelial and brain endothelial cells which expresses high levels of P-gp and MRP2. LPV is a protease inhibitor (PI) that is commonly indicated in highly active antiretroviral therapy (HAART) for the treatment of HIV infection. Inclusion of PIs in HAART has significantly improved clinical outcomes in HIV infected patients. However, a major problem associated with PIs is their inability to attain higher systemic concentrations following oral administration. LPV alone displayed poor oral bioavailability primarily due to high P-gp and MRP2 mediated efflux  and hepaVarious strategies have been employed to improve oral and brain absorption of P-gp and MRP2 substrates. One of the most common approaches employed have been the co-administration of P-gp and MRP2 substrates with efflux inhibitors. However, systemic administration of high doses of inhibitors required to inhibit P-gp and/or MRP2 may result in unacceptable toxicities. Moreover, these efflux proteins are also expressed in normal tissues and extrude xenobiotics and cytotoxic molecules thereby providing cellular protection. Therefore, inhibition of these efflux processes might result in cellular side effects. Prodrug based delivery strategies capable of circumventing P-gp and MRP2 mediated efflux could be valuable in such circumstances. Transporter targeted prodrug modification is one such promising strategy where the ligand coupled drugs can bypass efflux pumps by binding to influx transporters highly expressed on the intestinal epithelium and/or BBB.i.e., valine-quinidine and isoleucine-quinidine have demonstrated high potential in circumventing P-gp mediated cellular efflux -LPV (1 Ci/mmol) was purchased from Moravek Biochemicals  and used at 0.25 µCi/mL. MDCK cells, retrovirally transfected with human 2 tissue culture flasks in DMEM medium supplied with high glutamine and glucose concentrations. Culture medium was supplemented with 10% FBS (heat inactivated), penicillin (100 units/mL) and streptomycin (100 µg/mL) and maintained at pH 7.4. Cells were incubated at 37 °C in an atmosphere of 5% CO2 and 90% relative humidity. The medium was replaced every alternate day until cells reached 70%–80% confluency (5–7 days).MDCK-WT, MDCK-MRP2 and MDCK-MDR1 cells (passage 8–10) were grown in 75 cmAqueous solubility study of prodrugs was performed according to the published protocol from our laboratory . Saturat1/2) of prodrugs were determined at various pH. Approximately, 50 µM prodrug solution was added in 1.5 mL of DPBS adjusted to pH 5, 6 and 7.4. Microcentrifuge tubes were placed in shaking water bath (60 rpm) at 37 °C. Samples (100 µL) were withdrawn at predetermined time points and stored at −80° C until further analysis. Prodrug concentrations remaining were plotted versus time in order to calculate degradation rate constants.Chemical hydrolysis study of prodrugs was carried out according to the previously published protocols from our laboratory . Degrada. Following incubation, 20 μL of MTS stock solution was added to each well and incubated for 2 h at 37 °C. Cell viability was determined by measuring absorbance at 485 nm with the help of a plate reader . The quantity of formazan product as measured by absorbance is directly proportional to the number of viable cells in test samples.Cytotoxicity of prodrugs was determined in MDCK-WT cells with MTS assay based cytotoxicity kit. Briefly, cells were grown overnight in 96 well plates at a density of 20,000 cells per well prior to drug and prodrug addition. Medium was aspirated and replaced with 100 μL of serum free solution containing serial dilutions of test compounds (5–250 μM). Cells were then incubated for 4 h at 37 °C6 cells in 12 well culture plates. Medium was replaced every alternate day until cells reached confluency (6–7 days). Cellular uptake studies were performed on confluent cell monolayers according to previously published protocol from our laboratory -LPV in DPBS at 37 °C for 30 min. Following incubation, radioactive solution was quickly removed and plates were washed with ice-cold stop solution  to arrest the uptake process. One mL of lysis buffer (0.1% Triton-X solution in 0.3% NaOH) was added to each well and plates were stored overnight at RT. Later, 500 µL solutions were transferred to scintillation vials containing 3 mL of scintillation cocktail. The radioactivity associated with cells was analyzed with a scintillation counter . Uptake rate was normalized to protein count which was quantified with a BioRad protein estimation kit. To study the effect of efflux inhibitors, cells were treated with GF 120918 (MDCK-MDR1) and MK 571 (MDCK-MRP2) prior to the initiation of the uptake experiment.Confluent cells were then trypsinized and plated at a density of 3 × 10boratory ,21. Brie® inserts  with transparent polyester membranes were employed to study transepithelial transport of LPV and prodrugs. Transwell® inserts were coated with type 1 rat tail collagen (100 µg/cm2). Cells were seeded at a density of 250,000 cells per well. Transport studies were conducted across MDCK-MDR1 and MDCK-MRP2 cells in presence and absence of efflux inhibitors for a period of 3 h at 37 °C. Prior to the initiation of transport study, cell monolayer integrity was evaluated by monitoring transepithelial electric resistance (TEER), with an EVOM . TEER values of the cell monolayer were approximately 250 Ω*cm2. Monolayers were washed thoroughly with DBPS (pH7.4) for 15 min at 37 °C, three times. For determining A–B permeability, 0.5 mL LPV or prodrug solution (25 µM) was added to the apical chamber of 12-well Transwell® plates. Similarly, 1.5 mL LPV or prodrug solution (25 µM) was added to the basolateral chamber to determine B-A permeability. At predetermined time points , 100 µL sample was withdrawn from the receiving chamber and replaced with fresh DPBS to maintain sink conditions. Samples were stored at −80 °C until further analysis. To determine the effect of efflux inhibitors, cells were treated GF 120918 (MDCK-MDR1) and MK 571 (MDCK-MRP2) prior to initiation of the transport study.Transepithelial transport studies were carried out according to the protocols published from our laboratory . TransweRat liver microsomes were employed to determine the substrate affinity of LPV and prodrugs towards CYP3A4. One mL of microsomal solution was generated by adding microsomal protein (0.3 mg/mL), magnesium chloride (5 mM), glucose 6-phosphate (5 mM), b-NADP1 (1 mM), and glucose 6-phosphate dehydrogenase (1 U/mL) in phosphate buffer . LPV or prodrugs was added to the microsomal solution and incubated at 37 °C for 5 min. The metabolic reaction was initiated by adding the NADPH generating system. NADPH generating system was freshly prepared by adding G-6-P, G-6-P DH and NADP in DDW. At predetermined time points, metabolic reaction was arrested by adding equal volumes of ice-cold acetonitrile to the sample. Samples were stored at −80 °C until further analysis by LCMS/MS technique. Both time and concentration dependent stability studies of LPV and prodrugs were conducted. For time dependent metabolism studies, LPV or prodrugs (25 μM) were incubated with activated liver microsomal solution. This study was performed according to the previously published protocols from our laboratory . At predFor concentration dependent degradation studies, various concentrations of LPV or prodrugs (1–20 μM) were incubated with activated microsomal solution for 5 min. Enzymatic degradation was arrested by adding equal amount of acetonitrile and samples were stored in −80 °C until further analysis.Aqueous solubility and buffer stability samples were measured with reversed phase HPLC technique. Waters 515 pump  connected to a UV detector  was employed. A C(18) Kinetex column  was pumped with mobile phase (1:1) acetonitrile and water (0.1% trifluoroacetic acid) at a flow rate of 0.3 mL/min. Analytes were detected with UV detection at a wavelength of 210 nm. Ile-LPV, Met-LPV and Trp-LPV eluted at approximately 9.2, 8.0 and 10.2 min, respectively.Transport samples of LPV and prodrugs were analyzed with a fast and sensitive LC–MS/MS method. Samples were extracted with liquid–liquid extraction technique. About, 50 μL of verapamil (200 ng) was added as an internal standard to each sample. Analytes were extracted by vortexing with 800 μL water saturated ethyl acetate for 2.5 min. Following centrifugation at 10,000 rpm for 7 min, approximately 600 µL of ethyl acetate layer was collected and evaporated under reduce pressure for 45 min. The residue was reconstituted in 100 μL of acetonitrile (80%) and water (20%) containing 0.1% formic acid. Ten microliters of reconstituted samples were injected in LCMS/MS. Similarly, standard solutions were extracted from DPBS and quantified with LCMS/MS.® LCMS/MS mass spectrometer  connected to Agilent 1100 Series quaternary pump (Agilent G1311A), vacuum degasser (Agilent G1379A) and autosampler  was employed. Chromatographic separation of analytes was obtained with XTerra®MS C18 column 50 mm × 4.6 mm, 5.0 μm . Mobile phase (80% acetonitrile and 20% water containing 0.1% formic acid) was pumped at 0.3 mL/min flow rate and chromatographs are obtained over 4 min. LPV and verapamil eluted at 3 and 1.7 min, respectively. Ile-LPV, Met-LPV and Trp-LPV eluted within 1.7–1.8 min, respectively.To analyze transport samples, QTrapElectrospray ionization in the positive mode was employed and analytes of interest were detected using multiple-reaction monitoring (MRM) mode. Precursor ion of analytes was determined from spectra which are generated with the infusion of standard solutions to the electrospray source with the help of an infusion pump. Each of these precursor ions was then subjected to collision-induced dissociation to produce product ions. Precursor/product ions observed for LPV and verapamil were 629.398/155.183 and 455.020/150.050, respectively. Precursor/product ions observed for Ile-LPV, Met-LPV and Trp-LPV were 742.489/155.192, 760.547/155.159 and 815.499/155.151, respectively.We also optimized the turbo ion spray setting and collision gas pressure . MS/MS was performed using nitrogen as collision gas. Other operational parameters optimized included declustering potential (DP): 80 V; collision energy (CE): 62 V; entrance potential (EP) 8.5 V; and collision cell exit potential (CXP) 4 V. These parameters were generated by shooting analytes in quantitative optimization mode. Each analyte of interest produced with MRM method showed a significant linearity up to ng range. With this method, rapid and reproducible results were successfully obtained.Transepithelial permeability was determined by plotting cumulative amount of analyte transported against time. Linear regression of analyte transported as a function of time generated rate of transport (dM/dt) which was divided by the cross-sectional area (A) available for transport to generate steady-state flux as denoted in Equation (1):Transepithelial permeability was calculated by normalizing the steady-state flux with the donor concentration (Cd) according to Equation (2):t-test was applied to determine the statistical significance among groups. A difference between the mean values was considered to differ significantly if the p value is ≤0.05.Uptake, transport and buffer stability results were expressed as mean ± S.D. Student’s LPV prodrugs were synthesized according to previously published protocols with minor modifications . BrieflyBoc-isoleucine-LPV was treated with approximately 60% trifluoroacetic acid (TFA) in dichloromethane at 0 °C for about 1 h. The solvent was quickly evaporated under reduced pressure to obtain oily crude product. The product Isoleucine-LPV (Ile-LPV) was purified by recrystallization in cold diethyl ether. This oily product was dissolved in methanol and evaporated under reduced pressure at RT to obtain a final dried powder with a 90% yield. The final product (prodrug) was then stored in −20 °C until further use. The reaction scheme for the synthesis of Ile-LPV is shown in ® LC/MS/MS spectrometer—Applied Biosystems) was employed under enhanced mass mode to identify prodrugs. LC/MS (m/z); [M+H]+ observed for Ile-LPV, Met-LPV and Trp-LPV were 742.5, 760.5 and 815.5, respectively. The purity of prodrugs was determined using TLC and HPLC analysis. For TLC analysis, ethyl acetate was used as the solvent system. For HPLC analysis, C(18) Kinetex column  was employed. Two different solvents were used: A) acetonitrile and B) water. Both solvents contained 0.1% formic acid. The gradient employed was 0% A to 100% A for 30 min, followed by 100% A for another 10 min. Total runtime was 40 min. The flow rate of solvent system was 0.4 mL/min. The wavelength used for prodrug detection was 210 nm. Purity of all prodrugs was observed to be greater than 95%. 1H-NMR ((CD3)2S=O) analysis was performed to detect the main characteristic peaks supporting the formation of prodrugs. The ppm values (δ) observed are (a) Ile-LPV: 0.78 , 0.82 , 0.90 , 1.00 , 6.35 , 6.90–7.10 , 7.16–7.3 , 8.45 ; (b) Met-LPV: 0.70 , 0.82 (CH3-S), 1.22 , 1.45 , 3.60 , 6.35 , 6.90-7.10 , 7.16–7.30 , 8.60 ; (c) Trp-LPV: 0.7 , 0.82 , 3.65 , 6.35 , 6.90–7.10 , 7.16–7.30 , 8.45 , 11.1 .Prodrugs were characterized with LCMS/MS, TLC and NMR analysis. A hybrid triple quadrupole-linear ion trap mass spectrometer . Currently, LPV is administered to HIV-1 infected patients in the form of approximately 40% v/v alcoholic solution as it is practically insoluble in water. Such high alcohol concentrations may not be suitable for pediatric patients. Moreover, ethanol has been reported to augment CYP3A activity . An enhai.e., 4, 5.5 and 7.4. Results obtained from this study are summarized in i.e., 7.4.Stability of prodrugs was determined in DPBS at various pH values Cytotoxicity of LPV and prodrugs was determined in MDCK-WT cells with MTT assay. Medium having no drug or organic solvents was used as control. DMSO (10%) or Triton-X (0.1%) containing medium was selected as positive control. Results obtained from this study are demonstrated in in vitro cell culture model alternative to Caco-2 for high throughput drug screening -LPV uptake in presence of efflux inhibitors in MDCK-MDR1 and MDCK-MRP2 cells, respectively. These cell lines represent an excellent creening ,25. PrioWe carried out [3H]-LPV uptake in presence of increasing concentrations of prodrugs to determine the extent of prodrug interaction with efflux proteins. As demonstrated in To confirm the observation obtained in concentration dependent uptake studies, we carried out cellular uptake of unlabelled LPV and prodrugs in MDCK-MDR1 and MDCK-MRP2 cells. Briefly, cell monolayers were incubated with unlabelled LPV or prodrugs at 5 µM concentration for 30 min. Following incubation, LPV or prodrug solution was aspirated and the cell monolayer was washed with ice cold stop solution three times (each wash of 5 min) to terminate the uptake process. About 500 µL of DMEM was added to each well and plates were kept in −80 °C overnight for cell lysis. On the following day, LPV or prodrugs were extracted by liquid-liquid extraction technique as described in Cellular uptake of prodrugs was observed to be significantly higher relative to LPV in both MDCK-MDR1 and MDCK-MRP2 cells . Ile-LPVTo examine the effect of efflux proteins in modulating permeability of LPV and prodrugs, we carried out transepithelial transport across MDCK-MDR1 and MDCK-MRP2 cells from apical to basolateral  and basolateral to apical  direction. In MDCK-MDR1 and MDCK-MRP2 cells, P-gp and MRP2 are expressed on the apical membrane. A classical substrate of these efflux proteins would therefore generate higher transport rate from secretive relative to absorptive direction. Hence, the role of efflux proteins in limiting LPV transport would be apparent provided there is a considerable difference in A-B and B-A permeability rates.−6 and 5.8 ± 0.5 × 10−6 cm/s, respectively. Efflux ratio displayed by LPV from B-A to A-B direction was approximately 2.5. Importantly, LPV transport increased drastically in A-B direction in presence of cyclosporine A and GF 120918 (data not shown). A-B permeability values generated by LPV in presence of cyclosporine A and GF 120918 were 8.5 ± 0.4 × 10−6 and 9.0 ± 0.3 × 10−6 cm/s, respectively. These results clearly suggest that the transport of LPV is significantly limited by P-gp.Transepithelial LPV transport study in presence and absence of inhibitors/substrates was conducted to determine the role of P-gp in modulating the transport process. As observed in −6 and 6.0 ± 0.1 × 10−6 cm/s, respectively. Efflux ratio observed was approximately 2.2. Moreover, a marked augmentation in LPV transport was observed in presence MK 571 across MDCK-MRP2 cells (data not shown). A-B LPV permeability value exhibited in presence of MK 571 was 8.7 ± 0.5 × 10−6 cm/s. Based on these observations, it is apparent that LPV transport is significantly restricted by MRP2.Transepithelial transport of LPV across MDCK-MRP2 cells in presence and absence of MRP2 inhibitors was also carried out. Similar to previous results, LPV transport was observed to be significantly lower in A-B direction relative to B-A B. A-B anTo investigate the potential of amino acid prodrug modification in overcoming P-gp and MRP2 mediated cellular efflux, we conducted prodrug transport across MDCK-MDR1 and MDCK-MRP2 cells. Circumvention of P-gp and MRP2 efflux mechanism would be evident provided the absorptive transport rate of prodrugs is significantly higher relative to LPV. Previously, dipeptide prodrugs of LPV have been developed and examined for their efficacy in circumventing P-gp and MRP2 mediated cellular efflux . These d−6, 5.3 ± 0.4 × 10−6 and 4.2 ± 0.3 × 10−6 cm/s, respectively. A-B permeability of Ile-LPV, Met-LPV and Trp-LPV observed was about 3, 2.3 and 1.8-fold higher relative to LPV. These results indicate that prodrugs may not be recognized by P-gp relative to LPV.In this study, we examined if amino acid modified prodrugs of LPV could also circumvent P-gp and MRP-2 mediated cellular efflux. A-B permeability values obtained for LPV and prodrugs across MDCK-MDR1 and MDCK-MRP2 cells are summarized in −6, 5.1 ± 0.2 × 10−6 and 4.5 ± 0.4 × 10−6 cm/s, respectively. A-B permeability of Ile-LPV, Met-LPV and Trp-LPV was about 2.8, 1.9 and 1.7-fold higher relative to LPV. Such higher permeability values clearly suggest that prodrugs might have lower affinity towards P-gp and MRP2 relative to LPV.Similarly, prodrugs exhibited higher A-B permeability across MDCK-MRP2 cells relative to LPV. Apparent A-B permeability generated by Ile-LPV, Met-LPV and Trp-LPV across MDCK-MRP2 cells were 7.5 ± 0.3 × 10−6, 5.8 ± 0.2 × 10−6 and 4.4 ± 0.5 × 10−6 cm/s, respectively. A-B permeability values of Ile-LPV, Met-LPV and Trp-LPV observed in presence of cyclosporine A across MDCK-MDR1 cells were 7.7 ± 0.2 × 10−6, 5.6 ± 0.3 × 10−6 and 4.1 ± 0.2 × 10−6 cm/s, respectively. Permeability values of prodrugs across MDCK-MDR1 cells were almost similar in presence and absence of GF 120918 and cyclosporine A. A-B permeability values displayed by Ile-LPV, Met-LPV and Trp-LPV across MDCK-MRP2 cells were 7.9 ± 0.7 × 10−6, 5.5 ± 0.4 × 10−6 and 4.9 ± 0.2 × 10−6 cm/s, respectively. No significant improvement in the permeability of prodrugs was observed across MDCK-MRP2 cells in presence of MRP2 inhibitor. These results confirmed that prodrugs show lower affinity towards efflux transporters such as P-gp and MRP2 relative to LPV. Based on the results obtained in the present study, we anticipate that prodrugs might produce higher oral absorption due to lower affinity towards efflux proteins and improved solubility.To confirm this observation, we carried out A-B transport of prodrugs in presence of P-gp and MRP2 inhibitors. A–B permeability values generated by Ile-LPV, Met-LPV and Trp-LPV in presence of GF 120918 across MDCK-MDR1 cells were 6.9 ± 0.4 × 10LPV is primarily metabolized by CYP3A4 enzyme. Hence, we carried out metabolic stability studies in rat liver microsomes to determine the affinity of prodrugs toward CYP3A4 relative to LPV. Previously, dipeptide prodrugs of LPV have been observed to exhibit higher metabolic stability in rat liver microsomes relative to LPV . MoreoveIn the present study we investigated the efficacy of amino acid prodrugs to circumvent CYP3A4-mediated metabolism of LPV. Rat liver microsomes were employed at the concentration of 0.3 mg/mL. We first determined the stability of LPV and prodrugs in liver microsomal solution in presence and absence of ketoconazole (100 µM), a potential inhibitor of CYP3A4 enzyme. Liver microsomal solution was incubated with ketoconazole for 15 min prior to the initiation of stability study. Results obtained from this study are depicted in −3 min−1. Interestingly, degradation rate constants displayed by prodrugs were significantly lower relative to LPV. Degradation rate constants generated by Ile-LPV, Met-LPV and Trp-LPV were about 3.8 ± 0.5 × 10−3, 3.6 ± 0.4 × 10−3 and 3.5 ± 0.2 × 10−3 min−1, respectively. These results clearly suggested that amino acid prodrugs had slightly reduced affinity for metabolic enzymes compared to LPV. Half lives of Ile-LPV, Met-LPV and Trp-LPV were observed to be approximately 1.6 to 1.8-fold higher compared to LPV. Based on this result, we anticipate that prodrugs will display higher stability in liver microsomes which in turn will result in higher systemic levels relative to LPV.We performed time dependent stability study to determine degradation rate constant in liver microsomal solution. Degradation rate constant of LPV observed was 6.0 ± 0.5 × 10To confirm reduced affinity of prodrugs towards metabolizing enzymes, we carried out concentration dependent degradation study by incubating various concentrations (0.5–40 µM) of LPV or prodrugs in liver microsomes (0.3 mg/mL) for 5 min. The data obtained was fitted in Michaelis-Menten kinetic model to generate Km (affinity constant) and Vmax values. Prodrugs displayed lower affinity towards metabolizing enzymes relative to LPV. Km values observed for LPV was approximately 6.0 ± 0.9 µM. Km values generated by Ile-LPV, Met-LPV and Trp-LPV were about 14.3 ± 1.1, 13 ± 2 and 13.2 ± 1.9 µM, respectively. These results confirmed previous observation that prodrugs have lower affinity towards metabolizing enzymes relative to LPV. We further determined the intrinsic clearance (Vmax/Km) of LPV and prodrugs from the data obtained in concentration dependent degradation studies. LPV displayed higher intrinsic clearance with value of 1.4 mL/min/mg protein. Ile-LPV, Met-LPV and Trp-LPV exhibited lower intrinsic clearance with values of 0.96, 0.5 and 0.77 mL/min/mg protein. Results obtained from these studies clearly confirmed that prodrugs have better metabolic stability and significantly lower affinity towards CYP enzymes compared to LPV. Thus, these amino acid prodrugs might bypass first pass effect to a significant extent relative to LPV. We also anticipate that amino acid prodrugs might generate higher systemic levels of LPV upon oral administration due to reduced metabolic degradation by CYP3A4 enzymes.in vitro. Prodrugs generated better aqueous solubility and permeability across MDCK-MDR1 and MDCK-MRP2 cells relative to LPV. Moreover, prodrugs displayed higher metabolic stability and lower affinity towards metabolizing enzymes relative to LPV. This study indicates that amino acid prodrug approach might be a viable strategy in improving the pharmacokinetic properties of LPV. Evasion of efflux process might be crucial in improving LPV transport across P-gp and MRP2 overexpressing membranes and overcoming development of resistance from chronic LPV administration. In further studies, interaction with various influx transporters such as peptide transporters, plasma protein binding and oral and brain pharmacokinetics of LPV prodrugs will be reported.Amino acid prodrugs of LPV were synthesized and evaluated for their efficacy in circumventing efflux proteins and metabolizing enzymes


Buthus martensi Karsch, induces pain behaviors in rats. However, the subtypes of VGSCs targeted by BmK I were not entirely clear. We therefore investigated the effects of BmK I on the current amplitude, gating and kinetic properties of Nav1.8, which is associated with neuronal hyperexcitability in DRG neurons. It was found that BmK I dose-dependently increased Nav1.8 current in small-sized (<25 μm) acutely dissociated DRG neurons, which correlated with its inhibition on both fast and slow inactivation. Moreover, voltage-dependent activation and steady-state inactivation curves of Nav1.8 were shifted in a hyperpolarized direction. Thus, BmK I reduced the threshold of neuronal excitability and increased action potential firing in DRG neurons. In conclusion, our data clearly demonstrated that BmK I modulated Nav1.8 remarkably, suggesting BmK I as a valuable probe for studying Nav1.8. And Nav1.8 is an important target related to BmK I-evoked pain.Voltage-gated sodium channels (VGSCs) in primary sensory neurons play a key role in transmitting pain signals to the central nervous system. BmK I, a site-3 sodium channel-specific toxin from scorpion  Primary sensory neurons in dorsal root ganglia (DRG) play an essential role in transmitting pain signals by detecting noxious stimuli through their peripheral axons and sending them to the postsynaptic neurons in spinal cord via their central axons  were identified and functionally characterized. According to their pharmacological sensitivity to tetrodotoxin (TTX), VGSCs can be divided into two groups: TTX-sensitive (TTX-S) and TTX-resistant (TTX-R). Nav1.8 sodium channel produces TTX-R current with a high activation threshold and slow inactivation kinetics and contributes to the upstroke of action potentials in small diameter nociceptive DRG neurons , is considered to be the key contributor of extreme pain-evoked by scorpion envenomation  was significantly leftward shifted after BmK I treatment. The slope factor (km) was not affected. Steady-state of Nav1.8 inactivation current was evoked by a 50 ms depolarizing pulse of 0 at the pre-pulse potentials ranging from −60 mV to +40 mV for 50 ms. Representative current traces in the absence and presence of 500 nmol/L BmK I were shown in Fig. 1/2) was left shifted in the presence of 500 nmol/L and 1 μmol/L BmK I. Similar to the activation curves, the slope factor (km) remained unchanged before and after application of BmK I.G(V) relationship indicated that BmK I shifted voltage-dependent activation of Nav1.8 in a hyperpolarizing direction Fig. A. The mif) was left shifted in the presence of 500 nmol/L and 1 μmol/L BmK I, while the slope factors (kf) were not affected significantly. The slow inactivation of Nav1.8 was also affected by application of BmK I. Representative current traces were shown in Fig. s) was leftward shifted in the presence of 500 nmol/L and 1 μmol/L BmK I. In addition, 1 μmol/L BmK I increased the slop factor of slow inactivation (ks) to 7.67 ± 0.83 mV.Furthermore, we studied the voltage-dependence fast and slow inactivation by using protocols which with pre-pulse for different time duration (10 ms for fast inactivation and 200 ms for slow inactivation) under the impact of BmK I Fig. . RepreseThe detailed data were shown in Tables Nav1.8 current was elicited by a 100-ms test pulse to 0 from a holding potential of −60 mV in a small-sized DRG neuron which exposed to the bath solution with 500 nmol/L TTX. The current was increased significantly in the presence of 500 nmol/L BmK I  DRG neurons. Rheobase, action potential threshold, was the minimal injected current to evoke an action potential. Fig. Action potentials (APs) in response to rheobase (1×), 2 times (2×) and 3 times (3×) rheobase stimulation were recorded under the same conditions Fig. B. ApplicWe next determined whether the firing of frequency was massively increased by BmK I. There was approximate 8 fold increase in the gain of frequency-stimulation relationship in DRG neurons   contributed to BmK I-insensitive sodium channels  . The external solution contained 0.0005 mmol/L TTX, 140 mmol/L NaCl, 3 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 0.1 mmol/L CdCl2, 10 mmol/L D-Glucose and 10 mmol/L HEPES, pH 7.3 . For current clamp recordings, pipette solution contained 140 mmol/L KCl, 10 mmol/L NaCl, 0.5 mmol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L EGTA, 3 mmol/L Na2ATP, and 10 mmol/L HEPES, pH 7.2 . The external solution contained 0.0005 mmol/L TTX, 150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 2.5 mmol/L CaCl2,10 mmol/L D-Glucose and 10 mmol/L HEPES, pH 7.4 . Both external solutions were saturated with O2.Whole-cell path clamp recordings were performed on small-sized DRG neurons (diameter <25 μm). For voltage clamp recordings, the pipette solution contained 140 mmol/L CsCl, 1 mmol/L MgClWhole-cell path clamping experiments were performed by using an EPC-10 amplifier  at room temperature. Patch pipettes were fabricated from glass capillary tubes by PP-830 Puller  with the resistance of 3–5 MΩ. Data acquisition and stimulation protocols were controlled by Pulse/Pusle Fit 10.0 software (HEKA Elektronik). Capacitance transients and series resistance errors were compensated by 80%. Cells were discarded when the series resistance values were over 20 MΩ. Linear leakage was subtracted using P/4 protocol. Data were sampled at 50 kHz and low-pass filtered at 10 kHz.Nav1.8 currents and Nav1.8-meditated action potentials were isolated using TTX (500 nmol/L) in the bath solution and identified by Nav1.8 selective blocker A-803467 (5 μmol/L).r), where I is the peak current elicited upon depolarization, V is the membrane potential, and Vr is the reversal potential.Mean conductance (G) was calculated from peak current (I)–voltage (V) relationships using the equation G = I/(V − Vmax= 1/[1 + exp(V − V1/2)/km], where V1/2 is the voltage for half-maximum activation and km is the slope factor.Nav1.8 currents were elicited by 200 ms pulses depolarizing to +40 mV from a holding potential of −60 mV for inhibiting Nav1.9 currents. The voltage-dependent activation was fitted with the Boltzmann relationship, G/Gmax = 1/[1 + exp(V − V1/2)/k], where V is the membrane potential of the conditioning step, V1/2 is the membrane potential at which half-maximal inactivation is achieved, and k is the slope factor. The parameters for fast inactivation were characterized by the half-maximal voltage Vf and the slope factor kf; correspondingly Vs and ks for slow inactivation.Steady-state inactivation were evoked by a 50 ms depolarizing pulse of 0 at the pre-pulse potentials ranging from −60 mV to +40 mV for 50 ms. The voltage-dependent fast inactivation and slow inactivation were analyzed by using protocols which with pre-pulse for different time duration (10 ms for fast inactivation and 200 ms for slow inactivation), to potentials ranging from −60 mV to +40 mV with the increments of 5 mV followed by a test pules of 0 for 50 ms. Data were fitted to the two-state Boltzmann equation, I/Inorm= A/[1 + ([BmK I]/EC50)p ] + C, where Inorm is the measured and normalized peak current, E50 is the half maximal effective concentration, and p is the slop factor.The equation used for fitting the dose-response relationship was: In. Student’s paired or unpaired t-tests were used for comparisons.Data were analyzed using Origin 8.5 , and they were presented as means ± SEM. The number of cells examined was represented by Y.P., L.T. and J.Y.H. conceived and designed the experiments. Y.P., J.Y.L. and L.Z.W performed the experiments. Y.P. and J.F. analyzed the data. Y.P., J.Y.L, L.Z.W, H.L.M. and F.J. contributed reagents/materials/analysis tools. Y.P., L.T. and J.Y.H wrote the paper. All authors have reviewed the manuscript.


Children in the VI+nys group were divided into three training groups: an experimental PL group, a control PL group, and a magnifier group. They were seen before (baseline) and after 6 weeks of training. Children with NV were only seen at baseline. The feature search task entailed finding a target E among distractor E's (pointing right) with element spacing varied in four steps: 0.04°, 0.5°, 1°, and 2°. At baseline, children with VI+nys showed longer search times, shorter fixation durations, and larger saccade amplitudes than children with NV. After training, all training groups showed shorter search times. Only the experimental PL group showed prolonged fixation duration after training at 0.5° and 2° spacing, p's respectively 0.033 and 0.021. Prolonged fixation duration was associated with reduced crowding and improved crowded NVA. One of the mechanisms underlying improved crowded NVA after PL in children with VI+nys seems to be prolonged fixation duration.Perceptual learning (PL) can improve near visual acuity (NVA) in 4–9 year old children with visual impairment (VI). However, the mechanisms underlying improved NVA are unknown. The present study compares feature search and oculomotor measures in 4–9 year old children with VI accompanied by nystagmus (VI+nys [ Evidence is accumulating that perceptual learning (PL) can have a beneficial impact on a range of visual perceptual skills, from contrast sensitivity , but can also be associated with congenital visual abnormalities such as albinism, congenital cataract or optic atrophy  and children with VI accompanied by nystagmus (VI+nys). The second is that search times of children with VI+nys are longer than search times of children with NV. The third is that training induces shorter search times in all training groups. Finally, it is hypothesized that there are no changes in fixation duration after training in children with VI+nys.Thirty-three children with VI+nys and 29 children with NV participated. Inclusion criteria for both groups were: age between 4 and 9 years and a normal developmental level. Additional inclusion criteria for children with VI were: distance visual acuity (DVA) between 20/400 and 20/40, normal birth weight (at least 3000 grams), birth at term (at least 36 weeks), no perinatal complications, no additional impairments, and an intact visual field. Table C-test at 5 m . Objective refraction was obtained after cycloplegia and if necessary spectacle correction was prescribed or changed before the experiment and training period started.C-test, which is at least 30′ or 0.5° at 40 cm). Therefore, the control group was the PL uncrowded group (PLu group).Children were divided into one of three training groups matched on age and distance visual acuity (see Table The third training paradigm was a magnifier training in which children had to search for the inversed Landolt C within a 191 mm long search strip consisting of Landolt C's sized 0.5 mm (or −0.10 logMAR) with element spacing of 0.3 mm (the M group). The optotypes were too small to inspect with a bare eye, so an electronic handheld magnifier was used to find the inversed C. A game element was included to provide feedback and engage the children. Each training consisted of a maximum of 12 trials and these trials were coupled to tiles that could be placed on answer boxes. If all tiles were placed correctly, they formed a pattern that matched with the pattern in the upper right corner of the page. During training, children worked at a self-chosen distance.The training tasks are described in the section Training Task. Children with NV were seen once as a reference group at baseline. For children with VI, this baseline performance counted as their pre-test score. Training started within 2 weeks after the pre-test. During the training, children with VI were seen 2× a week for a period of 6 weeks . Each training session consisted of 30 min of practice on the training task described in the section above. Trainers visited children at their schools. Within 2 weeks after the last training session children performed the post-test.Children sat at a distance of 60 cm from the monitor wearing their best available optical correction. Viewing was binocular. Sixteen trials were presented to the children (4 (orientation) × 4 (spacing), see Figure Stimuli were presented on a 17″ monitor with integrated eye-trackers . Stimulus presentation was driven by Delphi software. Eye movements were analyzed using MATLAB . We did not fix the head positions of the children, because the Tobii has a large head movement box which means that it has a good ability to compensate for head movements (44 × 22 cm at 70 cm). Children stayed well within these limits, so head movements are not likely to cause data distortions. A rule was incorporated into the stimulus-presentation software, in order to assure that the children were seated at a proper viewing distance. When children came closer to the monitor than 60 cm, the stimulus disappeared from the screen, and reappeared if they were seated at 60 cm or more again. This rule was included to prevent children from reducing their viewing distance and to standardize our measurements. Eye movements were registered at a sampling rate of 60 Hz. Before the visual search task commenced a standard 5-point calibration procedure was performed. Fixations were detected offline and were defined as periods in which eye velocity remained below 50°/s for at least 50 ms. The velocity threshold was recalculated as 3.5× the standard deviation of the calculated noise of the eye velocity for each session. This recalculation is necessary, because of the diversity of the eye movements of our participants. For example, eye movement velocity might be seriously elevated in children with nystagmus and we might need a higher threshold than 50°/s to detect fixations. Data files with less than 35% valid data points were excluded from analysis.p-value in which the K refers to the number of groups (padj = p*K (K − 1)/2).There were 2 main outcome categories: visual search performance measures (accuracy and search time) and oculomotor measures . Accuracy measures were compared between children with NV and children with VI+nys with nonparametric tests (Mann–Whitney test and Friedman's test), because of skewed distributions and unequal variances. A correction for pairwise comparisons was used for the Friedman's test by reporting the adjusted t-tests for each training group to see under which condition changes in performance occurred. In case pre-post × spacing interactions were found, separate Repeated Measures ANOVA's were conducted to evaluate how spacing affected performance before and after training. Post-hoc tests were performed using the Bonferroni method to correct for multiple comparisons (familywise Type I errors). Alpha was set on 0.10 to enable the detection of trends in our data set and because of the small sample sizes.Search time showed near-normal distribution and oculomotor measures were distributed normally. These measures were analyzed with a Repeated Measures ANOVA. Group was entered as the between-subjects factor and spacing as the within-subjects factor. Training effects in children in the VI+nys group were evaluated by conducting a 2 (pre-post) × 4 (spacing) Repeated Measures ANOVA. Training group was then entered as the between-subjects variable. In case of pre-post × training group interactions, we conducted paired Figure Accuracy. No group differences were found in accuracy (see Table 2(3) = 22.25, p < 0.001; VI+nys: χ2(3) = 11.68, p = 0.009]. Children with NV showed lower accuracies at 0.04° and 0.5° (75%) than at 2° spacing (100%), resp. p = 0.026 and p = 0.005. Pairwise comparisons in the VI+nys group were non-significant  = 3.71, p = 0.059, partial η2 = 0.06]. Children with NV showed shorter search times (3.7 s) than children with VI+nys . No spacing or interaction effect was found.Fixation duration. There was a group × spacing interaction, F = 2.57, p = 0.057, partial η2 = 0.06. At 0.04°, children with VI+nys showed shorter fixation durations (255 ms) than children with NV (361 ms), F = 13.31, p = 0.001, partial η2 = 0.24. At 0.5°, children with VI+nys showed shorter fixation durations (268 ms) than children with NV (325 ms), F = 3.34, p = 0.075, partial η2 = 0.07. At 1°, children in the VI+nys group showed shorter fixation durations (245 ms) than children in the NV group (304 ms), F = 7.21, p = 0.010, partial η2 = 0.15. At the largest spacing (2°), no group differences were found. Children with NV adjusted their fixation durations to spacing, F = 7.45, p < 0.001, partial η2 = 0.28. Children fixated longer at 0.04° than at 1° and 2° spacing, p's 0.037 and 0.002  = 8.22, p = 0.006, partial η2 = 0.16. Children in the VI+nys group made larger saccades (9.2°) than children with NV  = 5.09, p = 0.002, partial η2 = 0.11. Saccade amplitudes were larger at 0.5° (9.3°) than at 2° (7.9°), p = 0.001. No interaction was found.n = 4; PLc, n = 9; PLu, n = 4). Figure We collected 17 valid eye movement recordings  = 5.00, p = 0.025; PLc: χ2(1) = 5.00, p = 0.025). Children in the PLu group did not show improved accuracy; they already showed a median accuracy of 100% at pre-test. At 0.04°, 1° and 2°, none of the groups showed improved accuracy after training.Search time. There was a spacing × training group interaction, F = 2.14, p = 0.057, partial η2 = 0.13. In the M group, a training effect was found, F = 10.31, p = 0.012, partial η2 = 0.56. Mean search time was 4.8 s before and 3.3 s after training  = 3.78, p = 0.024, partial η2 = 0.32. However, post-hoc tests were not significant. No pre-post × spacing interaction was found. In the PLc group, we also found a training effect, F = 4.99, p = 0.042, partial η2 = 0.26. Mean search times were 5.2 s before and 4.0 s after training  = 2.37, p = 0.096, partial η2 = 0.23. Paired t-tests showed that children did not show reduced search times at 0.04°, but did show reduced search times at all other spacings [0.5°: t(8) = 2.15, p = 0.064, 1°: t(8) = 1.88, p = 0.097, 2°: t(8) = 1.98, p = 0.083; see Figure Number of fixations. There was no main training effect on the number of fixations, but there was a pre-post × spacing interaction, F = 3.83, p = 0.071, partial η2 = 0.22. Children made more fixations before (10.0) than after training at 2° (7.1), t(16) = 2.23, p = 0.041. All other t-tests were non-significant.Fixation duration. There was a three way pre-post × training group × spacing interaction, F = 2.24, p = 0.058, partial η2 = 0.24. Paired t-tests were conducted for each spacing and training group to disentangle interaction effects. In the M group, no changes were found in fixation duration  = −2.58, p = 0.033; at 2°: t(8) = −2.87, p = 0.021, see Figure t(3) = −2.49, p = 0.089  = 2.12, p = 0.071, partial η2 = 0.23. Post-hoc analysis showed that in the M-group, spacing affected saccade amplitude, F = 4.21, p = 0.040, partial η2 = 0.58. Children tended to make larger saccades at 0.5° (10.4°) than 2° (8.7°), p = 0.055  = 4.51, p = 0.034, partial η2 = 0.60. However, post-hoc tests were non-significant.r's 0.65 and 0.52, p's 0.002 and 0.016.A correlational analysis was conducted to investigate the relations between changes in visual and oculomotor measures for children in the VI+nys group. As can be seen in Table The goal of this study was to evaluate the effect of PL on visual feature search performance and oculomotor measures in children with VI+nys. Different from what we expected, we found evidence that oculomotor parameters in children in the VI+nys group can be altered after a 6-week period of PL. We only observed significant increases in fixation duration in the PL groups. In the next sections our findings are discussed in the light of the postulated hypotheses.Our first hypothesis was that there were group differences in fixation duration at baseline. Children with NV showed longer fixation durations (278–361 ms) than children in the VI+nys group (242–268 ms). This finding is in line with results from a previous study from our group which reported shorter fixation durations in children in the VI+nys group compared to children with NV during serial visual search  than the saccade amplitude of children in the VI+nys group (9.2°). Saccade amplitudes were calculated by measuring the distance from one fixation point to a following fixation point. The finding that children with VI+nys make larger saccades than those with NV is a replication of what was reported in an earlier study .The group differences reported in the present study are much smaller than previously reported differences in search time between children with NV and children with VI. For example, in an earlier study where children had to search for a unique target in a grid with elements sized 2 times their threshold acuity, we found up to 5-fold slower search times for children with VI compared to children with NV  and 2° element spacing (from 222 to 294 ms). In addition, the PLu group showed prolonged fixation duration at 1° spacing, but the effect was not observed at other spacings in this training group and seemed to be less consistent than the increase in fixation duration in the PLc group.r = 0.65) and improvements in crowded NVA (r = 0.52). In an earlier study, we found that nystagmus frequency was positively related to mono- and binocular crowding ratios in children with idiopathic nystagmus (Huurneman and Boonstra, The prolongation of fixation durations in the PLc group is an unexpected and exciting finding, because individuals with nystagmus are considered to be unable to voluntary diminish their nystagmus and attempts to prolong fixation periods usually have a negative impact on nystagmus (Sheth et al., Three explanations may account for changes in fixation duration after training. The first is maturation of the nervous system, which may alter the sinusoidal and jerk nystagmus so that foveation time increases and vision improves (Van Vliet, The mechanism responsible for the extended fixation durations after training is not known, since we only recorded eye movements. A combination of psychophysiological and brain imaging techniques would be required to find out whether changes on a behavioral level have a specific neural origin. One might expect that the reticular formation is involved, a brain structure responsible for arousal, alertness and awareness (Role and kelly, The present study indicates that PL in children with VI showing nystagmus does not only transfer to improvements in NVA, but also induces improvements in feature search performance and oculomotor measures. The most striking finding observed in this study is that children with VI prolonged their fixation duration after training. This prolongation of fixation duration is significantly related with improvements in crowded NVA and reduction of crowding ratios.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.