Phenolic compounds with antioxidant properties are highly sensitive molecules, which limits their application. In response, extruded esterified starch has been proposed as efficient encapsulating material. In this work, we aim to describe the encapsulation of red sorghum phenolic compounds by spray drying using extruded phosphorylated, acetylated and double esterified sorghum starch as wall material. Their respective encapsulation yields were 77.4, 67.4 and 56.8%, and encapsulation efficiency 91.4, 89.7 and 84.6%. Degree of substitution confirmed esterification of the sorghum starch and Fourier transform infrared spectroscopy showed the significant chemical and structural changes in the extruded esterified starch loaded with phenolic compounds. Microcapsules from phosphorylated sorghum starch showed the highest endothermic transition (173.89 °C) and provided a greater protection of the phenolic compounds during storage at 60 °C for 35 days than the other wall materials. Extruded esterified sorghum starch proved to be effective material for the protection of phenolic compounds due to its high encapsulation efficiency and stability during storage. In vitro studies have shown that the exposure of cancerous cells of the colon and the oesophagus to polyphenolic sorghum extracts decreases or inhibits cell proliferation ; it has low production costs and multiple agroindustrial applications  to develop three different esterified sorghum starch types by reactive extrusion, (ii) to evaluate the encapsulation of red sorghum phenolic compounds by spray drying, and (iii) to evaluate the storage stability of microencapsulated red sorghum phenolic compounds.Therefore, it is clear that sorghum grain is emerging as a potential source of polyphenolic compounds with health benefits, in addition to being a cereal with high starch content. To our knowledge, there is no published information on the esterification of white sorghum starch by reactive extrusion for evaluation as a wall material. Also to our knowledge, there are no reports regarding encapsulation of red sorghum phenolic compounds using starch derivatives as wall materials. Moreover, to date and to our knowledge, no double modification of the starch has been reported to obtain a phosphorylated and acetylated starch by extrusion. Based on the latter, we are raising the following as aims in this paper: , Guanajuato, Mexico) was used for the isolation of starch according to the wet milling method , with a 20-mm inner barrel diameter. Barrel temperatures were maintained at 60, 130 and 170 °C at the feeding, transition and high-pressure extrusion zones, respectively. Screw speed was 80 rpm and feed rate was 70 g/min, while the compression ratio of the screw was 3:1. The resulting extruded samples were dried in the oven (40 °C), milled and sieved through a mesh with a 149 µm pore size. Powder samples of esterified starch were packed for subsequent characterization and microencapsulation of phenolic compounds.Phosphorus content and degree of substitution (DS) in phosphorylated sorghum and double esterified sorghum starch were determined by the method of Smith and Caruso (w(P) is the mass fraction of phosphorus (on dry mass basis) in the phosphorylated sorghum starch, 162 is the molar mass of the anhydroglucose unit, 3100 is the atomic mass of phosphorus multiplied by 100 and 124 is the molar mass of the phosphate substituent.where b/%) was calculated according to the following equation:The amount of acetyl groups and the DS of the acetylated sorghum and double esterified sorghum starch were determined by titration, as described by Wurzburg (Vcontrol and Vsample are the titration volumes for the blank and sample respectively (mL), msample is the sample mass (g), and 0.043 is the milliequivalent of the group CH3–C=O. DS was calculated as follows:where 3–C=O multiplied by 100 and 42 is the atomic mass of group CH3–C=O minus 1.where 162 is the molar mass of the anhydroglucose unit, 4300 is the atomic mass of group CHm/m). The spectrum was recorded in a range between 400 and 4000 cm–1.Fourier transform infrared spectroscopy (FTIR) analyses were performed to determine the structural changes in derivatized starch samples  compared to native starch with a spectrometer . The microparticles of the three types of starch were prepared by finely grinding the powder with KBr (1:100 V/V) HCl in 70% ethanol) (pH=2.5). After 1 h of extraction, the solution was centrifuged  for 10 min at 5000×g and filtered by vacuum filtration through a Whatman No. 1 filter paper to recover the solids. The process was repeated on the solids (a 250-mL volumetric flask containing 10 mL of acidified ethanol) and the extracts were combined. Total polyphenol content was estimated using the Folin-Ciocalteu method (Phenolic compounds were extracted from the red sorghum grain. A sample of finely ground sorghum grain (6.25 g) was stirred in 250 mL of acidified ethanol  were used for microencapsulation of red sorghum polyphenols by spray drying . Emulsions of 10 g of starch esterified by extrusion, 60 mL of the polyphenolic extract and 30 mL of distilled water were prepared. The internal phase was added at 30 °C to form the emulsion immediately prior to drying. Emulsions were homogenized with an Ultra Turrax T-25-SI homogenizer  at 14 000 rpm for 5 min. The dispersion was maintained under constant stirring with a magnet bar during drying. Drying conditions were as follows: inlet air temperature (150±1) °C, outlet air temperature (80±5) °C, nozzle diameter 0.5 mm, and a liquid flow rate 10 mL/min. The airflow equipment was set at 70 mV/V/V), stirred using a vortex mixer  for 1 min, ultrasonicated in a 20-kHz Branson ultrasonic bath  twice for 20 min, and then centrifuged at 112 000×g for 5 min. Finally, the residues of both total and superficial polyphenols were quantified according to the Folin-Ciocalteu method (Encapsulation efficiency (EE) and encapsulation yield (EY) were quantified by determining the concentration of superficial and total polyphenols  was used to investigate the thermal properties of microcapsules (loaded with sorghum polyphenols) of phosphorylated, acetylated, double esterified starch and Capsul® starch employing a DSC instrument  calibrated with indium. Each sample (7 mg) was heated on an aluminium pan at a rate of 10 °C/min at between 25 and 210 °C.The morphological characteristics of the microcapsules were evaluated using SEM  at an acceleration voltage of 3 kV. Dried microcapsule samples were previously sprinkled on a double-sided sticky tape placed on aluminium stubs and covered with thin graphite film.Microcapsules loaded with red sorghum phenolic compounds were stored at 60 °C in an oven with controlled temperature and in the absence of light for 35 days. Samples of 200 mg of microcapsules  and Capsul®  were transferred into 100 mm×150 mm clear glass vials. Determination of total polyphenols was performed to evaluate changes during storage. Triplicate vials were removed every 7 days.All experiments were performed in triplicate. The differences between mean values of the treatments were determined using analysis of variance (ANOVA) and the paired Tukey’s test (p<0.05) with IBM SPSS v. 20 statistical software  were 0.040 and 0.043 for phosphorylated and acetylated starch, respectively, and 0.017 and 0.037 for double esterified starch  (data not shown). In this regard, the percentages of acetylation and phosphorylation were higher for acetylated and phosphorylated starch, respectively. Double esterified starch had greater number of hydroxyl groups substituted by the phosphate and acetyl groups than acetylated and phosphorylated starch. This could be explained by the transformation of the crystalline regions into amorphous regions when the acetic acid is mixed with starch at high temperature, contributing to the contact between the OH groups of the starch molecules and the substituent groups, in this case, the phosphate and acetyl groups  and acetyl groups (1735 cm–1) of double esterified starch are observable; however, the signals are weaker, which is associated with the low DS obtained for each functional group . Nonetheless, these values show a greater degree of substitution (0.054) of hydroxyl groups.d starch , a new set al. . This polyphenolic extract was used as an internal phase during microencapsulation. Encapsulation yield (EY) indicates the amount of polyphenols recovered in the encapsulation process, based on the initial amount of polyphenols added to the emulsion per g of starch against the amount of polyphenols recovered per g of capsules. Encapsulation efficiency (EE) indicates the potential of the encapsulating material to maintain the polyphenols inside the capsule . As can –1 than the spectra of all of the starch esterified by extrusion . This sSEM micrographs in et al. . The highest thermal stability was evident in microcapsules with phosphorylated starch. This could be explained by the fact that the phosphate group provides greater structural stability due to a higher molecular mass. The addition of high-molecular-mass groups into chemical structures increases endothermic transitions. These results are similar to those reported by Gonçalves da Rosa et al. (The microcapsule stability during storage at 60 °C is shown in In this research, we obtained a satisfactory encapsulation of the polyphenolic compounds of red sorghum using phosphorylated, acetylated and double esterified sorghum starch. The extrusion technique represents an attractive approach for obtaining esterified starch that can be applied as encapsulating material for the conservation of red sorghum phenolic compounds. To the best of our knowledge, the double esterification of sorghum starch has been successfully developed for the first time and evaluated as an encapsulating material. The introduction of acetyl and phosphate groups into the starch molecules was confirmed by FTIR analysis. Phosphorylated starch was the best cover material because it offered the greatest thermal stability compared with the other materials. Esterified sorghum starch as encapsulating agent by a spray dryer can improve the stability of sorghum phenolic compounds and optimize routes for various applications.


To confirm the suitable synthesis process parameters of preparing bromoisobutyryl esterified starch (BBES), the influences of the synthesis process parameters—amount of 2-bromoisobutyryl bromide (BIBB), amount of catalyst (DMAP), reaction temperature and reaction time—upon the degree of substitution (DS) were investigated. Then, to produce a positive effect on the properties of graft copolymers of BBES prepared in the near future, a series of BBES samples were successfully prepared, and their sizing properties, such as apparent viscosity and viscosity stability, adhesion, and film properties, were examined. The BBES granules were characterized by Fourier transform infra-red spectroscopy and scanning electron microscopy. The adhesion was examined by determining the bonding forces of the sized polylactic acid (PLA) and polyester roving. The film properties were investigated in terms of tensile strength, breaking elongation, degree of crystallinity, and cross-section analysis. The results showed that a suitable synthesis process of BBES was: reaction time of 24 h, reaction temperature of 40 °C, and 0.23 in the molar ratio of 4-dimethylaminopyridine to 2-bromoisobutyryl bromide. The bromoisobutyryl esterification played the important roles in the properties of the starch, such as paste stabilities of above 85% for satisfying the requirement in the stability for sizing, improvement of the adhesion to polylactic acid and polyester fibers, and reduction of film brittleness. With rising DS, bonding forces of BBES to the fibers increased and then decreased. BBES (DS = 0.016) had the highest force and breaking elongation of the film. Considering the experimental results, BBES (DS = 0.016) showed potential in the PLA and polyester sizing, and will not lead to a negative influence on the properties of graft copolymers of BBES. It can ic bonds . Generalic bonds  or graftic bonds ,15. The ic bonds ,17,18. Tic bonds ,19,20.As is well known, starch has widespread applications in the textile field as a sizing agent and n paper making as a surface-sizing agent ,22. HoweFor example, due to the similarity with polyethylene terephthalate, easy processing as well as environment-friendliness, polylactic acid (PLA) acquired from renewable resources has been extensively explored for fiber applications . NowadayCurrently, there is little research about the synthesis process of starch macroinitiator, i.e., bromoisobutyryl esterified cornstarch (BBES). Therefore, an important aim of this work is to obtain suitable synthesis process parameters for preparing the BBES, as shown in Natural cornstarch with a moisture content of 12.8 wt. % and an apparent viscosity of 46 mPa·s, was purchased from Shandong Hengren Industry and Trade Co., Ltd. . Before use, the starch was refined to remove the protein substance  and subsThe BBES was synthesized by the reaction between ACS and BIBB in a THF medium.2 atmosphere. Subsequently, a certain amount of BIBB was added dropwise in an ice-cold bath under mechanically stirred. The reaction mixture was stirred for 1 h in the bath and heated up to a given temperature. Afterwards, the mixture was reacted at the given temperature for a certain time. Then, the final product was filtered, washed thoroughly with anhydrous ethanol, followed by freeze-dried in vacuum, powdered, and sieved with a 100-mesh sieve.Briefly, dried ACS (2 g) and a certain amount of DMAP were added into a 250 mL four-necked flask connected to a mechanical stirrer, and dispersed with 100 mL of THF under mechanical agitation. Then, the TEA was added into the dispersion, and the reaction system was mechanically stirred for 30 min under NB and 150 denote the percentage content (%) and molecular mass of 2-bromoisobutyryl substituents, respectively, and M and n are the moles of BIBB and anhydroglucose residues of starch, respectively.DS indicated the number of the hydroxyls per anhydroglucose unit substituted by the 2-bromoisobutyryl substituents. Back titration with HCl preceded by alkali saponification ,35 was e−1 with the running condition of 4 cm−1 spectral resolution.The Fourier transform infra-red (FTIR) spectra of ACS and BBES samples were acquired on an IRPrestige-21 FTIR Spectrometer  by using KBr disk technique to prove the successful introduction of new functional 2-bromoisobutyryl substituents in the starch molecules after bromoisobutyryl esterification. The spectra were collected in a wavenumber range of 500–4000 cm2 atmosphere. After the temperature of the reaction system had been cooled to 0–8 °C, certain amounts of BIBB and DMAP  were slowly added dropwise. The reaction was kept for 1 h at the above temperature and then heated up to 40 °C for 24 h. Finally, the final product was filtered, washed thoroughly with anhydrous ethanol, followed by freeze-dried in vacuum, powdered and sieved with a 100-mesh sieve.Dried ACS (120 g) and anhydrous sodium sulfate (30 g) were added into a 500 mL four-necked flask connected to a mechanical stirrer and dispersed with THF to form a 30% dispersion. Then, the TEA (molar ratio of TEA to BIBB was 2:1) was added dropwise into the dispersion, and the dispersion was mechanically stirred for 60 min under NThe surface morphologies of the granular ACS and BBES samples as well as the cross-sections of their film samples were observed by means of a scanning electron microscope . Prior to SEM imaging, the samples were sprayed gold to avoid charge accumulation.w/w)) was recorded using an NDJ-79 Rotary Viscometer  at 95 °C by the method described in the literature [The apparent viscosity of gelatinized starch paste  in China to determine the adhesion of a sizing material to fibers. The determination of the adhesion contains three steps  was used to determine the surface tension of cooked starch paste. A 1 wt. % starch aqueous paste was prepared by heating the 1 wt. % starch aqueous dispersion to 95 °C and maintaining at this temperature for 1 h. After cooling down, the tension was determined in duplicate at room temperature.w/w) starch aqueous paste (400 mL) was formed by preparing the starch aqueous suspension and heating the suspension to 95 °C for 1 h under mechanical agitation. Afterwards, the paste was cast onto a smooth polyester film (650 mm in length and 400 mm in width) spread on a glass plate and dried at the above ambient condition for forming the starch film.The film was formed by drying cooked starch paste that was cast onto a polyester film at 65% RH and 20 °C according to the method described in our previous work . BrieflyThen, the films prepared were cut into 200 mm × 10 mm strips. After storing at 20 °C and 65% RH for 24 h, tensile strength and elongation at break of the films were measured on a YG026D Electronic Strength Tester in an initial chuck-distance of 100 mm and a stretching speed of 50 mm/min in accordance with the ASTM D 882-02 method. For each set of data, the averages of test results of twenty strips were reported.The XRD patterns of ACS and BBES films were recorded on an XRD-6000 X-ray Diffractometer  equipped with a wavelength of 0.154 nm CuKa radiation at an X-ray generator setting of 40 kV and 30 mA. The scanning region of diffraction was registered with a speed of 4°/min and a 2θ angular range of 5° to 45° with an angular step of 0.02°.To ascertain the suitable synthesis process of BBES, this work will mainly carry out research on the influences of the synthesis process parameters—amount of BIBB, amount of catalyst (DMAP), reaction temperature, and reaction time—upon the DS.To confirm that the synthesis process of BBES in −1, which were mainly attributed to the decreases of the OH groups after the esterification. In addition, the FTIR spectrum of the BBES  of 2.8; the influence of the amount of DMAP (denoted by the molar ratio of DMAP to BIBB) on the DS is depicted in To avoid the gelatinization of starch granules in the aqueous dispersion, a reaction temperature range of 20–60 °C was selected for the study. The influence of reaction temperature on the DS is represented in After investigating the influences of the synthetic process parameters upon the DS, we obtain the suitable synthesis process parameters of BBES: reaction time of 24 h, reaction temperature of 40 °C, and 0.23 in molar ratio of catalyst (DMAP) to BIBB. Accordingly, to study the properties of BBES, such as adhesion-to-fibers and film properties, etc., a series of BBES samples with different DS values were prepared.SEM technique has become an important means of clearly recording the granule morphology of a modified starch . Figure Using the suitable synthetic process parameters, the DS values and reaction efficiencies of granular BBES samples prepared were measured and denoted by plotting the DS values and reaction efficiencies versus moles of BIBB, as illustrated in The influence of bromoisobutyryl esterification on adhesion of ACS to PLA and polyester fibers was estimated by plotting the bonding forces versus DS values, as shown in To ascertain the influence of bromoisobutyryl esterification on adhesion, the characteristics of the gelatinized starch paste must be given attention. It is generally known that starch granules in the aqueous dispersion heated at a high temperature will swell due to adsorbing the water, and the hydrogen bonds of starch will be disrupted; sequentially the crystalline structure of the granules will be destroyed to form an amorphous structure, subsequently forming a gelatinized starch paste . The pasUndoubtedly, steric hindrance generated by the 2-bromoisobutyryl substituents derivatized into starch molecules can obstruct the parallel fashion and closely approach between starch amyloses in the paste, and lower the co-crystallization between the amyloses and linear branches of amylopectin, thereby favoring the diminishing of paste retrogradation. Consequently, the 2-bromoisobutyryl substituents derivatized may be expected to improve the wetting and spreading of the paste on the fiber surfaces. In addition, as broadly accepted, wetting and spreading of an adhesive liquid onto a given solid surface is closely related to surface tension . A decreDuring the sizing process, a portion of starch paste forms a starch film around the surfaces of sized yarns . The filThe influence of bromoisobutyryl esterification on tensile strength and breaking elongation of ACS film is shown in During formation of the film, the amyloses in aqueous paste parallelly arrange and approach closely with each other, and subsequently form a three-dimensional network structure held together by hydrogen bonding between the hydroxyls . This isThrough the investigation to the influences of the synthesis process parameters—amount of BIBB, amount of catalyst (DMAP), reaction temperature and reaction time—upon the DS, we obtain suitable synthesis process parameters for BBES: reaction time of 24 h, reaction temperature of 40 °C, and 0.23 in molar ratio of DMAP to BIBB. Then, to study the properties of BBES such as adhesion-to-fibers and film properties, etc., for producing a positive effect on the properties of grafted BBES prepared in the near future, a series of BBES samples with the DS values of 0.009, 0.016, 0.027 and 0.036 and reaction efficiencies of 61.3%, 54.5%, 46.0% and 40.9%, were prepared according to the suitable synthesis process parameters. The successful introduction of the 2-bromoisobutyryl substituents was demonstrated by FTIR technique. It could be concluded that introducing 2-bromoisobutyryl substituents into starch molecules via bromoisobutyryl esterification of ACS with BIBB generating the positive effects on the properties of ACS, such as paste stabilities of above 85%, improvement in the adhesion of ACS to PLA and polyester fibers, and reduction of film brittleness. The stabilities of above 85% indicated that the introduction of the substituents could meet the requirement in the stability for warp sizing. The adhesion of BBES to both fibers was correlated with the DS, and the forces presented a trend that first increased and then decreased as increasing DS. When the DS was 0.016, the highest force was obtained. The breaking elongation of the BBES film was higher than the ACS film, and its tensile strength and degree of crystallinity were inferior to those of ACS film, indicating that 2-bromoisobutyryl substituents derivatized could lower the film brittleness . Considering the results of BBES with respect to paste stability, adhesion, and film properties, BBES with a DS of 0.016 showed potential in the applications of PLA and polyester sizing, and will not produce an adverse effect on the properties of grafted BBES prepared in the near future based on the BBES.


A rare complication after lower limb revascularization using a saphenous vein bypass graft in a crush injury patient where the saphenous vein graft was the sole supplying vessel to the leg is described; a pseudoaneurysm developed in the saphenous vein graft and caused active profuse bleeding through the surgical wound. The aetiology of this condition is uncertain but it could occur owing to slippage of ligature from one of the tributaries of the saphenous vein. The diagnosis was made by digital subtraction angiography. The pseudoaneurysm was successfully obliterated by glue embolization, which stopped the bleeding immediately, with preservation of distal flow, thereby salvaging the limb. Because the presentation of these conditions is generally dramatic and the mortality associated with an acute rupture is high, immediate treatment, either surgical or endovascular, is warranted.Pseudoaneurysms of coronary saphenous vein graft are unusual and difficult to treat.We report a case in which a pseudoaneurysm developed in the lower limb saphenous vein graft from the anterior tibial artery (ATA) to dorsalis pedis artery (DPA) and caused profuse life-threatening bleeding through the surgical wound. As an alternative to surgical management, we used emergency glue embolization to repair the pseudoaneurysm, a technique that we believe has not been used before in such a case.via the contralateral common femoral artery retrograde access using a 4 French sheath and 4 French Cobra catheter  which showed a pseudoaneurysm   by mixing 1 ml of glue with 2 ml of Lipiodol ] into the neck of the pseudoaneurysm after flushing the microcatheters with 5% dextrose solution. Soon a complete glue cast  was performed in the interventional radiology suite aneurysm  that hadaneurysm . As the aneurysm  was navilue cast  formed ilue cast . Local b4 Complications after surgical bypass include wound infection, necrosis, tissue loss, graft occlusion and bleeding.6Vein grafts are one of the most commonly used conduits for bypass grafting. The autologous saphenous vein is commonly used in vascular surgery as a bypass graft for relief of limb ischaemia and reconstruction of lower limb arteries such as the tibioperoneal and the DPA.In our case, in view of the absence of any cause of bleeding, DSA was advised, which showed the pseudoaneurysm arising from the saphenous vein graft, probably owing to slippage of ligature from one of the tributaries. CT angiogram might help in identifying the source of bleeding, its location, extent and relationship with adjacent structures along with the extent of injury.7Traditionally, pseudoaneurysms have been managed surgically but with the advancement of endovascular intervention, optimal treatment is usually determined by the personal experience of the clinician. Treatment options include ultrasound-guided compression, percutaneous thrombin injection and endovascular stent graft placement. The success rate of thrombin injection (90–96%) is much higher than compression (75–78%). Stent graft placement can be considered as a bailout procedure.8 Our case was complicated by the small size of the pseudoaneurysm neck, which was difficult to cannulate and get the microcatheter in a stable position. So owing to the risk of non-target embolization with use of pushable microcoils, we deferred coil embolization. We managed to keep the tip of the microcatheter just at the neck of the pseudoaneurysm and injected NBCA. NBCA is a permanent liquid embolic agent that turns radiopaque on mixing with Lipiodol or tantalum powder. It immediately polymerizes/solidifies on contact with an anion, such as hydroxyl moiety in water or various anions in blood.9Angiography and embolization in vein grafts is extremely challenging, as veins have thin and fragile walls compared with arteries. The risk of vein graft perforation owing to percutaneous coronary intervention is estimated at 0.1%.Intervention was thought to be successful when complete obliteration of the pseudoaneurysm sac was achieved and immediate stoppage of bleeding from the wound site was noted. Clinically, there was no further bleeding in the post-operative period. Clinical follow-up after 4 weeks also did not reveal any further bleeding and a good DPA pulsation was noted.This case demonstrates the complexity of managing a saphenous vein graft pseudoaneurysm, in which it is difficult to place coils, with the help of glue embolization. This has seldom been reported in the literature.Lower limb saphenous vein bypass graft pseudoaneurysm is a rare complication, the successful treatment of which with preservation of distal flow is critical, especially in patients with crush injury where the graft is the sole supplying vessel to the lower limb following surgery.The small size of the pseudoaneurysm neck was difficult to cannulate and get the microcatheter in a stable position. Thus, owing to the risk of non-target embolization, glue embolization was preferred over pushable microcoils.Awareness of ligature slippage as a common and important cause for pseudoaneurysm/bleeding when vascular grafts are used is required.Informed consent was obtained from the patient


Starch is an important nutrient in animal feed, and so its analysis is of considerable concern as it is one of the most relevant energy containing fractions. Method AOAC 996.11 was modified to exchange the enzymometric and colorimetric step full approach to a simpler HPLC amine-based column one. The method was optimized and validated for its application in animal feeds and silages.•We demonstrated that the method could be used for quality control for animal feeds and silages•We modified the final incubation time, the initial sample mass, the quantity of enzyme added and buffered, to pH 6.2, the medium to which α-amylase is added.•We applied a chromatographic analysis of the glucose that resulted from starch enzymatic hydrolysis, via a refractive index detector and amine-based chromatographic column. Starch, a frequently analyzed component of animal feedstuffs is incorporated into production animals diets such as beef and dairy cattle , chickenSM . This last essay is based on direct acid hydrolysis which is time-consuming and, depending on the compound feed ingredients, may lack specificity. Other quantification approaches include polarimetry , Megazyme kit, YSI analyzer and NIR [ISO 12099:2010]. Hall described a dietary Starch in Animal Feeds and Pet Food by an Enzymatic-Colorimetric Method , 5 μL were injected into the system. We got a complete chromatographic run for glucose under 8 min using an amine-based chromatographic column for analytical separation .The isocratic analysis was performed at 0.7 mL min5 to 2.3 × 106 which increase sensibility of the method and ease chromatographic integration. Similarly, we determined that our proposed procedure starch highest recovery was obtained when doubling enzyme units added to feed mixture concerning the reference method. When feed ingredients  which contain substantial quantities of starch , sample initial mass must be reduced to 0.25 g to account for method’s dynamic range. On cases in which the glucose chromatographic signal x + (0.036 ± 0.004) with an average coefficient of determination of 0.9992  demonstrate the applicability of the method  g/100 g], rice bran  and wheat middlings  corn meal  were in line with those reported elsewhere . The resulting general equation derived from five different calibrations curves was: f 0.9992 B. Sensiblsewhere . This daSilages present a singularity since the organic acids that are a result of the fermentation process suffered by the raw plant material generate solutions with low pH. The α-amylase will not work under these conditions. Hence, the preparation of the enzyme in the MOPS buffer, adjusted to pH 6.2, which is the optimum pH value for α-amylase assisted hydrolysis , circumvMethod 996.11 considers the determination of resistant starch which includes an initial DMSO solubilization step. Initially developed for cereals, the method has found success in the determination of resistant starch fractions in other feed ingredients such as cassava or square banana meals  or, as sTotal starch is calculated as follows:


Starch gels from the yellow variety were more stable to freeze-thaw and recorded 37.1% syneresis, compared to 56.5% after the first storage cycle. Pasting properties were significantly different (p < 0.05) among starch from the two tigernut varieties, with black recording higher peak viscosity, lower breakdown, and higher setback viscosity. Gels made from the yellow variety were clearer, softer, more adhesive, and more cohesive. Both gels showed a pseudoplastic flow behavior without thixotropy.The aim of this study was to characterize the physicochemical properties of starch isolated from two varieties of tigernuts. The results showed wide variations between the two types of tigernuts. Mean granule sizes were 11.1 and 6.1  Cyperus esculentus is an edible grass plant which produces nut-like tubers known as tigernut. The nuts, which are about 30 mm long , are characterized by a sweet and somewhat milky taste. It is cultivated and consumed in many tropical and subtropical countries. Two major varieties (black and yellow) have been identified in many growing areas. C. esculentus has significant levels of major storage nutrients as well as micronutrients. The tuber mainly contains carbohydrates (more than 60%), which to a large extent consists of starch and dietary fiber. Lipids are the second most dominant constituent (25%), while protein and ash make up 5% and 1.7% of the nuts, respectively [ectively . Additiotituent 2%, while α-1,4 glycosidic bonds, amylopectin is highly branched and therefore is much bulkier. These two components ultimately account for the behavior of starches in food systems.Tigernut is a very useful source of starch. Starch is a key component of most tuber and cereal crops, and it essentially consists of two glucose polymers: amylose and amylopectin. These polymers are contained in varying amounts, depending on the source of starch. Native starch is generally known to be composed of up to 25% amylose and 75% amylopectin. Whereas amylose is generally linear with glucose units linked by As described by Manek, Builders, Kolling, Emeje, and Kunle , tigernuThe two major cultivars of tigernut (black and yellow) were used in the study. The tigernuts were fully matured and freshly harvested. They were cleaned of adhering earth material, washed in clean potable water, and transported to the laboratory for starch extraction and analysis.μ mesh. The starch was allowed to settle after standing for 2 h and the supernatant was discarded. The procedure of resuspending, filtration (100 μ mesh) settling was repeated once again before the isolated starch was dried in an air oven at 40°C for 18 h. The dried starch was milled into fine powder and sealed air-tight in HDPE bags for analyses.Tigernut starch was isolated following the method of , with sl0 = 97.63, a0 = 0.31, and b0 = 4.63) before use. Starch samples were contained in a transparent pyrex petri dish and covered with same. Starch color was described using in L∗ C∗ h notation.Objective color of starches from the 2 major varieties of tigernut was measured using a Minolta-CR410 Chromameter . The instrument was calibrated against a standard white tile  of starch was weighed into a centrifuge tube and 10 mL of distilled water added [The procedure described by Simi and Abraham  was usedStarch flow behavior was measured with a DV2T Viscometer (Brookfield Engineering Inc.). The viscosity of starch gels  was determined at different spindle speeds (5-25 rpm) using an RV spindle 2, without the guard leg. Using an end condition of 10 points, viscosity data was recorded at 2 min intervals. Measurements were taken in a 250 mL beaker, 2 min after spindle was immersed in the starch gel. This was done in order to allow thermal equilibrium between test sample and spindle and to eliminate the effect of immediate time dependence, as directed by the manufacturer.Starch pasting characteristics were quantitatively determined on a 10% slurry, using the Brabender Viscoamylograph . Viscosity profile indices recorded were peak viscosity, pasting temperature, hot paste viscosity, cool paste viscosity, breakdown, and setback viscosity.plus, Stable Microsystems, Surrey, UK) with a compression cylindrical probe. Gels from the pasting experiments were kept at 4°C overnight before the TPA. A double bite compression cycle with the probe set to compress starch gels to about 75% of its height at a test speed of 1 mm/s during each cycle was used. Hardness of the gels was determined as the peak force required to compress it through 75% of its height. Adhesiveness, springiness, and cohesiveness of starch gels were also derived from the Exponent software.Texture Profile Analysis (TPA) was performed on starch gels from the pasting analysis using a Texture Analyzer . Starch samples were first coated with platinum before imaging at a voltage of 3.0 kV and X1200 magnification.All the analyses were done in triplicate. T-test was used to compare the means obtained from the two cultivars (Minitab 17.0.1).μm (mean 11.1 μm) and 3.3 to 12 μm (mean 6.1 μm) were, respectively, recorded for starch from yellow and black cultivars. Clearly, both starches largely consisted of spherical granules with smooth surfaces but black contained a fairly large number of smaller granules. Some oval shape types measuring nearly 10 μm on its major axis (black) and damaged granules and a few with faceted sides (yellow) were seen in the tigernut starches. Even though their granules were relatively smaller, the shapes and surface features of these tigernut starches closely resembled starches from other tropical tubers such as sweetpotato [Micrographs of starch granules show that the yellow cultivar had fairly equal amounts of loosely packed small and large granules, whereas starch from the black cultivar was dominated by small granules which were rather clustered and densely packed . Generaletpotato .p < 0.01) between amylose content of the two starches. The results suggest that products made from these starches may vary in some key attributes. This is because relative proportion of amylose and amylopectin is largely responsible for the functional behavior and nutritional properties of starches and starch-based food products. For instance, high amount of amylose enhances film-forming ability, slows digestibility, and results in higher expansion ratios in extruded products [The apparent amylose content of the tigernut starches ranged between 19 and 21% . Althougproducts  and compproducts .This range observed in the present study was consistent with the amylose content of native starches 15-30%) noted by Bertoft -30% note, and 10.p < 0.05) higher amounts of AM in the black cultivar resulted in lower SP compared to the yellow cultivar. Since AP is postulated to leach out at temperatures beyond 90°C [The ability of starch to swell and eventually produce a paste when heated in water is one of its essential features. Granule swelling is generally attributed to amylopectin, whereas amylose plays the role of a diluent and also restricts swelling . Starch ond 90°C  and 85°Cond 90°C .p > 0.05) but starch of the yellow cultivar was obviously the whiter and brighter of the two . Gel from the yellow variety recorded a higher transmittance and was therefore the most transparent of the two, probably as a result of its lower amylose content and larger granule size [Starch paste clarity is an essential property of starch gels, especially for industrial uses, and varies considerably among different botanical sources. As hypothesized by Craig, Maningat, Seib, and Hoseney , swollenule size . This maThe freeze-thaw behavior of gels from the two varieties of tigernuts was typical of most native starches of root and tuber origin . This prThe pasting pattern of tigernut starches was typical of many root and tuber starches, which are characterized by high peak and breakdown viscosities. Marked differences were observed in the pasting patterns of starches from the two cultivars . For insp < 0.05) as shown in The texture of the two gels varied widely. Hardness, adhesiveness, and cohesiveness of the gels made from the two starches were significantly different (p < 0.05) in starch isolated from the yellow cultivar. A narrow range (866-879 BU) of peak viscosities was observed in starch from the two cultivars. Retrogradation tendency was higher in black tigernut starch gel that made it harder and less cohesive among the two cultivars. Findings of the study suggest that starches from these two varieties of tigernut may be suited for different culinary or industrial purposes.The results showed wide variations in some physicochemical properties of starch isolated from the two tigernuts. Although both mainly consisted of spherical granules, the starch of the yellow cultivar had fairly larger granules compared to the starch of the black cultivar. Amylose content ranged between 19 and 21% for the two. Swelling and water solubility properties were significantly higher (


Starch is a plant storage polyglucan that accumulates in plastids. It is composed of two polymers, amylose and amylopectin, with different structures and plays several roles in helping to determine plant yield. In leaves, it acts as a buffer for night time carbon starvation. Genetically altered plants that cannot synthesize or degrade starch efficiently often grow poorly. There have been a number of successful approaches to manipulate leaf starch metabolism that has resulted in increased growth and yield. Its degradation is also a source of sugars that can help alleviate abiotic stress. In edible parts of plants, starch often makes up the majority of the dry weight constituting much of the calorific value of food and feed. Increasing starch in these organs can increase this as well as increasing yield. Enzymes involved in starch metabolism are well known, and there has been much research analyzing their functions in starch synthesis and degradation, as well as genetic and posttranslational regulatory mechanisms affecting them. In this mini review, we examine work on this topic and discuss future directions that could be used to manipulate this metabolite for improved yield. As starch is the major form of calories within plants, increasing starch concentrations in plant tissues can mean that less food or fodder has to be consumed to supply the same energetic value . It is aDue to its many roles, the pathway of starch metabolism has been studied intensively, and many enzymatic steps involved in its metabolism have been elucidated. This knowledge has been used to construct a detailed model of its metabolism, which has been described in several recent reviews . The resStarch polymer formation Figure  involvesStarch degradation Figure  is initiThe regulation of starch metabolism takes place at several levels, including both transcriptional and posttranslational mechanisms. A number of transcription factors affecting expression of genes encoding starch metabolizing enzymes have been identified, and their roles are currently being examined. At the posttranslational level, allosteric regulation  protein ADP-glucose pyrophosphorylase Figure  has beenIt has been known for many years that increasing AGPase activity leads to increased starch synthesis, and as was mentioned above, increasing starch in food and fodder crops would improve their calorific value , 2018. HIncreasing AGPase in storage organs can also increase starch amounts . Initialin vivo using gene editing  and/or glucose 6-phosphate. In dicots, ATP limitation of AGPase within plastids has been suggested , meaning amounts  while an amounts . That la amounts .A second strategy to influence ATP supply occurred through manipulation of a plastidial isoform of adenylate kinase, an enzyme that interconverts ATP with ADP and adenosine monophosphate (AMP) Figure . RepressThe work performed in altering substrate supply for AGPase has generally been performed in dicotyledonous plants. A recent study  has examAlthough most work has examined influencing AGPase as a method of increasing starch amounts, it may not be the only protein that can do this. The polymerizing enzymes SS and SBE Figure  are presThere are fewer reports of SBE overexpression; however, one study in potatoes demonstrated that this led to synthesis of starch with altered structure but did not report on an effect on yield . InteresPlant biotechnologists have generally attempted to increase starch yield through altering activities of enzymes involved in its synthesis. Degradative enzymes have often been ignored as targets for improving yield as mutations in them are often associated with reduced plant growth . Recent dpe2 mutant plants, which accumulate maltose, demonstrate reduced freezing damage (Dpe2 expression only at times when increased maltose would be advantageous. Simultaneously, β-amylases that produce maltose during starch degradation (Manipulating starch degradation Figure  may haveg damage ; howeverg damage . One strradation  could beAlthough alterations of individual enzymatic steps within the starch pathway can have a beneficial effect on yield, alterations in transcriptional control mechanisms allow the possibility to influence these in a beneficial manner through altering multiple steps simultaneously. A few transcriptional regulators affecting starch metabolism have been identified , and manPost-transcriptional regulation will also be influential in controlling starch metabolism. Many sugar sensing mechanisms involving 14-3-3- and SnRK proteins, trehalose metabolism, TOR kinases, and hexokinase are known to affect starch metabolism. Alterations in expression of some 14-3-3 and SnRK proteins can lead to improved starch accumulation or to the upregulation of enzymes involved in starch synthesis, in several species , 2018. Tin vivo has been the examination of AGPase and GWD, where redox insensitive proteins have been expressed in plants (Post-translational alteration of proteins involved in starch metabolism can involve protein phosphorylation  or reducn plants . Redox in plants . On the n plants , which an plants .Although much has been achieved over the past decades in the manipulation of starch metabolism, there are still improvements that can be made. Detailed analysis of both transcriptional and post-translational control mechanisms will help fine tune current attempts at manipulating the pathway. Perhaps most importantly is the rational integration of metabolic engineering simultaneously in leaves and storage organs. One attempt to accomplish this involved reducing starch synthesis in potato leaves in order to increase soluble sugar export, while simultaneously increasing substrate supply to starch synthesis in tubers through overexpression of two plastidial transport proteins . This apJL wrote the first draft and edited the manuscript alongside JK.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.


Calanus finmarchicus, suggesting broader ecosystem implications of phytoplankton phenology.The phenology of major seasonal events is an important indicator of climate. We analyzed multiple datasets of in situ chlorophyll measurements from the Gulf of Maine dating back to the early 20th century in order to detect climate-scale changes in phenology. The seasonal cycle was consistently characterized by a two-bloom pattern, with spring and autumn blooms. The timing of both spring and autumn blooms has shifted later in the year at rates ranging from ∼1 to 9 days per decade since 1960, depending on the phenology metric, and trends only emerged at time scales of >40 years. Bloom phenology had only weak correlations with major climate indices. There were stronger associations between bloom timing and physical and chemical variables. Autumn bloom initiation correlated strongly with surface temperature and salinity, and spring bloom with nutrients. A later spring bloom also correlated with an increased cohort of  For others, a conversion was necessary. For phytoplankton colour index (PCI) measured by the continuous plankton recorder (CPR) and for Forel-Ule (FU) scale, we used established relationships for conversions, which for PCI was categorical (−3 and FU), Continuous Plankton Recorder , Gulf of Maine North Atlantic Time Series . In the post-1960 period, most datasets were largely gap-free during the indicated sampling years, with the exception of FU measurements, which were sporadic throughout the time series (<1% of measurements). We averaged these data into a monthly time series, removing duplicates, spanning the time period 1912–2015.We aggregated datasets of in situ phytoplankton measurements from multiple public sources. We used chlorophyll  as an estimate of phytoplankton biomass and used previously published datasets. Chlorophyll time series in the interior Gulf of Maine vary together based on empirical orthogonal function analysis, separately from the coastal Gulf of Maine . This anegorical  and for egorical . These c3 and FU , includi3 and FU , and the3 and FU  . When the interpolation was tested for points surrounded by missing data, there was no significant correlation between interpolated and measured values, so we did not interpolate beyond one point.We tested the accuracy and limitations of a temporal cubic spline interpolation for missing data. We validated the interpolation by removing each data point, interpolating, comparing the interpolated value to the measured value, and comparing the phenology metric (described below) calculated from the interpolation to that calculated from the measured value. For an interpolation that only included points not surrounded by missing data, the interpolated values compared well to measured values  between the surface (0–50 m) and deep (50 m - bottom) layers 4, and PO4 measurements, using only those measurements that passed all quality control criteria  layers . Chemicacriteria . We alsoCalanus finmarchicus  of the bloom cycle across years. This helped to confirm that our phenology metrics were applicable, and also to detect changes in the bloom pattern across time. We clustered years based on the shape of the seasonal cycle of chlorophyll following the methodology of We produced time series of phenology metrics for spring and autumn blooms. There are many metrics for quantifying the timing of phytoplankton blooms, with most metrics targeting the timing of either bloom initiation or bloom peak . Bloom s 1.μm - Timing of maximum. Because the dataset is binned monthly, this is a coarse time series; values are midpoints of months. 2.μc - Center of mass. This metric computes the center of mass within the February-May time period (spring index) and of the August-November time period (autumn index), and provides a smooth metric. 3.μg - Midpoint of Gaussian fit. This metric fits a Gaussian curve to half of the year (for spring and autumn indices) and uses the fitted mean (µ) parameter as the bloom peak timing, providing a smooth metric. 4.τd - Timing of maximum increase. This is a coarse (monthly) index of the timing of bloom initiation. 5.τt - Threshold timing. This metric computes the timing at which the total value crosses a threshold that is some percentage of the year’s total . We computed this metric for multiple thresholds for both spring and autumn indices.p-value determinations and dichotomous thinking .We used the phenology metrics in time series analysis. We examined the time series for statistically significant trends. We then looked for long-term associations with physical drivers and with upper trophic levels to determine the potential causes and consequences of changing phenology. Analysis was performed using Pearson correlation and coherence analysis. The coarse spatio-temporal resolution of the dataset means that there is additional variance around time series and associated relationships, and correlation coefficients reflect that. We took steps to avoid over-reliance on single thinking . While wapproach . We alsoExcluding singletons, there were three main bloom types that emerged consistently over the ensemble of cluster calculations . The mosμg, shifting later with mean rates of 8.9 (p = 0.001) and 4.3 (p = 0.02) days per decade . Other metrics with noteworthy trends were spring μm , spring μc , autumn μc , and spring τt . Taking the average of metrics, trends were significant for both spring (p = 0.03) and autumn (p = 0.01), with rates of 3.7 and 3.8 days per decade, respectively. Over the past sixty-year period, where there are enough data to consistently capture the phenology metrics, these shifts equate to a roughly 10–50 day shift in timing. There is substantial inter-annual variance around these trends . These AMO correlations were also significant at lags of up to three years, with correlations peaking for spring τt at a three year lag (r2 = 0.19), and for autumn τt at a two year lag (r2 = 0.24). Coherence analysis . The western Gulf of Maine C. finmarchicus population is supplied by coastal waters, so we compared the phenology metrics to the C. finmarchicus in the eastern Gulf of Maine (> − 68∘) and found stronger and more consistent correlations: μm , μc , μg . Coherence analysis , rather than the reverse. The autumn bloom was more tightly associated with changes in physics. Low temperature and salinity are both associated with increased transport of Arctic-origin water via the Labrador Current, which altered autumn bloom patterns during the 1990s . The patIt is worth noting the secular trends in conditions, as well as correlations, focus on high-frequency variability and can overlook climate-scale changes. Spring conditions have shifted towards higher stratification and lower nutrients. The timing shift of the spring bloom is consistent with short-term shifts in the three Gulf of Maine zones described by ; they hyC. finmarchicus abundance is notable. If later timing of the center of the bloom is an indication of an extended bloom, then C. finmarchicus emerging from diapause could have a longer window for reproduction, and the first generation cohort would be more abundant than in a typical year. This offers one possible explanation for why the species has persisted in the western Gulf of Maine despite rapid warming and temperatures that should be detrimental to the population , chlorophyll is probably effective, but this caveat should be kept in mind. Second, the dataset includes measurements that use different methodologies. Measuring chlorophyll “methods” . Again, Although it seems intuitively reasonable that a sudden effect should have a sudden cause, ecological systems need not behave intuitively. ...[S]pring blooms can occur without any sudden changes in driving variables.” The Gulf of Maine, with its unprecedented rapid changes (The shift of both spring and autumn bloom timing toward later dates is surprising. The high range in rate estimates is indicative of the difficulty of quantifying bloom phenology, so the emphasis should be on the consistent directionality across metrics rather than any specific rate. Finally, while the analysis here is suggestive of possible drivers of the shift in bloom timing, a mechanistic explanation for bloom initiation has been an elusive goal in oceanography. As  changes  is often10.7717/peerj.6735/supp-1Dataset S1Click here for additional data file.10.7717/peerj.6735/supp-2Supplemental Information 1Click here for additional data file.


We have previously described a method to predict antigenic epitopes on proteins recognized by specific antibodies. Here we have applied this method to identify epitopes on the NS1 proteins of the four Dengue virus serotypes (DENV1–4) that are bound by a small panel of monoclonal antibodies 1H7.4, 1G5.3 and Gus2. Several epitope regions were predicted for these antibodies and these were found to reflect the experimentally observed reactivities. The known binding epitopes on DENV2 for the antibodies 1H7.4 and 1G5.3 were identified, revealing the reasons for the serotype specificity of 1H7.4 and 1G5.3, and the non-selectivity of Gus2. As DENV NS1 is critical for virus replication and a key vaccine candidate, epitope prediction will be valuable in designing appropriate vaccine control strategies. The ability to predict potential epitopes by computational methods significantly reduces the amount of experimental work required to screen peptide libraries for epitope mapping. Aedes aegypti, the major mosquito vector, is found [Dengue fever is the most important human arthropod-borne viral disease with dengue virus (DENV) infections representing one of the biggest public health burdens globally. More than 400 million infections and 100 million symptomatic cases occur annually, with as many as 3.6 billion people at risk of infection in areas where Dengue virus has four serotypes DENV1–4), all of which are responsible for the spectrum of disease ranging from benign dengue fever to severe DHF/DSS ,3. The D, all of Previously, we developed a new approach for determining linear antibody-binding epitopes of antigens ,11. ThisRecently, Jiang et al. used a phage display technique to identify epitopes on the NS1 protein of DENV2 recognized by a polyclonal antibody . Being aMore recently, the crystal structure of the DENV2 NS1 protein (PDB code: 4O6B) has been solved in both dimeric and hexameric configurations thereby revealing the molecular basis of their formation . The cryIn this work, we have used computational methods to predict the binding epitopes on DENV NS1 for three mAbs. We first showed DENV serotype specificity by ELISA. Secondly, we identified potential epitopes for the published mAbs 1H7.4 and 1G5.3 and compared these with published experimental results, providing validation of the prediction approach. Finally, we applied our approach to epitope prediction for a previously unpublished mAb, Gus2, which was shown experimentally to be cross-reactive against NS1 from all four DENV serotypes.The epitope identified experimentally for 1H7.4 is referred to as LD2 (residues 25–33) . FirstlyNo ACET minima were found due to the negatively charged nature of the surfaces. While only one MAMM minima was found on S2 interacting with Asp97 (H chain), 35 MGUA minima were distributed over S1 and S2, interacting with negatively charged residues Asp97 and Glu48 of the H chain. The best minima of MGUA were located close to Glu48 (H chain) of S2.Using the minima on the two surfaces S1 and S2, we constructed a sequence pattern for the peptides that could potentially bind to the antibody. The maximum distance between the two binding surfaces S1 and S2 is approximately 6.0 Å, indicating an adjacent position. While MGUA, IMIA, PHEN, and ACEM minima were found on both surfaces S1 and S2, only one MAMM minima was found on the surface S2. Therefore, the key sequence pattern for the binding epitope peptides can be defined as “XZ”, in which X = R, H, Y, Q, N, and Z = K, R, H, Y, Q, N. n = 1 to 11 in The sequence pattern was subsequently used to search for “binders”  from the peptide libraries derived from the sequence of the DENV2 NS1 protein. The seven libraries were searched for peptides matching the calculated sequence pattern of the binding epitope. There are 352 residues in DENV2 NS1, resulting in 50 × 7-mer peptides for each of seven libraries. Appendix S1 in the Supplementary Materials shows the identified binder peptides from each set of peptide libraries with the binders displayed in lower case characters. Overall, eleven peptides were identified with potential to bind to the antibody , experimentally identified previously [As a result, only six binder peptides are considered as potential epitopes for the antibody, i.e., eviously .1–4 and 6) were located at conserved sequence positions amongst all four serotypes (coloured in yellow/green), significantly, one epitope, epitope 5, was found only in DENV2 (colored in red). Epitope 1 overlaps with peptide LD2 and is conserved amongst all four DENV serotypes. However experimental binding data ([5 (unique to DENV2) potentially playing a previously unrecognized role in the 1H7.4 selectivity, especially as epitope 1 is considered to be close to the membrane in the dimeric complex of DENV2 NS1 [ng data  of the H chain, and a pocket B2 formed by L chain  and H chain  residues. The positively charged Arg106 of the CDR3 loop sits at the gate of the pocket, resulting in the charged nature of B2. Similar to 1H7.4, a groove is formed between the L and H chains by L chain  and H chain  residues. Compared to the pocket B2, this groove is comparatively narrow for antigen access. Therefore, we discarded the MCSS minima inside this groove.Based on the distribution of the important minima shown in n, n = 1 to 12 in I—residues 111–128 “LKYSWKTWGKAKMLSTES”; II—residues 233–241 “SNGVLESEM”; III—residues 282–291 “GTTVVVTEDC”; and IV—residues 303–311 “ASGKLITEWC”. IV overlaps with the epitope 24C (residues 299–309 “RTTTASGKLIT”) observed experimentally [Seven sets of peptide libraries were generated. Appendix S2 in the Supplementary Materials shows the identified binders from each of the seven sets of peptide libraries with the binder residues highlighted in lower case. In total, 12 peptides were identified with potential binding to the antibody 1G5.3  more variation. These two epitopes are in close proximity, located towards the C terminus, thus forming a potential binding site  found in DENV4. The selectivity of 1G5.3 to DENV2–4 could thus be mainly due to the sequence variability at epitope IV which is overlapping with the epitope 24C (13). In particular, residue Thr308 of epitope IV in DENV1 is replaced by the hydrophobic Leu in DENV2–4. This residue is completely exposed to solvent and could thus serve as the key residue for antibody-antigen recognition.Epitopes ing site C. IntereGUS2 is a previously unpublished antibody which binds to all four of the dengue virus serotypes, as demonstrated by ELISA against recombinant NS1 from each serotype . Here, wUsing the minima on the two sites C1 and C2, we constructed a sequence pattern for the peptides that could potentially bind to the antibody. The maximum distance between the two binding sites C1 and C2 is approximately 5.5 Å, indicating that there is no amino acid gap between the two sites. While MGUA, IMIA, PHEN, and ACEM minima were found on the surfaces C1 and C2, only one MAMM minima is found at C2. Therefore, the key sequence pattern for the binding epitope peptides can be defined as “XZ”, in which X = R, K, Q, N, Y, and Z = R, K, Q, N, Y, F, H or W. n = 1 to 10 in Appendix S3 in the Supplementary Materials shows the identified epitopes from each set of peptide libraries with the epitopes highlighted in lower case characters. In total, 10 peptides were identified as potential binders to the antibody GUS2 . Further analysis of the surface accessibility and conformations of these peptides  has previously been shown to bind to mAbs which are cross-reactive against all four serotypes of DENV [The epitope LX1  was coated per well on a Nunc MaxiSorp plate , in PBS at 4 °C overnight. The plate was washed three times with phosphate buffered saline (PBS) with 0.01% Tween20 (PBS-T). The plate was blocked using PBS with 2% skim milk powder (MPBS). After blocking, 200 µL of 3 µg/mL test antibody diluted in MPBS was added to relevant wells. Following one hour incubation, plates were washed three times with PBS-T and probed for one hour with 200 µL of HRP-conjugated goat anti mouse IgG H+L (Life Technologies) in MPBS at 0.1 µg/mL. Three final washes were performed with PBS-T. 100 µL TMB substrate  was added to develop the chromogenic signal. The reaction was stopped with 2N sulphuric acid and the absorbance at 450 nm was read using the Spectramax .http://blast.ncbi.nlm.nih.gov). For the 1H7.4 mAb, the most homologous sequences were from PDB entries “1SY6” , “1Z3G”Fragment ) and “3SFragment ). The VLFragment ), “3J1S”Fragment ), “4HC1”Fragment ), “4BKL”Fragment ), “2AAB”Fragment ), and “1Fragment ). The amFragment ), “3CFB”Fragment ), “1FOR”Fragment ), “1RVF”Fragment ), and “3Fragment ). The amFragment .http://www.computistresearch.com) implementing our MCSS approach was used to scan the preferred locations of functional chemical groups on the binding surfaces around the “Complementary Determining Regions” (CDRs). Eleven functional groups were used, each of which corresponds to the side chains of different amino acids [Using the homology model of an antibody, our quCBit software  is completely conserved across all four serotypes, despite the fact that 1H7.4 binds only to NS1 from DENV2. This implies that specific binding may be due to epitope recognition that is impacted by protein folding and conformational effects from other regions of the protein [5  is unique for DENV2 for the antibody 1H7.4, inferring the importance of this peptide on selectivity. The additional potential epitopes identified in this study for 1H7.4 antibody-antigen recognition will require further experimental validation.As well as the reproduction of known epitopes LD2 and 24C for the antibodies 1H7.4 and 1G5.3 , respect protein . This isIV or previously identified 24C (residues 299–309 “RTTTASGKLIT”) [III and IV, which are noticeably divergent amongst the serotypes. Moreover, these two epitopes are next to each other in the three-dimensional structure, forming an accessible recognition site (IV as the dominant site for recognition [IV) by Leu in only DENV1 is consistent with our experimental data that 1G5.3 is selective for DENV2–4.For the antibody 1G5.3 , the preSGKLIT”)  is locatSGKLIT”) . As showion site C. Previoognition , the repA–D) were predicted as the binding epitopes of GUS2, located at the regions of residues 27–36, 110–118, 124–132, and 239–264, respectively .Having verified the known epitopes and identified additional epitopes for the NS1 specific antibodies 1H7.4 and 1G5.3, and investigated the role that these individual epitopes play in binding, the method was then used to predict epitopes of antibody GUS2. The surface of the antibody around the CDR3 loop has a relatively flat topography with MCSS minima of different functional groups to those seen previously, with stronger interactions to the antibody . Four peectively . In the erotypes . Interestructure B. The coOverall, our calculations have identified several epitopes of the DENV NS1 protein to the antibodies 1H7.4, 1G5.3 and GUS2, and these peptides are in agreement with previously published (1H7.4 and 1G5.3) or experimentally validated (Gus2) results.Previously we have developed a simple qualitative method to search for epitopes of proteins that bind to specific antibodies. In this work, we have applied this method to identify the recognition sites of antibodies 1H7.4, 1G5.3 and GUS2 to the NS1 proteins of the four dengue virus serotypes (1–4). While our calculations have reproduced the epitopes LD2 and 24C identified previously for the antibodies 1H7.4 and 1G5.3, respectively, additional potential epitopes were also obtained comprising residues 97–115, 125–133, 143–151, 170–178, and 289–297 for 1H7.4 and at residues 11–128, 233–241 and 282–291 for 1G5.3. Rather than the previously identified LD2, the selectivity of antibody 1H7.4 to DENV2 is most likely controlled by the residues 170–178 which is only found in DENV2. The binding of 1G5.3 to DENV2–4 can be explained by the sequence differences between DENV1 and DENV2–4 of the recognition region formed by the epitope at residues 282–291 and especially residues 303–311. For antibody GUS2, four epitopes are predicted at residues 27–36, 110–118, 124–132 and 249–264. The non-selectivity of GUS2 against the dengue virus serotypes is the result of the highly conserved sequence homology of all the NS1 epitopes in DENV1–4 (with the exception of epitope 124–132), as well as the conformational flexibility of a long extended loop where the epitopes 110–118, and the more variable 124–132 are located. Our method provides a means to identify linear epitopes which bind to antibodies with desirable characteristics for virus neutralization.The method is based on sequence information only but the prediction accuracy is improved when 3D structural data of the antigen (and or antibody) is known. The method is based on an intuitive concept that does not critically depend on the knowledge of the exact fold of CDR loop regions as we have shown previously ,3. Our m


In this Editorial, the new Editor‐in‐Chief Professor Miguel A. De la Rosa introduces his plans for the journal. FEBS Open Bio, as it is with enormous pride that I announce the beginning of my tenure as Editor‐in‐Chief. Since its inception in November 2011, FEBS Open Bio has been under the helm of Mary Purton as Executive Editor, who has guided the journal to maturity and great success. FEBS Open Bio has recently published its 1000th article FEBS Open Bio, The FEBS Journal, FEBS Letters and Molecular Oncology. I wish her great success in this important role, and I look forward to working with her to ensure the journal retains its high standards.This issue marks a new chapter in the 8‐year history of As Editor‐in‐Chief, I will be working closely with the journal’s prestigious editorial board to ensure that we continue to meet the journal’s mission of publishing technically sound manuscripts. I also intend to appoint new editors to ensure that the journal is well placed to expertly review manuscripts on research areas across the life sciences, as befits a multidisciplinary mega journal. In particular, I hope to increase editorial representation from Latin America, Asia and Eastern Europe, to ensure that the board continues to represent the scientific community. Our editorial board will be supported by staff in our editorial office in Cambridge: I would like to welcome Duncan Wright as our new Editorial Manager, who will be responsible for managing the day‐to‐day operation of the journal. Duncan and I will continue to be ably supported by Jacob Weller in his role as Editorial Assistant. I would like to extend my thanks to all of our editors and the editorial office staff for their hard work on the journal, which is instrumental to the journal’s success.Bifidobacterium infantis may help maintain barrier function in the gastrointestinal tract of infants Downloads for the journal have increased dramatically over the last year, with downloads in October 2019 80% greater than the same month in 2018. Our most downloaded article ever is a Method paper published last year, which describes a protocol for rapid bacterial identification based on sequencing of 16S rRNA genes FEBS Open Bio mission.In 2017, FEBS Open Bio started an Education section, publishing original research articles on pedagogical approaches in biochemistry and molecular biology. To date, we have published six Education articles covering a range of topics, including monitoring the effect of changing teaching practices on student performance in an introductory biochemistry course FEBS Open Bio and the annual FEBS Congress  through various new initiatives. It is my hope that FEBS Open Bio will serve as the official journal of the Congress and that this will facilitate mutual promotion between the various activities of FEBS. I look forward to introducing this and other new developments over the upcoming year. On the behalf of the FEBS Open Bio editorial board and office staff, I wish all of our readers, authors and reviewers a happy and productive new year, and I hope you will continue to consider us when publishing your work.Finally, as part of my long‐term vision for the journal, I intend to increase ties between


No data are available on liver transplantation (LT) outcome and donor liver steatosis, classified as large droplet macrovesicular (Ld-MaS), small-droplet macrovesicular (Sd-MaS), and true microvesicular (MiS), taking into account the recipient Hepatitis C virus (HCV) status. We investigate the impact of allograft steatosis reclassified according to the Brunt classification on early graft function and survival after LT. We retrospectively reviewed 204 consecutive preischemia biopsies of grafts transplanted in our center during the period 2001-2011 according to recipient HCV status. P=0.003) and when including indexes of early postoperative graft function . Graft Sd-MaS>15% showed a risk for graft loss > 2.5-folds in both the models. Graft Sd-MaS>15% was associated with reduced graft ATP content and, only in HCV- recipients, with higher early post-LT serum AST peaks. The median follow-up after LT was 7.5 years (range: 0.0-16.7). In negative recipients (n=122), graft loss was independently associated with graft Sd-MaS, in multivariable Cox regression models comprehending only pre-/intraoperative variables (HR=1.03, 95%CI=1.01-1.05; In HCV-negative recipients, allografts with >15% Sd-MaS have significantly reduced graft survival and show low ATP and higher AST peaks in the immediate posttransplant period. Donors with >15% Sd-MaS have significantly higher BMI, longer ICU stays, and lower PaO2. The frequency of steatosis in donors for liver transplantation (LT) is increasing over time, showing similar trends as the general population. Most studies exploring the effect of steatosis on LT outcomes classify it as “macrovesicular” or “microvesicular” steatosis . Based oPoor clarity exists also on a clear histological definition of macro- and microvesicular steatosis. In this respect, an accurate classification of hepatic steatosis has been proposed by Brunt, classifying the steatosis as (a) large droplet macrovesicular (Ld-MaS), (b) small-droplet macrovesicular (Sd-MaS), and (c) true microvesicular steatosis . In the Another underestimated aspect to consider is the potential confounding role of HCV infection when we analyze the association between allograft steatosis and post-LT including (a) HCV interaction with lipid metabolism in the hepatocytes ; (b) indThe principal aim of the study was to investigate the impact of allograft steatosis, reclassified according to the Brunt classification, on early graft function and survival after LT. Separate analyses were done in HCV-negative (HCV-) and HCV-positive (HCV+) patients. The secondary aim was to evaluate the ATP levels in the donor livers according to the type and percentages of steatosis.During the study period , 233 consecutive adult (≥18 years) patients received a first, nonurgent, deceased-donor, whole-organ LT at Sapienza University of Rome Liver Transplant Center, Italy. Protocol preperfusion donor liver biopsies were prospectively collected for all patients and retrospectively evaluated for reestimating the donor steatosis according to the Brunt classification.As shown in All transplants were performed with a terminoterminal choledochocholedochostomy with T-tube placement. The immunosuppressive protocol was based on a triple therapy with methylprednisolone, mycophenolate mofetil, and calcineurin inhibitor . Methylprednisolone was rapidly tapered. Donor and recipient data were prospectively collected using an in-house database and retrospectively reviewed; donor information was supplemented by data held in the National Transplant Center database. Initial Poor Graft Function (IPGF) was defined according to Nanashima et al. . Early aPermanent histological sections were prospectively collected from allograft preischemia liver wedge biopsies performed on the left hepatic lobe. The liver tissue was immediately fixed in 10% formalin and within few days was embedded in paraffin and then stained with hematoxylin and eosin, to assess hepatic steatosis in all transplants. To grade the severity of ischemia-reperfusion injury (IRI), permanent histological sections in the recipient within 1 hour after complete revascularization of the allograft (postreperfusion biopsy) were obtained in 134 cases  with the same procedure.Frozen-section evaluation was performed in selected cases based on gross appearance of the graft only to decide whether to discard the graft. Two expert pathologists (AC and MR), blinded to clinical data and to the frozen-section evaluation, retrospectively reviewed and scored all the preischemia liver samples for steatosis, defined according to the Brunt classification , 14 Fig as follo U test or T test according to the variable normality. Categorical variables were expressed as count and percentages and compared by the chi-square or Fisher's exact test, as appropriate. As a value over 15% of graft Sd-MaS turned out to be relevant for graft survival in HCV- patients, we decided to categorize both Sd-MaS and Ld-MaS as nil steatosis (absence of steatosis), 1 to 15%, and >15% steatosis. We categorized the total histopathological IRI score in mild/moderate  and severe , the latter corresponding to the higher tertile in our population.Continuous variables are presented as median and interquartile ranges (IQR). After assessment of normality by the Kolmogorov-Smirnov test, the differences between groups were evaluated by Mann-WhitneyAll the analyses were performed separately in patients with HCV- and HCV+ liver disease.Survival rates were calculated using the Kaplan-Meier method. In order to calculate graft survival, patients alive and not retransplanted were censored at the date of last follow-up, while time to graft loss was measured from LT to patient death or retransplantation. Patient-, donor-, graft-, and transplant-specific risk factors for overall graft survival were investigated using univariable Cox regression analyses. Different multivariable Cox regression models were constructed, considering as covariates only pre-/intraoperative variables or both pre-/intraoperative and early postoperative variables. Hazard ratios (HR) and 95% confidence intervals (95%CI) were reported.To investigate donor factors independently associated with graft Sd-MaS >15% compared to a lower degree of Sd-MaS or nil Sd-MaS, we used logistic binary regression. P value <0.05 at univariate analyses were introduced as covariates in all the multivariable analyses.Variables with a P value <0.05 was considered statistically significant. Computations were carried out with SPSS software 24.0 for Windows . The study was approved by the Sapienza University of Rome Ethical Committee and patients signed written informed consent forms.AThe recipient, donor, graft, intraoperative, and early postoperative characteristics of the entire study population are shown in P=0.043).In the entire study population, median follow-up was 7.5 years (range: 0.0-16.7). Comparing HCV- versus HCV+ patients, the only difference was that Anti-HBc positive donors were less frequently allocated to HCV+ patients (P<0.001) or a categorized (>15%) variable (P=0.002). Other risk factors for graft loss were donor Anti-HBc positivity (P=0.001), longer time since transplantation (P=0.036), and the occurrence of IPGF (P=0.025) and EAD (P=0.008). On the opposite, Graft Ld-MaS, IRI severity, HCC, and other studied variables were not associated with overall graft survival . During the follow-up period, 28 grafts (22.9%) were lost for liver-related causes. In detail, we observed nine cases of delayed graft dysfunctions, six ischemic cholangitides, four primary nonfunctions, three HCC recurrences, two hepatic artery thromboses, one acute rejection, one chronic rejection, one recurrence of primary biliary cholangitis, and one portal thrombosis. Fifteen (12.3%) liver-unrelated causes for graft loss were observed . One-, 3-, and 5-year graft survival rates were 82.8%, 76.2%, and 71.3%, respectively. As shown in survival .P=0.001 in the pre-/intraoperative model and P=0.007 in postoperative model). Grafts with Sd-MaS >15% had a 2.5-fold increased risk for graft loss in both the models (P=0.008 in the pre-/intraoperative model and P=0.019 in postoperative model). When Sd-MaS was considered as a continuous variable, it was an independent risk factor for graft loss with HRs 1.036 (P=0.001) and 1.032 (P=0.003) in the pre-/intraoperative model and in that including postoperative variables, respectively.Two multivariable Cox regression models were created , the firP=0.015) and 3.157 (P=0.021) in the two models, respectively. Ld-MaS was not significant in these models.Although not significant in the univariate model, we decided to test the Ld-MaS in separate analyses, with the main intent to exclude a possible effect of coexisting Sd-MaS and Ld-MaS on graft loss. After having constructed the same multivariable models based on pre-/intraoperative and pre-/intra-/postoperative variables plus the variable Ld-MaS, Sd-MaS >15%, we confirmed its independent role of Sd-MaS >15% as a risk factor for graft loss, with HRs 3.311 (Kaplan-Meier curves reporting the graft loss rates stratified for Ld-MaS >15%  and Sd-MP<0.001). Similar results were observed after using grafts with Ld-MaS >15% (P=0.025) (P=0.025) .P=0.015), graft cold (P<0.001) and warm ischemia (P=0.017) times, occurrence of IPGF (P<0.001) and EAD (P=0.049), and the severity of graft histopathological IRI (P=0.004) were significant risk factors for graft loss. Graft Ld-MaS and Sd-MaS, HCC, and other studied variables were not associated with overall graft survival (The median follow-up was 7.1 years (range: 0.0-16.7). During follow-up, 32 grafts (39.0 %) were lost for liver-related causes. Specifically, we observed 19 recurrences of HCV-related cirrhosis, five delayed graft dysfunctions, two primary nonfunctions, two HCC recurrences, one hepatic artery thrombosis, one chronic rejection, one ischemic cholangitis, and one hepatic artery aneurysm. Ten (12.2%) grafts were lost due to liver-unrelated causes: five cerebrovascular accidents, one de novo malignancy, one sepsis, one acute myocardial infarction, one pulmonary embolism, and one intra-abdominal hemorrhage. One-, 3-, and 5-year graft survival rates were 75.6%, 67.1%, and 63.4%, respectively. As shown in survival .P<0.001) variable associated with graft loss in the pre-/intraoperative model. When also the postoperative variables were considered, the severity of graft IRI (P=0.002) and the occurrence of IPGF (P=0.009) were associated with graft loss.At multivariable Cox regression analyses , graft cNo statistical differences were found in terms of survival rates when the cohort of HCV+ patients was stratified according to Ld-MaS and Sd-MaS values Figures . Severe In HCV+ patients, serum AST peaks observed during the first 3 postoperative days did not differ according to both Sd-MaS and Ld-MaS distribution Figures .P=0.048), a shorter length of donor ICU stay (P=0.048), and a lower donor PaO2 (P=0.020) (P=0.023) and a lower donor PaO2 (P=0.019) were independent risk factors for Sd-MaS >15%  . At multMaS >15% .P=0.019). In addition, only grafts with Sd-MaS >15% in the preischemia biopsy significantly reduced ATP content in the postreperfusion biopsy when compared to preischemic results (P=0.028).A subanalysis was performed in a cohort of 42 grafts in which we measured preischemia and postreperfusion hepatic ATP content . Grafts In the present study we have investigated the impact on LT outcomes of donor liver steatosis evaluated using protocol preischemia biopsies and revised according to the Brunt classification. This classification identifies three different types of steatosis, namely, two subtypes of macrovesicular steatosis (Ld-MaS and Sd-MaS) and true microvesicular steatosis , 14. ThiWe have conducted separate analyses for graft survival in HCV+ and HCV- recipients. The main result of our study is that liver donor Sd-MaS is an independent risk factor for graft loss in HCV- patients, but not in HCV+ ones. In particular, graft Sd-MaS was associated with graft loss when considered as either a continuous variable or categorized using a cut-off of >15%.Although the accuracy of frozen liver sections is debated for steatosis assessment , our resTo date, only one recently published study by Choi et al. has analyzed the impact of Sd-MaS and Ld-MaS on LT outcomes, finding an association of Sd-MaS with acute and chronic rejection, but not with graft survival . The dis2 and short length of ICU stay; and (c) when transplanted to HCV- recipients, showed a higher early postoperative serum AST peak, compared to the other grafts. Thus, we hypothesize that donors with relevant Sd-MaS have a preexisting impaired mitochondrial function with low baseline ATP content, failure to recover ATP levels after reoxygenation and increased susceptibility to ischemia-reperfusion injury [With regard to the mechanisms through which allograft with relevant Sd-MaS have a poor outcome in recipients with HCV- liver disease, we found that these grafts (a) had low ATP content in the preischemia biopsy, suffering a further significant reduction of ATP after reperfusion; (b) were associated with low donor PaOn injury –26. The n injury , 28. As n injury .In our present study we did not find a negative impact of Sd-MaS on graft survival and early postoperative AST peak in HCV+ patients. Although we do not have a clear explanation for this latter observation, we should underline the fact that HCV is known to strictly interact with lipid droplets into the hepatocytes, redirecting autophagy by inducing lipid-selective autophagy , 30. In With regard to predictors of graft loss in our HCV+ patients, we found that, as previously reported, severe IRI was associated with cirrhosis due to HCV recurrence .The definition of Ld-MaS in the present study is concordant with the term macrosteatosis/ macrovesicular steatosis used in the literature; according to several studies, when macrosteatosis occurs in more than 60% of hepatocytes, poor outcomes are observed –4, 13. HThe observed result that donor Anti-HBc positivity was connected with poor graft survivals in HCV- patients is in line with previous studies . HoweverThere are some limitations of our study that should be addressed. First of all, this is a monocentric study needing an external validation of our results. The study was performed in a long time frame. However, this possible bias was corrected adding the period of transplant as a covariate in our analyses. Although we performed only one liver biopsy of the left hepatic lobe to assess preischemia steatosis, previous studies have shown minimal steatosis variability between left and right lobe sampling . Lastly,In conclusion, using protocol preischemia liver graft biopsies, we observed that the presence of Sd-MaS >15% is associated with lower graft ATP content, severe early hepatocellular damage, and reduced graft survival in HCV-negative patients. These data may play an important role in modifying the organ allocation process, especially nowadays with the spreading of NASH and the reduction of HCV-RNA positive recipients thank to DAAs, but need to be validated in other studies.


For more than 40 years, under the leadership of four editors and two publishers, The International Journal of Aging and Human Development (IJAHD) has featured multidisciplinary scholarship related to aging processes and older adults. With the publication of eight issues a year, with over 800 pages of scientific content, the IJAHD places emphasis upon psychological and social studies of aging and the aged. However, the Journal also publishes research that integrates observations from other disciplines that illuminate the "human" side of gerontology. A more recent focus includes midlife development, as well. About half (47%) of the publications in the IJAHD are from international colleagues. This presentation will discuss tips for both international and US-based scholars for ensuring timely reviews and positive decisions for manuscript submissions, including such areas as key words, suggesting unbiased reviewers, formatting, writing mechanics, clearly-articulated methods, and a sound theoretical basis.