Trunk stiffness is an important parameter for trunk stability analysis and needs to be evaluated accurately. Discrepancies regarding the dependence of trunk stiffness on the direction of movement in the sagittal plane suggest inherent sources of error that require explanation. In contrast to the common assumption that the muscle stiffness remains constant prior to the induction of a reflex during position perturbations, it is postulated that muscle-stiffness changes of nonneural origin occur and alter the experimental trunk stiffness, causing it to depend on the sagittal direction. This is confirmed through reinterpretation of existing test data for a healthy subject, numerical simulation, and sensitivity analysis using a biomechanical model. The trunk stiffness is determined through a static approach (in forward and backward directions) and compared with the model stiffness for assumed scenarios involving deactivated muscles. The difference in stiffness between the opposite directions reaches 17.5% without a preload and decreases when a moderate vertical preload is applied. The increased muscle activation induced by preloads or electrical stimuli explains the apparent discrepancies observed in previous studies. The experimental stiffness invariably remains between low and high model-stiffness estimates based on extreme scenarios of the postulated losses of muscle activation, thereby confirming our hypothesis. The trunk stiffness is an important parameter used in studying trunk postural control and stability –4. SpinaThe sagittal directional dependence of the trunk stiffness, which was demonstrated in , was notThe measured trunk stiffness is a combination of varying components that must be dissociated to obtain a rigorous interpretation of test data. The transient stiffness is generally determined during short transient trunk position perturbations , 7, 18, During position perturbation, even before the induction of a paraspinal reflex, an activated muscle may undergo a stiffness reduction due to excessive shortening or a high shortening velocity , 27, 28.The trunk experiment, which provided the data used in the present study, was performed in the Kevin P. Granata Biomechanics Laboratory at Virginia Tech and described in . It deteThe subject held an emergency safety button to shut down the servomotor at any time during the test. The experimental trunk ESS was calculated using the force data recorded for the anterior and posterior position perturbations.FUB as a function of the muscle length L and maximum active force at rest F0max. The minimum active force FLB, i.e., the tonus, and maximum force F0max of a muscle are constants that are proportional to its largest physiological cross-sectional area ))FUB as ac—a constant given in  is the system stiffness matrix, which depends on {Uk} and {F}; ΔUik and ΔUjk are the displacement increments relative to preceding equilibrium position; and UijLB, and UijUB, are the lower and upper bounds on relative displacement [Here, the symbols are defined as follows: lacement  between quadprog function.This quadratic programming problem is solved using the MATLAB The experimental trunk ESS introduced in To determine the forward and the backward trunk stiffness separately, the unilateral behavior of the muscle-tendon complex is taken into consideration. Clearly, the tendon cannot withstand compression. It is stretched while carrying tensile forces; in their absence, it is relaxed, and the active force in the corresponding muscle vanishes. Additionally, the tension vanishes when the maximum shortening velocity is exceeded, even for active muscles . The tenL0 as the reference. In this study, the reference length is defined as the preperturbation length, on an approximate basis.In the following, the “state” of the tendon or muscle refers to whether it is stretched or unstretched and whether it is subjected to tension or is force-free. The stretching is defined relative to a generic reference length that depends on the activation and contraction dynamics. It is common  to consiAt the outset of perturbation, the body is in a natural state where muscles are all activated and in a state of tension. The muscles are activated at least at the tonus level. After perturbation, there are three possible muscle-tendon states. In the first state, the muscle remains stretched and fully maintains its active force and stiffness. In the second, the muscle remains stretched, but the active force decreases because of the high shortening velocity. In the third state, the tendon is relaxed owing to shortening; thus, the muscle loses its stiffness, and the force in it vanishes.The trunk stiffness of the model is determined as the ratio of a horizontal force increment to the calculated induced displacement. For a given preperturbation trunk state, the calculation involves two steps. First, the recruitment problem is solved for the muscle activations consistent with the given trunk state. Second, a specified horizontal force increment is added to the forces of Step 1. Then, the equilibrium problem is solved using the muscle stiffness derived from the preperturbation activations obtained in Step 1, while treating the T10 displacement as free. To approach the test conditions, the force increment is set to a value that induces a 3 mm displacement. For a more realistic estimation, the muscle-stiffness values should be those recorded immediately after perturbation (preceding the induction of the reflex), because the trunk settles in the static postperturbation state. In this end state, the muscle active forces and elongations remain at their eventual unknown postperturbation levels, which determine the trunk reaction to be used in the model trunk stiffness calculation.An approximate assessment of the end state of the trunk muscles is performed first by running the recruitment problem subject to the preperturbation conditions. The muscle-tendons that are shortened during the displacement perturbation are identified and are considered as potentially inactive. Depending on the investigative purpose, some of these candidates are retained as inactive, with the others being treated as active at their preperturbation levels. In the subsequent equilibrium problem, the inactive muscles are considered to withstand no forces and to no longer develop stiffness. The equilibrium problem is solved subject to the force increment while considering only the retained active muscles, followed by a straightforward calculation of the trunk stiffness.By adopting appropriate criteria for defining the postperturbation status of muscles, lower and upper estimates of the trunk stiffness can be obtained. For instance, assuming scenario 1 for all muscle-tendons preserves the muscle stiffness, leading to an upper estimate. Alternatively, an overprediction of tendon relaxation contributes to underestimating the trunk stiffness. An extreme scenario is one where all shortening muscles are deactivated. Interestingly, the idea of selective muscle deactivation provides a quantitative indicator of the influence of an individual muscle or muscle group, which is obtained by calculating its contribution to the trunk stiffness.q which is widely reported in the literature [Kf,exp and Kb,exp are obtained. The upper estimates correspond to the maximum trunk stiffness values Kf,max and Kb,max, determined with all the muscle-tendons remaining activated during perturbation. The lower estimates correspond to the reduced trunk stiffness values Kf,min and Kb,min, determined while excluding from the calculation the muscle-tendons undergoing shortening during perturbation. Because the upper estimates Kf,max and Kb,max are calculated according to the same set of active muscles, they are identical. They are both denoted as Kmax .The muscles' insertion points are determined according to the graphic localization specified in , 32. Theterature , 34. It terature  and founIf the true set of activated muscles for a given direction of movement were known and used in the model, the resulting  trunk stiffness estimate would necessarily lie between the maximum and minimum ones. These two extreme estimates are said to form a bracketing of the realistic estimate. Moreover, the trunk stiffness calculated according to a realistic set of deactivated muscles should be a closer approximation of the experimental stiffness. Thus, the latter is expected to be bracketed by the minimum and maximum model estimates, provided that the model is sufficiently accurate. This bracketing of the experimental stiffness can serve as an indicator of the quality of the numerical model. It is used here to assess the choice of muscle tonus levels treated as control parameters. Therefore, it is relevant to analyze the sensitivity of the trunk stiffness results to these parameters.2 are assigned to class C3. The muscles with a PCSA of 120 mm2 or less and length greater than 200 mm are classified as C2, and the remaining muscles belong to class C1. The retained model tonus is a combination of three tonus ratios (associated with the three classes), verifying that the intervals  and  are larger than the 99% confidence interval for the experimental stiffness  lies between 1.12 and 1.16 mm . Many groups are found to shorten during extension only, and others undergo shortening during movement in either direction. The cumulative contribution of groups 1 and 2 represents 37% of erector spinae, with most shortenings between 0.7 and 0.9 mm. Their contribution to the rearward stiffness amounts to 39%. Furthermore, the multifidus alone accounts for 6.3% of the trunk stiffness, with most shortenings between 0.31 and 0.94 mm. Muscle groups 3–5, which undergo shortening in both anterior and posterior perturbations, together contribute less than 1% in either direction. The remaining muscles, which are omitted from Muscles that shorten exclusively during backward perturbation belong to the For the subject carrying no load, the experimental trunk stiffness is 17.5% larger in the forward than in the backward direction. As the load is increased, the difference between the rearward and the forward stiffness decreases and reaches zero at a carried load of approximately 200 N. Beyond this load, the rearward stiffness becomes larger than the forward stiffness.The trunk experiment was designed such that the perturbation occurs while neural activations remain unchanged. Because the perturbation is preceded by a sustained state of equilibrium, all muscles are active prior to perturbation. Thus, during muscle-tendon shortening, the tendon condition may belong to either scenario 1 or 3. Model results indicate that most preperturbation muscle forces are less than 50 N for all loading levels. The elongations due to a 50 N force remain under 0.5 mm for most tendons –37. Thispsoas major, which is usually recruited for thigh and hip flexion, makes the largest contribution, possibly because it interferes with spine flexion despite the subject's hip attachment. Likewise, the quadratus lumborum is recruited for extension, lateral flexion, and thoracic rotation. Both groups are clearly dissociated from flexural behavior. The muscle contributions should be considered as approximate sensitivity indicators. The cumulative contributions are calculated as sums of individual contributions, ignoring the interactions that may lead to an overestimation of the actual contribution of multiple groups acting simultaneously.The lists of deactivated muscles  based onmultifidus muscles help maintain spine stability but are also recruited during extension. The muscle groups that shorten during both anterior and posterior perturbations together contribute only 1% of the extensional stiffness. Groups 3 and 5 are known for their secondary role in spinal extension, which explains their deactivation in backward perturbation. Their contribution is minimal but exceeds that of the psoas major, which plays no role in extension.Muscles that shorten exclusively during posterior perturbation belong to groups 6–10, which are responsible for extension. They contribute over a third of the rearward trunk stiffness. They are naturally expected to shorten while undergoing concentric contraction. The ith combination, i.e., Ti, the forward (backward) minimum stiffness is denoted as Kf,min,Ti , and the maximum stiffness is denoted as Kmax,Ti. The sensitivity results are presented in Figures 2The tonus combination  labeled T1, which is associated with classes C1, C2, and C3, respectively, yields a narrow bracketing  of the eFor all the tonus values, the experimental and the extremum model stiffness increases with the supported load, while the experimental stiffness is always between the extreme model values. The sensitivity of both the maximum and the minimum stiffness to the tonus almost vanishes for class C2 in both forward and backward perturbations. It also vanishes for the minimum backward stiffness for class C1. The sensitivity is low for the minimum forward and backward stiffness for class C3. For the remaining cases, i.e., the maximum stiffness for classes C1 and C3 and minimum forward stiffness for class C1, the model stiffness is significantly sensitive to the tonus. Among the explored tonus combinations, T4 provides the narrowest bracketing of the experimental forward trunk stiffness.As the trunk stiffness is a key parameter in the study of spine stability, accuracy and rigor in its evaluation are important. Discrepancies in trunk test data with regard to the dependence of the trunk stiffness on the direction in the sagittal plane –5, 17 raSupport of this hypothesis involves numerous assumptions associated with the experimental protocol and the numerical model. A major assumption is that the sagittal trunk-stiffness dependence on the direction is not intrinsic but is associated with measurement. Ideally, the trunk stiffness is posture-specific and should be indifferent to position perturbations. In this sense, it would be independent of the perturbation direction despite the anatomical asymmetry. However, its experimental evaluation requires measurements at distinct time instants and postures where stiffness is likely to be different. It is common to assume the trunk stiffness to remain constant before the induction of the paraspinal reflex , 17, 21.The adopted static approach has the merit of isolating the stiffness component from the inertial and damping effects. It is based on the assumption that the pre- and postperturbation states are static for the trunk as a whole. The fact is that, while the attachment at T10 is at rest, the movement of the other parts of the trunk (the head and the suspended masses) is not restrained. Hence, preventive measures are taken to minimize undesirable movements. For this reason, long ropes are used to suspend the masses, and the subject is instructed to stiffen his neck to avoid unwanted head oscillations. The preperturbation state can justifiably be considered as wholly static because it is preceded by a phase involving no stimulus. The force signal is composed of the constant preperturbation static reaction in addition to the noise. As for the postperturbation state, its departure from a wholly static one is not excluded. Any persistent dynamic effect should be reflected in the reaction force at T10. The postperturbation force signal is composed of the static reaction superposed to the noise and any oscillatory reaction force. Before the induction of a paraspinal reflex, the latter component exclusively represents the reaction associated with the trunk dynamic effects, captured at T10. From inspection of the recorded T10 force signals, the variable part of the signal is found to be similar in pattern and magnitude to the preperturbation noise. In other words, the postperturbation dynamic effects are practically insignificant, which justifies the assumption of the static state. Further confirmation of the static assumption can be obtained through displacement measurement at nonrestrained locations using sensors or image processing –40.The multiplicity of preload cases enlarges the set of relevant data used in the study. However, the data being specific to a single subject represents a limitation of the study at this stage. The participation of a statistically representative number of subjects in future experiments will provide for statistical relevance.Examination of the influence of lifted loads on the difference between forward and rearward stiffness is pertinent for the comparison with previous work. The lifted loads are known to generally increase the activation levels in muscles , 41 likeIn this study, in contrast with the common assumption of muscle-stiffness invariance, stiffness reductions of nonneural origin during the trunk test were demonstrated to affect trunk stiffness values significantly and to lie at the root of the directional dependence of the sagittal stiffness. The investigation is based on the retreatment of trunk test raw data (separately for forward and backward perturbations), combined with simulations performed using a numerical model specific to the same subject. The static technique is employed to separate the stiffness component from the dynamic effects. The results elucidate the apparent discrepancies in the reported differences , 5 betwe


This study aimed to produce starch esters by roasting potato starch with apple distillery wastewater at various temperatures and aimed to determine the effects of esterification conditions on selected properties of the modified preparations. Apple distillery wastewater was concentrated, mixed with starch (30 g of dry matter per 100 g of starch), dried, and roasted at temperatures of 110, 130 or 150 °C for 3 h. The resulting preparations were rinsed 30 times with a 60% ethanol solution, dried, and disintegrated. After that, the following analyses were performed: content of substituted acids  with high performance liquid chromatography (HPLC); thermal characteristics with differential scanning calorimetry (DSC); swelling power and solubility in water at 80 °C; color changes with a colorimeter; rheology of the pastes based on the plotted flow curves; and the pastes’ resistance to amyloglucosidase. Starch treatments with apple distillery wastewater at 130 and 150 °C caused significant changes to its properties when compared to the control samples of native starch and starch roasted without wastewater, including: a lower temperature and heat of pasting, lower swelling power and solubility in water, darker color, higher resistance to amyloglucosidase, and the formation of pastes with a lower viscosity. Starch, as the main component of cereal grains and tubers, is one of the most abundantly available, inexpensive, and biodegradable polymers, commonly used in the food, pharmaceutical, textile, biomass energy, and chemical industries ,2,3. FurAt present, different methods have been applied to prepare RS, including chemical, physical , and enzymatic modification methods . Among tHowever, the use of chemical components in food production raises anxiety in a growing number of consumers. This is due to their increasing awareness of the risks and impacts such products have on the human body. The use, for starch esterification, of organic acids that occur naturally in plant materials may offer an alternative to its chemical modification. An example for this could be apple distillery wastewater, generally known as stillage or slops, which is a by-product of apple brandy production and which is generated in large quantities. It is wet (3–6% d. m.) and acidic waste (pH 3.5–3.9), in which various organic acids  have been identified ,14. AlonThe initial study material was Superior Standard potato starch produced by PEPEES S.A.  in 2018 and apple distillery wastewater obtained as a by-product after the distillation of fermented apple juice in a laboratory distillation device  at the Faculty of Food Technology Osijek.Apple distillery wastewater was concentrated (to 50 °Bx), mixed with potato starch (30 g of dry matter per 100 g of starch), dried at 35 °C for 12 h, and then roasted at temperatures of 110, 130 or 150 °C for 3 h in a laboratory air dryer  in a thin layer with occasional stirring. The resulting preparations were rinsed 30 times with a 60% ethanol solution, dried, and disintegrated. Native starch was also roasted without apple distillery wastewater with the application of the same roasting procedure. The modified preparations were named: R 110 °C, R 130 °C, R 150 °C—roasted starch without wastewater, and RW 110 °C, RW 130 °C, RW 150 °C—roasted starch with wastewater.The chromatographic analysis of organic acids was performed after refining the starch esters and after their alkaline de-esterification, which allowed for the assumption that all free acids were removed from the sample. Namely, a dose of starch roasted with wastewater (20 g per dry matter basis) was transferred to a conical flask with 300 mL of distilled water. The flask was placed on a magnetic stirrer, and the suspension was brought to the boiling point and then stirred for 10 min. After cooling, 1 L of rectified ethanol was added to the flask to enable starch precipitation. The sample was left for 24 h, and the excess of clear solution was decanted from above the precipitate. The refining process (dissolving in water and precipitation with ethanol) was carried out three times. Finally, the precipitated starch was dried in an air dryer at 35 °C, milled, and sieved through a 400-mm sieve.−1, using a Supelcogel TM C–610H column (30 cm × 7.8 mm) with a pre-column . The absorbance was measured at a wavelength of 210 nm with a diode array detector (DAD). Calibration curves (R2 = 0.9990) were plotted in triplicates, using organic acid standards by Sigma-Aldrich . The degree of substitution was expressed as the percentage content of acid residues in the preparations, according to Zdybel et al. [Afterwards, alkaline de-esterification was conducted. For this purpose, 2 g of the refined starch sample (per dry matter basis) and 100 mL of 0.5 M NaOH were mixed in a magnetic stirrer at a temperature of 35 °C for 12 h. Then, 400 mL of rectified ethanol was added to the homogenous solution to enable starch precipitation. The obtained filtrate was concentrated by evaporation on an evaporator  to a volume of 10 mL. Next, a 1-mL aliquot of the concentrated solution was filtered through a Millipore membrane filter with a mesh size of 0.45 μm and analyzed with the technique of high-performance liquid chromatography (HPLC)  according to the method of Hernández et al. . The anal et al. .In the case of apple distillery wastewater, it was only centrifuged for the analysis, and after that, 1 mL of aliquot was passed through a 0.45 μm Millipore filter and was then used for the same HPLC procedure. The results were expressed as the concentration in g per 100 mL of sample.g. The separated supernatant was determined for the dry matter content with the gravimetric method at 105 °C, and the precipitate left in the centrifuge tubes was weighed [A water suspension was prepared from a starch preparation that contained 1 g of starch per 100 g of solution. The suspension was shaken at a temperature of 80 °C for 30 min. Afterwards, the sample was cooled to 20 °C and centrifuged for 30 min using a Biofuge 28RS centrifuge  with an acceleration of 22,500× Thermal properties were determined with a differential scanning calorimeter DSC 822E , following the methodology provided by Gryszkin et al. . Before −1. The flow curves were described by the following equations [−1), K—consistency coefficient (Pa sn), τ0 (τ0C)—yield stress (Pa), ηC—Casson’s plastic viscosity (Pa s), n—flow behavior index.For the determination, the prepared suspensions contained 5 g of starch per 100 g of solution and were then heated at a temperature of 96 °C for 30 min with continuous mixing. The analysis was performed using a Haake Rheostress 6000 universal rheometer , according to the method of Gryszkin et al. . The floquations :(1)Ostwag for 5 min. The supernatant was collected from the centrifuged sample and mixed with the reagent GLUCOSE (containing glucose oxidase and peroxidase) from the Biosystem company , and was then incubated at 20 °C for 15 min. Afterwards, the absorbance was measured using a CECIL CE 2010 colorimeter  at a wavelength of λ = 500 nm. The measurements were conducted against a blank sample constituted by a reagent with an acetate buffer. The quantity of glucose was read out from a standard curve that was plotted as above using glucose (p.a.) solutions. The degree of saccharification was computed with respect to the theoretical quantity of glucose produced from the complete saccharification of a weighted portion of starch. The result of the hydrolysis was considered final when three consecutive read-outs of the absorbance did not differ from one another [The prepared suspensions contained 0.36 g of starch per 100 g of solution. They were heated to the boiling point with continuous mixing and then cooled to a temperature of 37 °C, at which point hydrolysis with amyloglucosidase (Amigase by Genecor)  was conducted. Acetate buffer (pH = 4.5) was added to the prepared suspension of the starch preparation at a ratio of 1:1. The flask was placed in a water bath with a shaker at a temperature of 37 °C , and 4 mL of an enzyme solution was added (at an enzyme-to-buffer ratio of 1:4). The concentration of the enzyme was selected so as to enable the complete saccharification of gelatinized starch after 120 min of the process. Every hour, 1 mL of the hydrolysate was collected and mixed with 95% ethanol (20 mL) to stop enzymatic digestion. These solutions were centrifuged  at a speed of 1825×  another .The color difference (darkening) was calculated from the results obtained in the CIE-Lab system by measuring L* (whiteness/darkness), a* (redness/greenness), and b* (yellowness/ blueness). The measurement was performed with a Chroma Meter CR-200  by using granular materials attachment. The total color change (ΔE) was calculated with the following equation :(3)ΔE=. Based on the statistical calculations , the Duncan’s least significant differences (LSD) test was determined for selected relationships at Cider is an alcoholic beverage made from apple juice, which, in addition to ethanol, contains: unfermented sugars, glycerol, organic acids , and smaThe concentrated apple distillery wastewater (used in the experiment) contained 15.96 g/100 mL of malic acid, 3.34 g/100 mL of lactic acid, and trace amounts of succinic, malonic, and oxalic acids (results not presented).The content of organic acids in fruit varies widely and depends, among other things, on the type, variety, degree of ripeness, vegetation conditions, time, and storage conditions . Most frStarch roasting with apple distillery wastewater allowed for the production of a starch ester mainly substituted by malic acid residues and trace amounts of other acids . The heaAs a result of roasting, native starch undergoes thermal depolymerization and transglycosidation . ConsequThe thermal characteristics of gelatinization depend on the roasting temperature of starch . The chaConsidering the functionality of starch preparations, the rheological properties of pastes that are made from them play a key role in the food production process. The obtained results are presented in the form of flow curves . It is eoc) and plastic viscosity (ηc), described with the Casson’s model. The yield stress is a measure of the maximum paste stress at the null shear rate, whereas Casson’s plastic viscosity indicates the paste viscosity at the final stage of shearing [shearing . Among tshearing . The passhearing ,78,79,80The resistance of roasted native starch to the activity of amyloglucosidase increased significantly in comparison to the native potato starch, with a more significant impact at higher roasting temperatures , which wAccording to the results obtained for the color, it is evident that all the measured parameters changed significantly with the increase of the roasting temperature . Higher The study results lead to the conclusion that it is feasible to produce starch esters via the roasting of native potato starch with a natural waste product—in this case, apple distillery wastewater—and that the properties of the modified preparations predispose them for use in the food industry, as well as in pharmacy or medicine. Namely, it is known that esterified starches have various applications, such as being used as thickeners, additives, stabilizers, emulsifiers, flavoring ingredients, or as fillers, coatings, binders, etc. The preparation obtained by roasting at 110 °C, characterized by an approx. 30% resistance to amyloglucosidase activity and the formation of pastes with a relatively high viscosity, could be used as a novel type of resistant starch with texture-forming properties in the production of new food products. Finally, it is important to emphasize that the obtained results represent a significant scientific contribution, since no research to date has been published on apple distillery wastewater application in starch modification processes. The production of resistant starch (RS) using natural raw materials combined with physical modifications could have a positive effect on the health-promoting value of food products and, in doing so, would satisfy consumer expectations. This research is part of one project: in the future we plan to investigate other similar by-products from the food industry for potential applications in the modification procedures of different starches. Furthermore, many other conditions and analyses will be included in order to obtain a better insight into the properties and potential applications of obtained starches.


JSR.Introducing two new Main Editors of  Journal of Synchrotron Radiation.Dibyendu Bhattacharyya of Bhabha Atomic Research Centre and Kristina Kvashnina of the Rossendorf Beamline – The European Synchrotron have recently been appointed as Main Editors of Dibyendu Bhattacharyya completed his undergraduation and post-graduation studies at University of Calcultta, Kolkata, India, and obtained his PhD degree from Jadavpur University, Kolkata, in 1995 based on his work at the Indian Association for the Cultivation of Science, Kolkata. After spending a few years at Newcastle Photovoltaics Applications Centre, University of Northumbria, Newcastle Upon Tyne, UK, as post-doctoral researcher, Dr Bhattacharyya joined Bhabha Atomic Research Centre, Mumbai, India, as Scientific Officer in 1997 where he is presently heading the Synchrotron Science and Multilayer Physics Section of the Atomic and Molecular Physics Division. Trained as a thin film multilayer device researcher, Dr Bhattacharyya moved to the area of synchrotron research in 2004, when the construction of India’s first hard X-ray synchrotron source Indus-2 began, and took a leading role in building up the two X-ray absorption spectroscopy beamlines. Over the years, Dr Bhattacharyya built up a highly professional group consisting of scientists, engineers and students which led to a very successful utilisation of these facilities leading to a large number of publications in reputed international journals and also contributing to product-oriented industrial research. Dr Bhattacharyya has over 260 publications in international journals and three book chapters to his credit with more than 4500 citations. He is recipient of several awards including the Homi Bhabha Scientific and Technical Excellence Award in 2014  and DAE-SRC Outstanding Investigator Award in 2012. Dr Bhattacharyya also holds the position of Professor in the Physical Sciences at Homi Bhabha National Institute, Mumbai, and has guided several PhD students.Kristina Kvashnina is ERC Group Head at the Rossendorf Beamline (BM20) of the European Synchrotron (ESRF), Grenoble, France, supported by Helmholtz-Zentrum Dresden-Rossendorf, Germany, and also Professor at the Department of Chemistry, Moscow State University, Russia. She completed her undergraduate degree in theoretical physics at Ural State University  in 2000 and her PhD degree in experimental physics at Uppsala University  in 2006, while carrying out experiments at the Advanced Light Source of Lawrence Berkeley National Laboratory, USA. She used soft X-ray absorption (XAS) and X-ray emission spectroscopy (XES) methods together with resonant inelastic X-ray scattering (RIXS) to study rare-earth containing materials. She then moved to the ESRF, where she conducted pioneering research on actinide systems using advanced high-energy-resolution X-ray spectroscopy methods (HERFD-XAS and RIXS) at the actinide M4,5 edges. In 2017, Kristina received a European Research Council (ERC) Grant to support her research on actinide and lanthanide nanomaterials aiming to better understand f-electron systems at the atomic level. In 2019 she received a Megagrant from the Ministry of Science and Higher Education, Russia. Kristina has published more than 140 papers in the area of X-ray spectroscopy, cited more than 2200 times, and has served as key note speaker, chair and co-chair, organizer and program committee member at numerous conferences in her area of expertise. Kristina is a member of several advisory groups and project review panels in Germany, Italy, UK, Russia, Switzerland and Hungary.Journal of Synchrotron Radiation since 2019, Dibyendu is a new addition to the editorial board. The two existing Main Editors (Yoshiyuki Amemiya and Ingolf Lindau) and Editor-in-Chief of IUCr Journals (Andrew J. Allen) are delighted to welcome Kristina and Dibyendu to the team, and look forward to working with them to further develop the journal for the synchrotron radiation and free-electron laser communities. They would also like to thank Professor Dr Ilme Schlichting  for her many years of service as a Main Editor of JSR, and wish her well as she steps down at this time.While Kristina has been active as a Co-editor of


Starches exhibited different effect on the overall quality of the yogurt due to their origin and amylose content. Regardless of the number of storage period duration, all of the samples, including the control behaved as pseudoplastic materials (n < 1) with various levels of pseudoplasticity. Yogurts with corn and tuber starches had the highest consistency coefficient (k), which indicated higher viscosity. The yogurt sample with chickpea starch exhibited the highest G´, making the gel more solid like. Therefore, the influence of tuber starches (potato and sweet potato) on G´ was different when compared to corn or legume starches. The behavior of the starches changed with storage time, where some starches performed better only at the beginning of the storage period duration. Wheying-off was significantly reduced, regardless of starch type. The pH of the yogurt remained unchanged through storage. Sensory evaluation showed a preference for starch-containing samples as compared to the control, regardless of the starch type. The variation in yogurt quality as a function of starch type could be attributed to the starch granule structure, gelatinization mechanism, or amylose content. This study was conducted to investigate the effect of various native starches on the rheological and textural properties of non-fat set yogurt. The yogurt samples were prepared while using five types of starches . The physical properties of the prepared yogurt were analyzed while using shear viscosity, viscoelasticity, and texture analysis. The tests were performed after 0, 7, and 15 days storage. The effect of these starches on the yogurt viscoelastic properties, texture, syneresis, and sensory evaluation were determined under optimum conditions. The results showed that adding 1% starch could significantly ( Streptococcus thermophiles and Lactobacillus [Nowadays, yogurt is considered to be one of the most popular fermented milk products and it has gained widespread consumer acceptance as a healthy food . It is pbacillus . Yogurt bacillus . The consumption of whole fat products  has declined due to the awareness of the probable harmful effect of fat on consumer’s health, thus the dietary habits of consumers have changed and market interest moved in favor of low or nonfat dairy products . The CodIn yogurt products, milk fat plays a major role in the texture, flavor, and color development of the final products . TherefoFood hydrocolloids, such as starches, are usually used in the food industries as thickeners, stabilizer, gelling agents, syneresis controller, and emulsifiers ,10. On tNonfat milk powder  of Nino brand was purchased from a local supermarket. Fresh potato and sweet potato were obtained from the produce market . Chickpea and Turkish beans were purchased from local store, whereas ARASCO  donated the corn starch.v/v) for 3 min. while using kitchen aid blender at medium speed . The slurry was filtered through a muslin cloth and the overs were re-suspended in distilled water (1:2 v/v), blended, and filtered in the same way, and finally the filtrate is sieved while using 200 mesh sieve. Starch was allowed to settle for 1 h at room temperature and the supernatant was discarded. The starch was re-suspended in distilled water and then centrifuged at 2000× g for 15 min. After centrifugation, the top dark layer was removed and the white material at the bottom of the bottle is reconstituted in distilled water and then centrifuged until pure white starch fraction is obtained. The isolated starch is dried, ground, and stored as in air tight jars at 5 °C.The potato or sweet potato starch was extracted according to . The tubw/w) in heavy duty blender  for 5 min. at medium speed. The slurry was passed through 200 mesh sieves and the filtrate was centrifuged at 2000× g for 15 min. [Whole meal of Turkish beans was prepared by crushing the dry beans in the blender at fast speed for 3 min. Slurry was prepared by blending the whole meal of chickpea or Turkish beans in distilled water  and starch. The blends were prepared by replacing 1.0 g of the skim milk with 1.0 g of starch. So as to ascertain complete solubility, dry starch and milk powder were mixed first. To the dry ingredients, water was added to finally maintain 14% total solids and the mixture was preheated to 60 °C for 30 min., cooled to 42 °C, and the yogurt starter was added at 3.0% of the dry ingredients. The microbial starter included Streptococcus thermophiles and Lactobacillus bulgaricus. The mixture (14 g) was divided into plastic cups (100 mL) and incubated at 42 °C until coagulation occurs or the pH reached 4.6  [The total solids content of yogurt samples were determined according to the Association of Official Agricultural Chemists AOAC Method (940.09) . Yogurt The ash content of the yogurt samples was determined according to the AOAC Method (942.05) , where 5The crude protein content was estimated according to the Kjeldahl method, as described in the AOAC method (992.15) . The yogThe crude fat content was determined while using the Gerber Method and the percent crude fat was determined by directly reading calibrated butyrometer  [The carbohydrate contents were determined by the difference method given using the expression given below:−1), n is the flow behavior index, and k is the consistency index (Pas). The values for the flow behavior index n were obtained from plotting the log of shear stress versus log of shear rate and the slope of the line (if the dependence is sufficiently close to a linear one) is simply equal to the flow index (n).The yogurt viscosity measurements were carried out at ambient temperature (25 °C) while using Brookfield viscometer  with spindle number 63. The disk spindle was selected because of the nature of the yogurt sample, which allows for readings within the instrument sensitivity. According to the manufacturer (Brookfield), samples should be kept in a thermostatically controlled water bath at 25 °C for about 10 min. before measurements. The first measurements were taken 2 min. after the spindle was immersed in the sample to allow for thermal equilibrium to eliminate the effect of immediate time dependence effect. The data were collected every 40 sec and the measurement was duplicated and the average value was considered. The experimental data obtained was converted to shear rate/shear stress and fitted to the power law model.The dynamic viscoelastic properties of nonfat yogurt were determined while using TA Instrument Discovery Hybrid Rheometer (HR-1) that was installed with parallel plates system . The samples were transferred to the plate and rheometer was calibrated at 25°C for one minute and excess sample was wiped off with spatula. Dynamic shear data were obtained at frequency sweeps ranging from 0.1–100 rad/s and 0.5% constant strain at 25 °C. Experimental data were collected using the software that was provided by the manufacturer. The storage moduli (G′), loss moduli (G″), and viscosity are the parameters obtained for every run. A strain-sweep experiment was performed to establish that all measurements were done within the linear viscoelastic rage of the experiment. Linear viscoelasticity indicates that the measured parameters are independent of shear strains. To determine the liner viscoelastic range (LVR), a stress sweep was increased from 0.1 to 50.0 Pas at a constant frequency of 0.1 Hz (0.628 rad/s). Frequency sweeps between 0.1 to 10 (rad/s) were implemented within the LVR of the yogurt samples at a constant stress of 1.0 Pas. The frequency range used here is typically used for frequency sweep to ascertain that G′, G″, and η* were within the linear region. The behavior of all the measured materials in this study was in the linear range below 1% strain. Measurement was repeated three times with fresh samples. The relative errors were within the range of ±10%. All of the calculations were performed with the Rheology Advantage Data Analysis software  that was provided by the manufacturer. The viscosity profile was used to provide information regarding the possibility of slippage, where the viscosity profile as a function of shear rate of duplicate runs was plotted in the same graph to ascertain the repeatability and slippage behavior of the material. No slippage was recorded. The double compression test was performed while using texture analyzer  equipped with a software. Plastic cylinder  is attached to crosshead moving at speed of 70 mm/min. in both upward and downward directions. The yogurt sample was placed on a flat holding plate and the plastic cylinder was inserted 20 mm below the surface of the yogurt sample. The firmness of the yogurt was calculated according to the method that was described by Steffe 1996 .The volume of the separating whey (wheying-off) on the surface of the yogurt sample was collected as an indicator for wheying-off (g/100 g yogurt) according to the siphon method. The level of spontaneous whey separation of undisturbed set yogurt was determined while using a siphon drainage method . In thisThe sensory evaluation was carried out by a group of 10 trained sensory assessors. The evaluation of the yogurt samples included the following sensory attributes; appearance, color, texture, aroma, taste, aftertaste, and overall acceptability. The scale used was nine points hedonic scale, where 1 represents dislike extremely, 5 for neither dislike or like, and 9 for extremely like.p ≤ 0.05 was used to compare means while using PASW® Statistics 18 software .Statistical analysis was performed while using SPSS . All of the measurements were done in triplicate and then subjected to analysis of variance (ANOVA) using factorial design. Duncan’s multiple range tests at Three different sets of temperatures were selected, 36–38 °C, 40–42 °C, and 44–46 °C, in order to determine the appropriate incubation temperature. The yogurt made at all three sets of temperature was tested for its texture, viscosity and wheying-off. Unlike the other temperatures, the yogurt that was prepared at 40–42 °C had good texture and viscosity as well as less wheying-off. Therefore, yogurt with or without starch was prepared at 42 °C. In addition, the pH was monitored throughout the incubation time and was found to decrease with the same rate in the presence of starch when compared to the control. Williams et al. (2003)  reportedThe proximate composition of the yogurt showed no significant difference between the control and the yogurt/starch blends with respect to the total solids, lipids, and ash. However, lower carbohydrates and higher protein content were recorded for the control. The percent amylose content for potato, sweet potato, corn, chickpea, and Turkish bean starches were 21.8, 22.9, 20.4, 32.2, and 17.5, respectively. k and n), where the data clearly fit the power law, because of the values of the high value of r2 (>0.95). The Y-intercept represents the K and the slope represents the n. Regardless of the number of storage days, all the samples  [n values, yogurt sample containing Turkish beans starch was the least pseudoplastic, especially at longer storage time. This behavior was also reflected for the yield stress. Potato and sweet potato starches exhibited the most pseudoplasticity after seven days storage. This difference can be attributed to the different amylose content of these starches, where the lowest amount of amylose showed the smallest pseudoplasticity. Conversely, chickpea with the highest amylose content started as the least pseudoplastic, but it become more pseudoplastic as a function of storage time ).  samples , includi. (2012) ; Yu et a. (2012)  noticed age time . When cotructure . The k vk and n values reported above.The dynamic rheological testing provides useful information regarding the internal structure of the yogurt, which is represented by storage modulus (G′) and loss modulus (G″) that denote the elastic and the viscous behavior, respectively, as well as the complex viscosity η* and the phase angle tan δ. The yogurt samples exhibited a weak gel behavior because G′ > G″ over the range of 0.1 and 50 Pas. A linear viscoelastic region was observed from 0.1 to 10 Pas. Therefore, 1.0 Pas stress was chosen for the frequency sweep test. These data are in agreement of other researchers who reported similar results for yogurt enriched with milk solids and inulin . The lonp < 0.05) after seven days, but the effect of corn starch dropped after 15 days. This could be attributed to weaker corn starch amylose network, which allows for the water to be free after it has been trapped in the gel network. Yogurt containing tuber starches (sweet potato and potato) exhibited the hardest gel through most of the storage time  in wheying-off, regardless of starch type for the same storage time  between the starches with respect to sensory texture, but it was significantly better than the control  if it stays on the tongue or flows slowly and is swallowed with difficulty, while the visually thick sample can be measured by tilting the spoon and observing how slow the sample will flow. The statistical analysis of this work underlined that there was no significant difference ( control . The senStabilizers are important ingredients in manufactured yogurt or other dairy products due to their capacity to improve viscosity and sensory properties, and to decrease wheying-off during storage. The results showed that adding 1% starch gave better results than the control for the syneresis, sensory evaluation, and viscoelastic properties. The outcome of this work showed that tuber starches can behave differently from cereal or legume starches. In addition, the data indicated that the storage time of yogurt could affect starches behavior. The variation in the effects of starches on yogurt quality could be due to the starch granule structure, gelatinization mechanism, and amylose content. The overall results obtained indicate tuber starches or chickpea starch as the most promising stabilizer of non-fat yogurt during storage.


M. tuberculosis MmsA in immunological regulation of type I IFN response by targeting the central adaptor STING in the DNA sensing pathway. We identified STING-interacting MmsA by coimmunoprecipitation-mass spectrometry-based (IP-MS) proteomic analysis and showed MmsA interacting with STING and autophagy receptor p62 via its N terminus and C terminus, respectively. We also showed that MmsA downregulated type I IFN by promoting p62-mediated STING degradation. Moreover, the MmsA mutant R138W is potentially associated with the virulence of M. tuberculosis clinical strains owing to the modulation of STING protein. Our results provide novel insights into the regulatory mechanism of type I IFN response manipulated by mycobacterial MmsA and the additional cross talk between autophagy and STING in M. tuberculosis infection, wherein a protein from microbial pathogens induces autophagic degradation of host innate immune molecules.It is unclear how the type I IFN response is regulated by mycobacterial determinants. Here, we characterized the previously unreported role of  Mycobacterium tuberculosis persistence and disease pathogenesis. M. tuberculosis has evolved a number of mechanisms to evade host immune surveillance. However, it is unclear how the type I IFN response is tightly regulated by the M. tuberculosis determinants. Stimulator of interferon genes (STING) is an essential adaptor for type I IFN production triggered by M. tuberculosis genomic DNA or cyclic dinucleotides upon infection. To investigate how the type I IFN response is regulated by M. tuberculosis determinants, immunoprecipitation-mass spectrometry-based (IP-MS) proteomic analysis was performed to screen proteins interacting with STING in the context of M. tuberculosis infection. Among the many predicted candidates interacting with STING, the M. tuberculosis coding protein Rv0753c (MmsA) was identified. We confirmed that MmsA binds and colocalizes with STING, and the N-terminal regions of MmsA (amino acids [aa] 1 to 251) and STING (aa 1 TO 190) are responsible for MmsA-STING interaction. Type I IFN production was impaired with exogenous expression of MmsA in RAW264.7 cells. MmsA inhibited the STING-TBK1-IRF3 pathway, as evidenced by reduced STING levelS and subsequent IRF3 activation. Furthermore, MmsA facilitated p62-mediated STING autophagic degradation by binding p62 with its C terminus (aa 252 to 455), which may account for the negative regulation of M. tuberculosis MmsA in STING-mediated type I IFN production. Additionally, the M. tuberculosismmsA R138W mutation, detected in a hypervirulent clinical isolate, enhanced the degradation of STING, implying the important relevance of MmsA in disease outcome. Together, we report a novel mechanism where M. tuberculosis MmsA serves as an antagonist of type I IFN response by targeting STING with p62-mediated autophagic degradation.Type I interferon (IFN) plays an important role in  Mycobacterium tuberculosis, is a fatal chronic infectious disease. According to the latest global tuberculosis report, this disease affected 10 million individuals in 2018, and it is estimated that approximately 1.7 billion individuals have a latent tuberculosis infection (LTBI)  24. The pa (LTBI) 2. A hyper (LTBI) 2. Furtherimmunity . Howevernfection , 12.M. tuberculosis infection  to activate the STING-TBK1-IRF3 pathway in response to different M. tuberculosis strains, such as Erdman and CDC1551  is an important adaptor molecule involved in innate immunity, linking cytosolic DNA sensing and cell activation. It also senses cyclic dinucleotides by itself, leading to the induction of type I IFN –15. STIN–nfection –20. In 2nfection . Further CDC1551 –20. MycoCDC1551 –. Accordinfection . It has rculosis . Subsequrculosis , 19.M. tuberculosis  and host-derived noncanonical CDNs to evade the immune system  gene was first reported in the study of whole-genome sequencing of M. tuberculosis virulence strain H37Rv (M. tuberculosis culture filtrate (M. tuberculosismmsA was upregulated in 28-day and 50-day cultures but not in 14-day cultures (mmsA homologs belong to the coenzyme A (CoA)-dependent aldehyde dehydrogenase (Aldedh) subfamily, catalyzing the NAD-dependent oxidation of methylmalonate semialdehyde and malonate semialdehyde into propionyl-CoA and acetyl-CoA in a wide variety of organisms, ranging from bacteria to mammals. The MmsA from bacteria species such as Streptomyces coelicolor is involved in valine catabolism  and promotes the Th1 immune response during M. tuberculosis infection (–M. tuberculosis MmsA and host STING and the regulation of the type I IFN response. Coimmunoprecipitation (co-IP) and mass spectrometry were performed to identify the mycobacterial STING-interacting proteins, revealing MmsA as a promising candidate to interact with STING and blunt the type I IFN response via p62-mediated autophagic degradation.The in H37Rv . It was filtrate . A previcultures . The mmstabolism . M. tubenfection , and thenfection –34. HoweM. tuberculosis genomic DNA and cyclic dinucleotide c-di-AMP  also had a high binding score (see M. tuberculosis Flag-MmsA could bind Myc-STING (Mycobacterium smegmatis (M. smegmatis MmsA) in HEK293T cells for co-IP experiments. Our finding showed that M. tuberculosis MmsA, but not M. smegmatis-MmsA, could bind STING, suggesting the binding specificity between STING and M. tuberculosis MmsA  or pFlag-MAVS (B), with or without pMyc-MmsA, for 48 h. Cell lysates were immunoprecipitated with the anti-Myc antibody, followed by immunoblotting with the indicated antibodies. (C) HEK293T cells were transfected with pFlag-MS-MmsA or pFlag-MmsA (M. tuberculosis) together with pMyc-human-STING for 48 h. Cell lysates were immunoprecipitated with the anti-Flag antibody, followed by immunoblotting with the indicated antibodies. Download FIG S1, TIF file, 0.6 MB.Copyright © 2020 Sun et al.2020Sun et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03254-19.5TABLE S1Table S1, DOCX file, 0.01 MB.Candidate STING interacting proteins identified by IP-MS approach. Download Copyright © 2020 Sun et al.2020Sun et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Mycobacterium smegmatis, has been widely used as a model for mycobacterial functional studies due to its fast growth and safety (M. smegmatis expressing M. tuberculosis MmsA (M. smegmatis::MmsA). To examine MmsA-STING colocalization in the context of mycobacterial infection, we infected the RAW264.7 cells with M. smegmatis::MmsA. Immunofluorescence assay showed that the endogenous MmsA expressed by M. smegmatis::MmsA indeed colocalized with STING  truncation mutant, STINGC-Region  of STING contains four transmembrane domains for the cellular location, such as endoplasmic reticulum and mitochondrion. A central globular domain contains a dimerization region (aa 153 to 177) and a cyclic dinucleotide binding domain (aa 178 to 341). The C-terminal tail aa 342 to 379) is required for downstream signaling –41. To i– to 379 iC-Region . InteresC-Region . We thenth STING . Thus, wM. smegmatis::MmsA-infected RAW264.7 cells compared to that in the M. smegmatis::Vector-infected cells (M. tuberculosis MmsA (RAW-MmsA) and control RAW-Vector cells  RAW-MmsA and RAW-Vector cells were treated with poly(dA:dT) (2 μg/ml) for the indicated time, and IFN-β production was measured by qPCR. GAPDH used as a loading control. (B) HEK293T cells were transfected with plasmids expressing pMyc-human-STING and vector or pFlag-MmsA for 48 h. Cells were lysed and then immunoblotted with the indicated antibodies. The results are shown as means ± SEM. **, Copyright © 2020 Sun et al.2020Sun et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To examine which of the steps in type I IFN production are affected by MmsA, we transfected plasmids expressing MmsA and key molecules involved in STING signaling, such as STING, TBK1, and IRF3, together with an IFN-β luciferase reporter plasmid, into HEK293T cells. The inhibition of IFN by MmsA was observed in STING- and TBK1-overexpressing cells. However, the production was no longer inhibited by the overexpression of constitutively active IRF3/5D, suggesting that MmsA suppresses the STING-mediated IFN signaling pathway upstream of IRF3 .The STING-TBK1-IRF3 pathway is critical for type I IFN production, and activated STING recruits the kinase TBK1 to stimulate the phosphorylation of IRF3 ; MmsA miM. smegmatis::MmsA-infected RAW264.7 cells compared to those in M. smegmatis::vector-infected cells  Stable transfected cell lines RAW-MmsA and RAW-Vector were analyzed by qPCR. STING mRNA was measured and GAPDH used as a loading control. The results are shown as means ± SEM. NS, not significant. (B) HEK293T cells were transfected with empty vector or the plasmids expressing Flag-M. tuberculosis-MmsA or Flag-MS-MmsA, along with expression plasmids of pMyc-human-STING for 48 h. Cell lysate was subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. Download FIG S3, TIF file, 0.4 MB.Copyright © 2020 Sun et al.2020Sun et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mmsA gene exists in the hypervirulent clinical Beijing-like isolates and that this mutation was considered likely to affect the protein function, as scored by PROVEAN (It has been reported that STING undergoes ubiquitination and can be degraded via the proteasome-dependent pathway –48. Ther– PROVEAN . To dete PROVEAN  or endog PROVEAN . Taken t10.1128/mBio.03254-19.4FIG S4M. tuberculosis MmsA mediated autophagy-dependent degradation of STING. (A) RAW-MmsA and RAW-Vector were treated with DMSO or rapamycin (100 nM) for 6 h and analyzed by immunoblotting with the indicated antibodies. (B) HEK293T cells were transfected with empty vector or the plasmids expressing Flag-MmsA, along with expression plasmids of Myc-human-STING, for 48 h. Cell lysate was subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. Download FIG S4, TIF file, 0.4 MB.Copyright © 2020 Sun et al.2020Sun et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the SQSTM1) functions as a major autophagy cargo receptor that delivers proteins for selective autophagosome degradation  of MmsA is critical for its interaction with p62  also reduced the intracellular bacterial numbers  mice  mice , 19, 23.WT) mice . In addifections , 57. Her studies , 60.M. tuberculosis infection. Evidence showed that cGAS functions upstream of STING to activate autophagy during M. tuberculosis infection  supplemented with 10% fetal bovine serum (HyClone), 1% penicillin-streptomycin solution. Both cell lines were cultured in humidified air containing 5% COM. tuberculosis H37Rv and M. smegmatis mc2155 were grown in Middlebrook 7H9 broth medium (BD Biosciences) supplemented with 0.05% Tween 80, 0.5% glycerol, and 10% albumin-dextrose-catalase (BD Biosciences) at 37°C. The recombinant M. smegmatis expressing M. tuberculosis MmsA (M. smegmatis::MmsA) was generated by electroporating pMV261-MmsA into M. smegmatis mc2155. The successfully recombinant M. smegmatis was selected using kanamycin and was further confirmed by Western blotting. The control M. smegmatis::Vector was generated by electroporating pMV261 vector plasmid into M. smegmatis mc2155. M. smegmatis::MmsA and M. smegmatis::Vector were grown in Luria-Bertani medium supplemented with 0.05% Tween 80 and kanamycin. All mycobacteria were counted based on CFU in Middlebrook 7H11 broth medium (BD Biosciences) supplemented with 0.5% glycerol and 10% oleic acid-albumin-dextrose-catalase (BD Biosciences) at 37°C.M. tuberculosis virulent strain H37Rv for 6 h at a multiplicity of infection (MOI) of 10. The cells were washed with phosphate-buffered saline (PBS) and lysed with immunoprecipitation lysis buffer containing 100 μM phenylmethylsulfonyl fluoride . The cell lysates were subjected to immunoprecipitation with 50 μl protein A-agarose and anti-STING (1:50) antibody (Ab) or normal rabbit IgG (1 μg/ml). The candidate proteins interacting with STING were identified by mass spectroscopy (Applied Protein Technology).RAW264.7 cells were left uninfected or were infected with the N-Region, and pSTINGC-Region. Human p62 cDNA from THP-1 cells was also cloned into pMSCV-puro-Myc vector and named pMyc-p62. The coding sequences of M. tuberculosis MmsA and its truncated mutants (aa 1 to 251 and 1 to 455) were amplified from H37Rv genomic DNA and cloned into pFlag-CMV2 vector to get the pFlag-MmsA, pFlag-MmsA1-251aa, and pFlag-MmsA1-455aa plasmids. Flag-MmsA was also cloned into the retroviral expression vector pMSCV-EGFP to get pMSCV-EGFP-Flag-MmsA for the generation of retrovirus expressing Flag-MmsA. M. tuberculosis MmsA was also cloned into pMV261 and pEGFP-C1 vectors to get the plasmids pMV261-MmsA and pEGFP-MmsA. MmsA was also cloned into pMSCV-puro-Myc vector and named pMyc-MmsA. M. smegmatis MmsA was amplified using M. smegmatis genomic DNA and cloned into pFlag-CMV2 vector to generate pFlag-M. smegmatis MmsA. These plasmids were constructed using the ClonExpress II one-step cloning kit  or standard molecular biology techniques. The pFlag-MmsA-R138W plasmid expressing M. tuberculosis MmsA-R138W mutant was generated by the site-directed mutagenesis method using Phanta Max super-fidelity DNA polymerase . Plasmids expressing Flag-STING, Flag-TBK1, Flag-IRF3, and Flag-MAVS were gifts from Fangfang Zhou (Soochow University). The primers used for clones and subclones are provided in The human STING cDNA from GENECHEM, Ltd., murine STING cDNA from RAW264.7 cells, as well as the truncated human STING, aa 1 to 190, and STING, aa 191 to 379, were cloned into pMSCV-puro-Myc vector and named pMyc-human-STING, pMyc-murine-STING, pSTING10.1128/mBio.03254-19.6TABLE S2Table S2, DOCX file, 0.02 MB.List of all primers for plasmid construction. Download Copyright © 2020 Sun et al.2020Sun et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 5 RAW264.7 cells/well were seeded in 12-well plates and then transfected with p62 siRNA (50 nM) or control siRNA using Lipofectamine RNAiMAX (Life Technologies).For knockdown experiments, the siRNAs targeting to human p62 coding sequence were designed and synthesized by RiboBio. A total of 2 × 10+ cells were sorted by a FACSAria III cell sorter (BD Biosciences). High purity of GFP+ cells was obtained by two or three rounds of sorting and termed Raw-MmsA cells. Similarly, RAW264.7 cells transduced with MSCV-EGFP retrovirus were termed RAW-Vector as a control.HEK293T cells were seeded in 60-mm dishes and transfected with 2 μg of packing plasmid pCL-Ampho  and 4 μg of pMSCV-EGFP-Flag-MmsA using Lipofectamine 3000. The virus-containing medium was harvested 48 and 72 h posttransfection, centrifuged at 4°C, 3,000 rpm, for 30 min, and filtered through a 0.45-μm filter (MILLEX GP). RAW264.7 cells were infected with the above-described retrovirus in the presence of Polybrene , and the culture plates were centrifuged at 2,500 rpm for 30 min at 30°C for high efficiency of infection. The cells were further cultured in fresh medium for 72 to ∼96 h, and the GFPPoly(deoxyadenylic-thymidylic) acid [poly(dA:dT)] (P0883) was from Sigma. MG132 (S2619), chloroquine (CQ) (C6628), and rapamycin (A606203) were from Selleck, Sigma, and Sangon Biotech, respectively.M. tuberculosis MmsA expressed by Escherichia coli.The following antibodies were used for immunoblotting. Anti-STING (13647), anti-TBK1/NAK (3013), anti-phospho-TBK1/NAK (Ser172) (5483), anti-IRF3 (4302), anti-β-tubulin (2128), anti-His-tag (12698), and anti-Myc-tag (2276) were from Cell Signaling Technologies. Anti-Flag (F1804), anti-Myc (C3956), anti-phospho-IRF3 (pSer385) (SAB4503926), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (G9545) were from Sigma. Anti-DDDDK-tag (PM020) and anti-p62/SQSTM1 (PM045) were from MBL. Anti-calnexin (10427-2-AP) was from Proteintech. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (4030-05) or anti-mouse (1034-05) IgG was from SouthernBiotech. Mouse polyclonal antibody against MmsA antiserum was raised against recombinant Anti-STING , normal rabbit IgG , protein A-agarose , cell lysis buffer for Western blotting and IP , EZview red anti-c-Myc affinity gel , and EZview red anti-Flag M2 affinity gel  were used for immunoprecipitation.The following antibodies were used for immunofluorescence: anti-Flag , anti-Myc , anti-STING , DyLight 549-goat anti-mouse IgG , DyLight 488-goat anti-rabbit IgG , DyLight 488-goat anti-mouse IgG , and DyLight 549-goat anti-rabbit IgG .Cells were lysed on ice for 30 min using lysis buffer containing 100 μM PMSF and PhoStop phosphatase inhibitor (Roche). Equal amounts of protein from every sample were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membrane was blocked with PBS containing 0.1% Tween 20 and 5% skim milk for at least 60 min at room temperature (RT). These membranes were further incubated with the appropriate primary antibodies and HRP-conjugated secondary antibodies. The membranes were detected by Super Signal West Pico chemiluminescent substrate (Thermo Fisher Scientific), and the chemical luminesce was visualized using an Amersham Imager 600 . The intensity of the bands was quantified by ImageJ software.For native polyacrylamide gel electrophoresis, cells were lysed in nondenatured lysis buffer  containing 100 μM PMSF. In brief, the 7.5% native PAGE gels were prerun with 25 mM Tris and 192 mM glycine, pH 8.4, containing 1% deoxycholate in a cathode chamber for 30 min at 70 V on ice. Samples in native loading buffer  were separated at 70 V for 180 min on ice and then transferred to PVDF membranes for immunoblotting.6 cells were plated onto a 60-mm dish and transfected with the indicated plasmids. Forty-eight hours posttransfection, the cells were washed three times with 1 ml cold 1× PBS and lysed with 1 ml cell lysis buffer (Beyotime) containing 1% Triton X-100 and 100 μM PMSF for Western blotting and immunoprecipitation. The cell lysates then were incubated with 20 μl EZview red anti-Myc/Flag affinity gel for 4°C with overnight shaking. The Sepharose beads were eluted with 2× protein loading buffer after five washings with lysis buffer and boiled for 10 min before immunoblot analysis. For the semiendogenous co-IP, the lysates of RAW-Vector and RAW-MmsA were incubated with anti-Flag antibody and analyzed by immunoblot analysis with anti-STING antibody.For immunoprecipitation in HEK293T cells, 2 × 10M. smegmatis::MmsA or H37Rv at an MOI of 10. At 24 h postinfection, the cells were fixed and processed for the staining of anti-MmsA mouse Ab and anti-STING rabbit Ab. For subcellular localization, RAW264.7 cells were infected with M. smegmatis::MmsA for 12 h. The cells were processed as described above and immunostained with anti-calnexin rabbit antibody and anti-MmsA mouse antibody. After washing, the cells were stained with DyLight 488/549-goat anti-mouse IgG and DyLight 549/488-goat anti-rabbit IgG secondary antibodies. Colocalization images were captured using a laser scanning confocal microscope (Nikon A1).To determine MmsA and STING colocalization, HEK293T cells were grown on live cell imaging culture dishes (Livefocus) and transfected with pFlag-MmsA and pMyc-murine/human-STING. The cells were washed with PBS 36 h posttransfection, fixed with ice-cold 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 PBS. The cells were then blocked with PBS containing 3% bovine serum albumin  and incubated with anti-Flag mouse Ab and anti-Myc rabbit Ab at 4°C overnight. After washing with PBS, cells were incubated with DyLight 549-goat anti-mouse IgG and DyLight 488-goat anti-rabbit IgG (human STING) or DyLight 488-goat anti-mouse IgG and DyLight 549-goat anti-rabbit IgG (murine STING) for 1 h at RT, and the cellular DNA was stained by 4′,6-diamidino-2-phenylindole (DAPI) (Sigma) for 10 min at RT. To examine the colocalization of STING and MmsA in the context of mycobacterial infection, RAW264.7 cells were infected with 5 cells/well. The cells/well were then transfected with 500 ng the indicated plasmids, 200 ng IFN-β-Luc plasmid, and 40 ng Renilla luciferase vector as an internal control. The cell lysates were assayed for luciferase activities 24 h posttransfection using the Dual-Luciferase reporter assay system  according to the manufacturer’s instructions. The luminance was detected by a microplate reader .HEK293T cells were seeded into 12-well plates at a density of 3 × 10M. tuberculosis clinical isolates was extracted via ultrasonication for 10 min  before adding RNAiso plus. All gene transcripts were quantified by real-time PCR with PowerUP SYBR green master mix  using an ABI Q6 instrument (Applied Biosystems). GAPDH was used as a reference in RAW64.7 cells, and SigA was used as an internal control for the normalization in M. tuberculosis. The primers were used for qPCR as shown in Total RNA was isolated using RNAiso plus  according to the manufacturer’s instructions and subjected to reverse transcription with a PrimeScript RT reagent kit . RNA from 10.1128/mBio.03254-19.7TABLE S3Table S3, DOCX file, 0.01 MB.List of primers for qPCR analysis. Download Copyright © 2020 Sun et al.2020Sun et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 5/well) were infected with H37Rv at an MOI of 10. The infected cells were changed into DMEM containing 200 μg/ml amikacin and 1% antibiotic/antimycotic solution (Gibco) to kill extracellular bacteria. The infected cells were lysed with 1 ml sterile water containing 0.05% Triton X-100 0, 2, and 4 days postinfection. For CFU assay, 50-μl lysates were added to Middlebrook 7H10 agar plates and cultured for 3 weeks at 37°C.For the detection of bacteria survival within macrophage, RAW-Vector and RAW-MmsA cells seeded in 6-well plates , <0.01 (**), and <0.001 (***) were considered significant. Correlations were performed using the Pearson test.All data are shown as the means ± standard errors of the means (SEM) and were evaluated with a two-tailed, unpaired Student's


The influence of starch concentration (3–15 wt%) and solvent content (20–80 wt%) on gel formation was also studied. It was demonstrated that the gelation of starch in binary mixtures of solvents can be rationalized by Hansen Solubility Parameters (HSP) revealing a crucial hole of hydrogen bonding for the gel’s strength, which is in agreement with rheological measurements. Only the addition of water or propylene glycol to starch/DMSO solutions resulted in strong gels at a minimum starch and solvent content of 7.5 wt% and 50 wt%, respectively. The resulting aerogels showed comparably high specific surface areas (78–144 m2 g−1) and low envelope densities (0.097–0.203 g cm−3). The results of this work indicate that the HSP parameters could be used as a tool to guide the rational selection of water-free gelation in starch/DMSO systems. In addition, it opens up an attractive opportunity to perform starch gelation in those solvents that are miscible with sc-CO2, avoiding the time-consuming step of solvent exchange.In this work, the ability of several solvents to induce gel formation from amylomaize starch solubilized in dimethyl sulfoxide (DMSO) was investigated. The formed gels were subjected to solvent exchange using ethanol and dried with supercritical carbon dioxide (sc-CO Starch is one of the most abundant biopolymers and the major carbohydrate reserve in higher plants. In vegetal tissues, starch it is stored as granules, and the major compounds (98–99 wt%) are amylose and amylopectin. Amylose is a linear polymer composed of α-D-glucopyranose linked α-1,4), while amylopectin is highly branched at the positions α-1,6) besides the linkages in α-. Although starch can be found in almost all vegetables, the main commercial sources are corn, wheat, potato, cassava, and rice. This important biopolymer is largely applied as thickening and gelling agent in food and non-food products ,4, while. Moreove besides 2) [Starch-derived aerogels, low-density mesoporous solids with high specific surface area, are commonly prepared in three steps: hydrogel formation, solvent exchange using ethanol, and supercritical drying with carbon dioxide (CO2) ,7. Aerog2) ,8. Starc2) ,10,11, t2) ,13,14, t2) ,16, as t2) , and ads2) .Starch gels are predominantly formed in aqueous media under thermal gelation through the so-called gelatinization and retrogradation processes . Dependitδ), or the so-called total Hildebrand solubility parameter: To rationalize the outcome of the gelation experiments, we employ the Hansen Solubility Parameters (HSP). In the field of polymer science, the HSP are a powerful empirical tool largely used to predict polymer solubility. More recently, it has been successfully employed to predict organogels formation ,27,28. I+δp2+δh2 . To predIn this scope, we set the goal to explore phase transitions in DMSO solutions of starch when combined with several solvents. The influence of starch concentration, type of solvent, and solvent-to-DMSO mass ratio on gel formation were discussed in terms of visual appearance, solubility parameters, and rheological measurements. Textural properties of aerogels were characterized through nitrogen adsorption/desorption technique and Scanning Electron Microscopy (SEM).w/w, using stock solutions of 30–37 wt% starch. For samples containing 15 wt% starch, the solvent-to-DMSO mass ratios used were 20:80, 30:70, and 50:50. This was because the stock solutions with starch contents larger than 40 wt% were too viscous to guarantee good homogeneity of mixing at higher solvent-to-DMSO mass ratios.The ability of several solvents to induce gelation in DMSO solutions of starch was first investigated. Samples containing starch concentrations of 3, 5, 7.5, and 10 wt% were prepared with the following solvent-to-DMSO mass ratios: 20:80, 30:70, 50:50, 70:30, and 80:20 The effect of the solvent nature, starch concentration, and solvent-to-DMSO mass ratio on gel formation is shown in mδ) and the gelation behavior obtained for all solvent-to-DMSO mass ratios. The tabular data are presented in mδ, there is a tendency to form gel-like and strong gel structures, indicating a reduction in the affinity of starch to DMSO and an increase in the polymer–polymer chain interactions. Following this tendency, it would be expected that glycerol presents a better ability to form strong gels than propylene glycol, but the opposite is observed. As pointed by McGrane et al. (2004) [2-OH group of glycerol is unlikely to bond to a third amylose molecule due to steric restriction; (ii) all the three alcohol groups form intramolecular bonds with amylose molecules, resulting in an irregular alignment of amylose chains and preventing the gel formation. Reducing the number of hydroxyl groups from three in glycerol to two in propylene glycol seems to favor the polymer–polymer interactions up to a certain point, when starch concentration and propylene glycol-to-DMSO mass ratio were 15 wt% and 50:50 (strong gel appearance), respectively.. (2004) , there amδ for binary mixtures of DMSO with sulfolane and 2-dimethyl ethanolamine are between 24.3 and 26.6 MPa1/2 , which is the case of sulfolane, dδ = 18.4 MPa1/2  were plotted in the 3D Hansen space for all solvent-to-DMSO mass ratios . We can hypothesize that the most suitable solvents to promote gel formation in a starch/DMSO system should be those with high values of tδ  is presented in Based on the visual inspection and rheological measurements, seven samples were chosen for aerogel production, which are assigned with codes and summarized in 2 g−1, but no clear influence of starch concentration and solvent-to-DMSO mass ratio is observed. On average, we can conclude that solvent-induced gelation with water yielded aerogels with a specific surface area of 113 ± 20 m2 g−1 (n = 6). The results for the sample S15_PG50 gelled with propylene glycol also fall within this range.The influence of starch concentration and solvent-to-DMSO mass ratio on the textural properties of aerogels was investigated, and the results are presented in 7.5_W70 and S7.5_W80. Visual inspection of images reveal that the aerogels present a net-like structure with nonuniform pore sizes and no sign of remaining granules. The structure is essentially similar to starch aerogels prepared by pasting at 140 °C followed by a retrogradation step at 6 °C [2 g−1) reported by other authors [2 g−1) have been reported by [ at 6 °C . The spe authors ,15,31,32orted by ,33, whictδ) or hydrogen bond interactions (hδ) will be carried out with the aim to find a gelation sphere for starch in the 3D Hansen space. This will provide information for predicting starch gelation in solvent systems that are miscible in sc-CO2 and would allow the direct drying of the gel. In addition, different starch types and amylose/amylopectin ratios could have an influence on the gelation profile of starch and will be considered in our future work with a long-term-goal to extend the list of biopolymers suitable for the water-free solvent-induced gelation.In this work, phase transitions from liquid to gel, in starch/DMSO mixtures, were systematically investigated as a function of starch concentration, type of solvent, and solvent-to-DMSO mass ratio. Based on the HSP and rheological measurements, it was demonstrated that hydrogen bonding and starch concentration play a crucial role on gel formation. We demonstrated that starch gels could be obtained from the solvent-induced gelation route, including a water-free system, and converted into aerogels. The resulting aerogels possessed comparably high surface areas and a well-connected fibrillar network, indicating that DMSO efficiently promoted the starch granules disruption. Further work evaluating other solvents with high values of total solubility parameters  was kindly provided by Ingredion . DMSO and anhydrous ethanol  were purchased from Carl Roth GmbH . Propylene glycol was provided by Sigma Aldrich . Glycerol was purchased from Merck . Sulfolane was obtained from Alfa Aesar  and 2-dimethyl ethanolamine was obtained from Fluka Analytical . Deionized water was used in all experiments. Carbon dioxide (CO2) was supplied by AGA Gas GmbH .Corn starch (Hylon VIIw/w). The solvents used in this study were selected by their capacity to form hydrogen bonds and included water, glycerol, propylene glycol, sulfolane, and 2-dimethyl ethanolamine.To investigate the gelation behavior of starch, several samples were prepared with different starch concentrations  and solvent-to-DMSO mass ratios . Then, the stock solution was stored at room temperature for 24 h to stabilize before use.Before the experiments, a stock solution of starch solubilized in DMSO was prepared according to the methodology of Han and Lim (2004)  with somThe samples were prepared as follows: the stock solution was mixed with pure DMSO and the solvent in the respective amounts to achieve a target starch concentration and a target solvent-to-DMSO mass ratio. The mixture was homogenized for 5 min at room temperature using magnetic stirring. After that, the solution was poured into glass tubes and stored at room temperature during 24 h for gel hardening.After the hardening period, the glass tubes were tilted and the flow behavior of the samples were evaluated and classified as liquid (fluidly flowed sample), thick liquid (slowly flowed sample), or gel-like (not flowed sample). Gel-like samples were further studied: they were again prepared as described in The gelation behavior of starch was investigated by oscillatory measurements using a rheometer Kinexus pro+ . Isothermal analyses were conducted for 2 h at 20 °C with a fixed frequency of 1 Hz and a constant deformation of 0.5% using a cone-plate geometry of 20 mm diameter and 0.5° angle. The gap was adjusted to 0.8 mm. Tests were performed at least in duplicate.t,mδ) was calculated according to Equation (1), where V is the volume fraction  were calculated similarly, according to Equations (2)–(4):The individual solubility parameters of the mixtures  for 6 h (first step) and then 18 h (second step) at room temperature. The solvent exchange was completed when the solution density presented a value near to pure ethanol density (0.789 g cm−3), which was monitored by using a density meter   [Aerogels in the form of monoliths were prepared as follows: gel monoliths  were sub., 2015) .2) using the equipment previously described by [2. The autoclave was kept at 50 °C using a heating jacket. Preheated CO2 (40–50 °C) was introduced to reach 12 MPa. The outlet micro-metering valve was adjusted to a continuous CO2 flow rate of 40 g min−1. Constant pressure of 12 MPa was maintained by feeding fresh carbon dioxide into the autoclave. The extraction was run for 3 h, followed by a depressurization at the same CO2 flow rate within 30–40 min [After the solvent exchange, the monoliths were dried with supercritical carbon dioxide , where The envelope density of aerogel samples was determined by their weight and volume with accuracies of ±0.0001 g and 0.005 mm, respectively. The specific surface area was determined by Brunauer–Emmett–Teller (BET) method using the nitrogen adsorption/desorption technique . Prior to measurements, samples were degassed at 40 °C under vacuum for at least 24 h. Internal morphology of the aerogels after sputtering with gold was analyzed by Scanning Electron Microscope (SEM)  operated at 5 keV.


Aortoenteric fistula is a rare complication following endovascular abdominal aortic aneurysm repair. However, there is a significant morbidity and mortality associated with this complication. Patients can present with gastrointestinal hemorrhage, fever, or nonspecific features of chronic infection. Extra anatomic bypass with complete graft explanation is the standard management. Endovascular repair of abdominal aortic aneurysms (AAA) is an alternative to open repair especially in high risk patients. Aortoenteric fistula (AEF) after open AAA repair is a well-known complication but this is rarely reported following endovascular repair. We present a 73-year-old male patient who underwent endovascular repair of an infrarenal AAA, which was complicated with an AEF and graft infection. He underwent axillary-bifemoral bypass and graft explanation but eventually succumbed due to multiple postoperative complications.A 73-year-old patient who was on follow-up for diverticulosis was incidentally detected to have an infrarenal AAA with a maximum anteroposterior diameter of 5.5 cm. He was on treatment for type 2 diabetes, hypertension, and hypercholesterolemia. In 2016 July, he underwent endovascular repair of the aneurysm using a bifurcated aortic stent graft system. His recovery was uneventful, and he was discharged 3 days after the procedure. Afterwards, he was lost to follow-up.In 2019 August, the patient was readmitted to the hospital with severe malaena. No source for acute bleeding was detected on upper and lower GI endoscopy.In 2019 October, the patient developed fever with chills and rigors and required another hospital admission. Blood investigations showed neutrophil leukocytosis with high CRP and ESR. An exhaustive search for the source of infection was done but all the investigations were negative. Despite broad spectrum antibiotics, the fever persisted, and the inflammatory markers remained elevated.During the same hospital stay, he developed sudden onset pain and weakness of the left lower limb with absent popliteal and pedal pulses. Acute limb ischaemia (ALI) was suspected, and a contrast CT of the abdomen and a CT angiogram of the lower limbs were done. There was a localized filling defect seen in the left popliteal artery with poor distal run off. The CECT abdomen showed a 5.7 cm size infrarenal AAA with an intraaortic stent graft. There was multiple gas loculi within the sac of the aneurysm . ContrasInitially, he underwent a left popliteal embolectomy to restore limb perfusion. Culture of the retrieved embolus yielded a growth of coliform organisms. This was indicative of septic embolism from the infected aneurysm sac.rd part of the duodenum and the infrarenal aorta. The tract appeared to communicate with the true aortic lumen which was not covered by the endograft. Clamps were placed on bilateral common iliac arteries and the infrarenal aorta. A longitudinal aortotomy was made. The iliac limbs of the graft were pulled out. The hooks of that anchor the proximal graft to the aortic wall were cut using scissors, and the proximal graft was pulled out  bleeding endoscopy is the usual first line investigation but a negative study does not rule out a possible AEF. In the MAEFISTO study, 25% of the patients had negative UGI endoscopy findings . HoweverContrast CT is the first imaging study done on most of the patients who are suspected with this complication. CT scan clinches the diagnosis in 33-80% of the patients , 5. CT wMultiple aetiologic mechanisms have been proposed as to how a patient may develop an AEF following EVAR. The most likely possibility is erosion of the aortic wall by the stent graft. Stent migration, fracture, and kink may exert pressure on the aortic wall leading to its erosion. The fixing hooks of the graft can directly damage the bowel wall .rd part of the duodenum and his native aorta which was not covered by the stent graft so theoretically, we can consider this as a primary AEF due to type II endoleak.Progressive sac expansion due to endoleak or endotension can result in erosion of the aorta and the adjacent bowel. Some authors have considered fistulae occurring by this mechanism as primary AEF.  Our patiAEFs have been reported following coil embolization for endoleaks and erosion of the coils in to the duodenum that has been cited as the possible mechanism in this setting . FistulaManagement of AEF following EVAR is challenging, and it has a mortality rate of 50-100%. The complexity of the surgery itself, systemic sepsis, and the poor physical condition in the majority who undergo surgery has been cited as causes for this high mortality. Most of the patients require supracoeliac or suprarenal aortic clamping with a great toll on the physiology . Due to Complete graft explanation with extra anatomic reconstruction is the gold standard of management . AxillarOperated and conservatively managed patients require antibiotics preferably directed by culture sensitivity. A minimum duration of 8 weeks is recommended. Otherwise, antibiotics should be generally continued until clinical and laboratory parameters of infection have subsided. Lifelong antibiotics are recommended in conservatively managed patients, those with incomplete graft explanation and highly virulent organisms .AEF after EVAR is a rare complication. However, the associated morbidity and mortality are extremely high. Conservative management with long-term antibiotics is an option in stable patients with a frail physiology but the results are disappointing. Operative management with graft explanation, vascular reconstruction with extra anatomic bypass, or in situ graft are the best management options. Patients usually require long-term antibiotics and lifelong close surveillance.