Hundreds of people are killed or injured annually in the United States in accidents related to ice formation on roadways and bridge decks. In this paper, a novel embedded sensor system is proposed for the detection of black ice as well as wet, dry, and frozen pavement conditions on roads, runways, and bridges. The proposed sensor works by detecting changes in electrical resistance between two sets of stainless steel poles embedded in the concrete sensor to assess surface and near-surface conditions. A preliminary decision algorithm is developed that utilizes sensor outputs indicating resistance changes and surface temperature. The sensor consists of a 102-mm-diameter, 38-mm-high, concrete cylinder. Laboratory results indicate that the proposed sensor can effectively detect surface ice and wet conditions even in the presence of deicing chlorides and rubber residue. This sensor can further distinguish black ice from ice that may exist within concrete pores. Surface ice  is a thin layer of frozen water that can form on the roadway surface. This transparent layer is commonly referred to as black ice because the pavement surface can be seen while the ice may not be visible. Black ice can typically form when moisture  comes in contact with a surface that has a temperature below freezing. Black ice affects aircraft on runways and taxiways, cars and trucks on the roads, and pedestrians and cyclists on sidewalks or walkways. As a layer of ice forms on a surface, the contact friction decreases drastically. This condition significantly increases the slippage hazard for pedestrians and vehicular traffic. Surface ice needs two main components to form: moisture and low temperatures. There are many potential sources of moisture for the formation of black ice such as rain, snow, hail, accidental water discharge, sleet, freezing fog, or blowing and drifting snow . Black iwww.icyroadsafety.com, black ice causes more weather-relevant deaths and injuries annually than all other severe weather conditions combined [According to combined . Ice hazcombined ,4,5.Current methods for the detection of ice on roadway surfaces include several non-embedded and embedded sensing systems. Non-contact sensing systems typically employ infrared technologies and use cameras that are mounted on elevated supports to assess their surrounding areas ,7. ThereIn general, the systems described above typically contain complex and hard-to-maintain hardware and software components. Existing embedded systems involve utilization of sensing materials that are different from the surrounding material. In such cases, there is potential for a thermal island effect . Furthermore, there is no distinction made between ice that may exist within the near-surface pores of the concrete material without surface ice (herein referred to as “frozen” condition) and the surface ice condition. High initial and long-term costs, complexity, and accuracy issues have prevented the wide-spread use of such systems on road networks nationwide.The primary objective of this research program was to develop a new low-cost embedded sensor system for the detection of surface ice, water, frozen pavement, and dry conditions on roadways, bridges, and runways. The proposed methodology is based on near-surface and surface resistance measurements in the concrete sensing material as well as surface temperature measurements. To achieve the research objective, a set of laboratory tests was performed on two prototype sensors to verify the sensor’s effectiveness in detecting various environmental surface conditions . A preliminary set of algorithms is proposed to determine the surface conditions based on the laboratory tests performed to date. Concrete was used as the sensor material in this initial development of the proposed ice sensor because surface ice can develop more rapidly on bridge decks compared to the approaching roadway, and most bridge decks in the U.S. have exposed concrete surfaces. Concrete pavements are also prevalent on U.S. roads and runways. It is envisioned that a similarly designed sensor can be developed for asphalt surfaces. However, this development was not within the scope of the study reported here.The proposed intelligent sensor system is designed to offer the following advantages: ability to detect/distinguish a variety of surface conditions; relatively low cost; simple detection technology; suitability for mass production; and potential for a variety of wireless or wired hazard communications through local warning signals, web site information, transmission to autonomous vehicles, etc.In this paper, a novel low-cost embedded sensor system is proposed that can detect surface ice, wet, dry, or frozen (ice in concrete pores) conditions on concrete roadways, runways, and bridge decks. This system utilizes measured changes in near-surface electrical resistance of concrete and the concrete surface temperature to determine surface conditions. Unlike other embedded sensors for ice detection, this sensor has a sensing material (concrete) that is compatible with the surrounding concrete and thus does not create a thermal island on the roadway or bridge deck.The proposed sensor takes advantage of the fact that near-surface resistivity of concrete changes significantly when there is an ice layer or moisture at or near the surface. This sensor is designed to detect ice as it starts to form. The sensor could be designed to transmit warnings and other information including its location, surface temperature, and indications of black ice, frozen condition (ice in near surface concrete pores without surface ice), dry surface, or wet conditions.The detection algorithm is based on measurements of electrical resistance of concrete (between embedded poles) and surface temperature measurements. The sensor is in the shape of a cylinder made with a concrete mix that is similar to those used in roadway pavements or bridge decks. It should be noted that the sensor is designed such that typical variations in concrete mix designs (concrete properties) could not mask the effects from ice or water conditions in the sensor output. Electrical resistance is measured between two sets of stainless steel poles embedded in concrete. Resistance measurements are made using two unbalanced Wheatstone bridge circuits (one for each set of poles). Two different types of poles are used:“Look-Up-and-Side” (LUS) poles measure near-surface (within 3.2 mm of surface) electrical resistance changes in the concrete between the surfaces of the two poles in direct contact with concrete (“Sides”) as well as surface ice that may exist above the surface (“Up”).“Look-Up” (LU) poles measure resistance changes directly above the surface of the concrete between the two poles. Under dry conditions, the resistance between LU poles is theoretically infinite as the space above the surface between LU poles could consist of air only.The LUS pole was the only pole type used in the first prototype of the sensor. However, as discussed later, test results indicated the need for an additional type of pole (LU) in the sensor to reduce uncertainties under certain exposure conditions.The proposed cylinder-shaped concrete sensor has a diameter of 102 mm and a height of 38 mm and includes a 76-mm-diameter opening at the bottom of the sensor, as shown in The LU poles are included to address the ice formation on the top of their cross-sectional area only, since they are electrically exposed from the top only . On the other hand, LUS poles are electrically exposed to changes in the near-surface concrete resistance as well as changes above the surface as shown in In addition to serving as resistance measurement poles, the stainless-steel rods are also used for making electrical connections to the circuit board (instead of wires). This is done to eliminate the potential for long-term corrosion of conventional wires. The circuit board and other electronic components are housed within the sensor. They include two separate Wheatstone bridges  to avoid moisture penetration. A cover is used to seal the opening and prevent possible damage to the electrical circuits.The proposed sensor can be used in new constructions or retrofitted on existing roads or bridge decks. The installation (embedment) of this sensor in existing concrete pavements or bridge decks involve using a 102-mm-diameter core drill bit to remove a core and replacing it with the concrete sensor. This is a common diameter for cylinders used in quality control tests to determine the concrete’s compressive strength. Coring machines with this bit size are widely available and accessible to concrete contractors, thus making the field installation relatively easy. A portland-cement-based mortar (regular or fast-setting) should be used to bond the concrete sensor to the existing concrete pavement or bridge deck to avoid any thermal incompatibility or thermal island effect.There are several options available to the installer to provide a flat transition between the existing surface and the sensor. One such option involves the temporary attachment of a mounting (or leveling) plate on the top surface of the sensor during installation and hardening of the cement mortar . The mouThe concrete mix used to make the sensor prototypes consisted of the following: 15.6%  Portland cement (Type I), 31.2% sand, 46.9% pea gravel, and 6.3% water. The water/cement ratio was 0.4, as specified by the Wisconsin Department of Transportation’s standard specifications for concrete Grade A. No other admixtures or additives were used. The specimens were stripped from their plastic mold two days after casting of the concrete and were then wet cured in the laboratory for 7 days. SP-I was the first sensor prototype used in this research. SP-I was similar to the proposed sensor described earlier except that all four poles used were LUS poles . Additionally, the diameter of the opening on the bottom of the sensor was slightly smaller than the proposed sensor. Four stainless steel nuts were embedded in the sensor mold prior to casting of the concrete. These poles were flush with the top surface of the sensor. Electrical wires were placed in the nuts before tightening the threaded rods. Unlike the proposed sensor that incorporates stainless steel poles for electrical connections, common electrical wires were used to connect the nuts (poles) to the Wheatstone bridge in the SP-I prototype.This sensor prototype had the same external dimensions as the SP-I with a 76-mm-diameter opening at the bottom. Two 6–32 stainless steel threaded rods with 6–32 stainless steel nuts were used as LUS poles similar to those used in SP-I. Two 8-mm-diameter stainless steel threaded rods were used as LU poles. The LU poles were included to better distinguish the frozen condition from surface ice, especially when the surface is contaminated with deicing chlorides and rubber shavings. All poles were insulated with a layer of epoxy paint and two layers of electrical shrink tubing throughout the embedded part of their length. This was meant to electrically isolate the LU poles from the concrete throughout the exterior surface of the pole .In SP-II, stainless steel rods were used in lieu of wires that were used in SP-I. Stainless steel rods were chosen to address the long-term corrosion problem caused by exposure to moisture.As discussed earlier, the output of an unbalanced Wheatstone bridge was used in this research as an indicator of resistance changes between two opposite poles in each sensor. The resistance between the two sensor poles was included as a leg of the Wheatstone bridge shown in The Wheatstone bridge has been used to measure resistance changes in a wide variety of different applications including load cells and strain gages. It offers a passive low-cost option to assess fluctuations in electrical resistance. 1, R2, and R3). Rx was the resistance of concrete being determined. The output voltage (VG) of an unbalanced Wheatstone bridge can be calculated using the following equation: In a Wheatstone bridge, there are four legs, each consisting of a resistor. Three of these were constant precision resistors (R1 thru R3) used in the prototypes were as follows: Vs = 6 VDC; R1 = 150 kΩ; R2 = 100 kΩ; R3 = 10 MΩ. This choice of resistors was made after many trials to achieve a desired wide range of output for various conditions, in which high-resolution voltage measurements were not needed. With this choice of resistors and an excitation voltage of 6 V, the lowest and highest sensor output voltages ranged from approximately −2 (for fully wet conditions) to under +2 V for fully dry conditions.The excitation voltage (Vs) and resistors on the three known legs of the Wheatstone bridge  was useTests on five different surface conditions were conducted. These include dry surface, wet surface, icy surface (black ice), frozen surface , and frozen-icy surface (combination of frozen and black ice conditions). A 457 mm by 457 mm concrete slab surface was used for this test as shown in For the frozen test, the slab was placed face down in a water-filled tray for 24 h. The specimen surface was then wiped to remove free water, and was then placed in the freezer for another 24 h. The concrete specimen was tested again to determine the BPN under frozen conditions. Finally, the process for the frozen condition was repeated, but this time water was sprayed on the surface of the concrete slab, and a frozen-surface ice condition was created for testing. Six readings were taken for each condition, and an average value was calculated.Results indicate that the presence of ice inside concrete pores without surface ice poses a far less safety risk than the surface ice condition. This demonstrates the importance of distinguishing the two forms of ice in the sensing system.1, R2, R3, and VG, the values of Rx could be calculated. The development and verification aspect of this work involved building and testing two sensor prototypes (SP-I and SP-II). These two prototypes were used to generate test data for the development of the preliminary decision algorithm and to establish the feasibility and effectiveness of the proposed sensor concept through laboratory testing. A Wheatstone bridge circuit built on breadboard was assembled for testing purpose. Using Equation (1) and knowing the values of RTo understand and determine the possible range of resistance, tests were performed under different environmental conditions. In all tests described below, the sensor output was monitored every few minutes as the surface temperature increased from below freezing temperatures up to room temperature.G. The following environmental conditions were tested:In the laboratory experiments, the surface temperature was monitored using an infrared non-contact temperature sensor even though the actual sensor in a field installation would have an embedded temperature sensor at the surface. This was done so that multiple measurements could be made at different locations and averaged. The recorded temperature was the average of 4–6 readings at different spots on the surface of the sensor. The temperature was measured along with VFor DR tests, the sensor was first placed in an oven to be dried for approximately 24 h at a temperature of 48 °C. After that, the sensor was taken out of the oven and allowed to cool down. Subsequently, it was placed in the freezer for 24 h at a temperature of −20 °C. The sensor was taken out of the freezer and connected to the Wheatstone bridge to monitor the output of the sensor. For the SP-I prototype (with LUS Poles), the dry sensor was placed in a freezer (−20 °C) overnight. Just prior to the beginning of testing, the sensor was removed from the freezer and sprayed with room-temperature water from a spray bottle. The surface ice was then formed , and meaFor the SP-II prototype (with LU Poles), a similar procedure was used for the SI tests. The sensor was dried out by placing it in the oven with temperature of 35 °C for 24 h. The sensor was then put in the freezer for another 24 h. While still cold, water was sprayed on its surface to form surface ice. The LU poles were connected to the electrical circuit to monitor the changes in the resistance associated with the changes in the temperature.For tests on the SP-I prototype (LUS poles), the top of the sensor was immersed in water for a few hours to allow its near-surface pores to be filled with water. Subsequently, the sensor was removed from water and the excess surface water was wiped off. The sensor was then placed in the freezer (temperature of −20 °C) overnight to allow water in the pores to freeze. The sensor was removed from the freezer just before testing. Unlike the SI test, water was not sprayed on the surface. This condition represents the situation in which the pavement is saturated before freezing without formation of surface ice. Temperature and sensor measurements were taken until the temperature of sensor reached the room temperature. It should be noted that the FR test would also provide results under wet conditions. As the temperature increases, the ice in the pores changes to water.For tests on the SP-II prototype (LU poles), a slightly different procedure was used. The LU poles were first covered with electrical tape to avoid contact with moisture. Water was sprayed on the surface several times for about an hour to make sure the pores were filled with water (without standing water). The sensor was then put into the freezer for 24 h, and then moved back to the lab to monitor the change in output voltages as temperature changed. This condition is a combination of the SI and FR conditions described above. The FR procedures were followed, but, after removal from the freezer, room-temperature water was sprayed onto the top surface to form a surface ice layer on top of the frozen surface.There were several other tests performed on the SP-I prototype to address the potential of surface contamination due to deicing salt or rubber residue from tires. Finally, a test involving the placement of crushed ice on the sensor was performed to simulate the effects of unbonded ice (not fully adhered to concrete) and snow. Finally, the resistance of a thin ice layer (without concrete) was also measured.In this test, the sensor was placed in the freezer for about 24 h. Subsequently, it was moved to the laboratory and its surface was sprayed with water to form surface ice. The test was started and readings were taking until the temperature increased up to −4 °C, then saltwater (SW) was sprayed on the sensor, and readings were taken as the temperature increased. The saltwater was a 6% NaCl solution at room temperature.The SP-I prototype was submerged in water for about 2–3 h, and then placed in the freezer for 24 h. The sensor was already contaminated with salt due to the previous test. Readings were taken after the sensor was removed from the freezer and connected to the Wheatstone bridge. Readings were taken as the temperature increased from −15 to −10.5 °C, after which saltwater was sprayed on the surface.In the previous test, saltwater was sprayed onto the sensor, and that caused the sensor to be contaminated with chlorides. As sodium chloride penetrates the concrete pores, the output voltage and resistance are reduced. In this test, a condition when the sensor is previously contaminated with chlorides was simulated. The dry sensor was placed for 24 h in the freezer with a temperature of −20 °C. The sensor was then moved to the laboratory and was connected to the Wheatstone bridge circuit. Water was sprayed on its surface to create an icy surface. Two tests were done using this approach.Rubber residue from passage of tires can be present on the roadway surface. In this test, an attempt was made to simulate the condition where there is contamination due to rubber residue on the surface of the sensor. Powdered rubber (from an unrelated wear test on concrete) was rubbed on the surface of the sensor prior to this test. This sensor was previously contaminated with chlorides as well. The SI procedures were then followed.The SP-I prototype was placed inside in the freezer (−20 °C) for 24 h. While it was in the freezer, crushed ice was placed on the top surface of the sensor. The sensor was then moved to the laboratory inside an insulation box to slow the temperature increase. The sensor output voltage was monitored as the temperature increased and ice melted.To better understand the electrical properties of the ice at different temperatures, tests were performed on an ice layer without concrete. A plastic container, which had served as a mold for casting of concrete sensors, was filled with 1.6 mm (1/16 in) of water. Stainless steel poles were placed in the same arrangement as those used in the sensor. S = 6 V) was on the order of 1.75 V (or resistance of 22–23 MΩ).Three DR tests were performed on the SP-I sensor . The sensor was dried in the oven for 24 h prior to DRI-1. In tests DRI-2 and DRI-3, the sensor was not dried in the oven before testing. The sensor was then placed in the freezer for 24 h. In the DRI-2 test, the condensation affect was larger (degree of condensation was related to the humidity of the laboratory). The dry resistance was again on the order of 22–23 MΩ. The DRI-3 test did not differ significantly from DRI-2, except for a more pronounced condensation effect. It should be noted that, at cold temperatures below −8 °C and at warmer temperatures above +8 °C, the voltage output for the three DR tests were similar and approximately equal to 1.75 V. The DRI-1 results began to diverge from DRI-2 and DRI-3 within a temperature range from −6 to +10 °C. The presence of moisture due to condensation drives the output voltage and resistance to lower levels. As the moisture disappears at higher temperatures, the output voltage and resistance values for all DRI tests converge again.This test was conducted to understand the effect of the presence of frozen water in the concrete pores and to see how the change from pore ice to pore water would affect the electrical resistance between poles. The voltage and resistance results are shown in x was 23 MΩ.The resistance of concrete is related to the presence of moisture in the pore structure. Therefore, the measured resistance and voltage both decrease as water content increases. Voltage output for frozen concrete ranged between +0.1 and −0.8 V (7.0–3.5 MΩ), while moist concrete output voltage was on the order of −1.4 V (2.0 MΩ). The wide gap in measured voltage and resistance values from dry to wet conditions indicates that typical variations in concrete properties may not be sufficient to mask the effects from ice or water. Spragg et al.  report cSurface ice is one of the most dangerous pavement condition as indicated in the friction tests reported earlier. The focus of interest in this research, therefore, was on proper detection of surface ice conditions.The voltage (resistance) and surface temperature readings for the SI-I tests (LUS poles) are shown in In the first test on the SP-II prototype (LU poles), the output voltage and resistance was monitored as the surface temperature of the sensor increased from −11.5 to 14 °C. The first four readings were taken while the sensor was cold and dry without moisture on its surface. Water was then sprayed on the surface and surface ice started to form . Ice forThe SI-SW I-2 test was performed on the same sensor following conclusion of the SI-SW I-1 test. Therefore, it is thought that the deicing salt used in the first test was absorbed by the concrete and stayed in its pores. This caused the sensor to be contaminated with salt, which caused a difference between results of SI-SW I-1 and SI-SW I-2. For the temperature above 2 °C, the SI-SW I-1 test showed the normal trend of concrete having melted ice on its surface. Test SI-SW I-2, however, shows the effect of salt contamination within the concrete pores and on the surface where voltage and resistance were lower prior to saltwater spray.In the FR-CC test, there was a rapid increase in temperature and a rapid decrease in output voltage because of the addition of saltwater. At temperatures above 0 °C, the output voltage was on the order of −2.0 V, and the resistance was on the order of less than 1.0 MΩ. The lower resistance under wet conditions is due to chloride contamination. The presence of salt affects the conductivity of concrete and the overall resistance decreases. However, there is still a significant difference in output voltage between surface ice and wet condition under chloride contamination. This allows the sensor to work even in chloride-contaminated environments. Nevertheless, the presence of salt affects the output, and this is considered in the preliminary decision algorithm proposed for the sensor.It is thought that the difference in the results of the two tests may be due to the amount of chloride contamination of the sensor in the two tests. As the ice fully melts, and temperature increases up to the room temperature, resistance and voltage results for both tests (SI-CC I-1 and SI-CC I-2) fall within a range from −1.75 to −2.0 V (≈1 MΩ).In the SI-RC tests, since the sensor was already contaminated with salt from previous tests, the response was like the chloride-contaminated tests described earlier. Overall, the rubber shavings on the sensor surface did not appear to change the results drastically. Outputs stayed within the range of icy surface results in clean or chloride-contaminated tests.In the crushed ice test, as surface temperature increased (crushed ice melted), the output voltage and resistance decreased. At lower temperature, the output voltage level was similar to the dry condition even though there was crushed ice on the surface. It is believed that, due to the voids between the crushed ice/snow particles (ice not fully adhered to concrete), the electrical resistance will not be similar to that under the surface ice condition. In fact, the output voltage was close to the that of the dry condition when the crushed ice had not melted. This pattern could be seen in the temperature range from −10 to −3 °C. As temperature increased and ice converted into liquid form, water became ice again because of the cold sensor. The output voltage reached −0.94 V (resistance ≈ 3.0 MΩ) for temperatures above 1 °C.Tests on the ice layer alone (without concrete) showed that the ice resistance has a linear relationship with temperature. This was seen in surface ice tests on the sensor as well. As the thickness of ice increases, the output voltage decreases.The presence of chlorides did not affect the overall shape of the voltage–temperature response. However, it drove the resistance and voltage values lower. Therefore, a range of voltage or resistance should be found for the sensor’s output that would include the effect of salt presence and the surface condition that is associated with it . There was also no interference between the conditions of interest  and the dry condition under the same temperature or range of temperatures. There was, however, one possible interference area between surface ice and frozen conditions when chloride contamination was considered. Since tests indicated that friction values for the frozen surface was only slightly lower than those under the dry condition (and much higher than those under the surface ice condition), the frozen condition should be clearly distinguished from surface ice or frozen-surface ice conditions. Friction for the frozen-surface ice combined was the worst (lowest), so the decision algorithm should be designed to fully separate various conditions. Since the LUS pole results could fall within the zone of interference for salt-contaminated surfaces, the LU poles were developed to conclusively separate the two conditions.When surface temperature is above −2 °C and the output voltage is within a range from −2.25 to −0.75 V, the surface condition is wet. As the output voltage approaches −2.25 V, the wetness increases.The proposed sensor system can be further developed through field testing and enhancement of the detection algorithm. In this study, the sensor output was related to surface and near-surface conditions . In the future, the sensor output could potentially be used for determining surface friction as a direct measure of safety risk. Durability assessments should also be made, both in terms of long-term concrete wear and freeze–thaw scaling, and with respect to battery life. Various communication and transmission systems including those intended for local warning, for traffic operations centers, and for autonomous vehicles could be developed in the future. A similar sensor system for asphalt pavements should also be considered in future sensor development work. This research aimed to develop a novel sensor system for detection of ice and water conditions on roadways, runways, bridge decks, sidewalks, and other surfaces. Several tests were performed using two sensors prototypes under different surface conditions  to simulate the effect of weather conditions on sensor output. The resistance across two sets of stainless steel poles, embedded at the surface of the concrete sensor, was monitored to develop a decision algorithm based on measurements of surface temperature and electrical resistance. Laboratory results have shown that the sensor output could be an effective basis for detection and reporting of surface ice, frozen concrete, and wet conditions.Two sets of poles were used: the LUS (Look-Up and Side) poles and LU (Look-Up) poles. Information obtained from both pole types were used to detect and confirm various conditions. Moisture presence drives the resistance (output voltage) lower, and the thickness of the ice layer affects the resistance (resistance decreases when the thickness of the ice increases). On the other hand, chloride contamination from deicing salt reduces the resistance of the concrete.The results of several tests conducted on LUS and LU poles under frozen and surface ice conditions indicated that these two conditions are distinguishable. A decision algorithm based on the measured surface temperature and output voltage of both sets of poles has been developed. The findings of friction tests indicated that the most dangerous conditions (from the friction standpoint) are surface ice and a combination of frozen concrete and surface ice conditions.The decision algorithm is designed to distinguish and identify surface conditions and to issue different warning levels accordingly. The proposed decision algorithm considers the effects of salt contamination on the roadway surface.The developed sensor system would have two embedded Wheatstone bridge circuits, a long-term battery, a logic controller, and a local-area signal transmission capability. The signal could either be used for local site warning signal/lights/messages or be relayed to a transportation control center, displayed on a web site, or communicated to vehicle information systems. The sensor could also transmit coordinates of its location. The warnings could be received by drivers, transportations authorities, and vehicle control systems to warn drivers of surface ice formation.Ultimately, the findings of this study could be used in many different fields and applications. The proposed sensor relies on low-cost and simple technologies that could be applied on a mass scale.


Some can be used for diagnostic purposes; specific IgE to allergen extracts is widely available; specific IgE to allergen components is used in most specialist centers, and the basophil activation test is becoming increasingly used clinically. IgE to allergen peptides, T-cell assays, allergen-specific/total IgE ratios, and allergen-specific IgG4/IgE ratios are currently reserved for research. Different factors can modulate the likelihood of IgE-mediated food allergy of a given allergy test result, namely, the patients' age, ethnicity, previous allergic reaction to the identified food, concomitant atopic conditions, and geographical location, and need to be taken into account when interpreting the allergy test results in the clinic. The importance of the specific food, the clinical resources available, and patient preferences are additional aspects that need to be considered when deciding whether an oral food challenge is required to reach an accurate diagnosis of IgE-mediated food allergy.Various  Credit can now be obtained, free for a limited time, by reading the review articles in this issue. Please note the following instructions.Method of Physician Participation in Learning Process: The core material for these activities can be read in this issue of the Journal or online at the JACI: In Practice Web site: www.jaci-inpractice.org/. The accompanying tests may only be submitted online at www.jaci-inpractice.org/. Fax or other copies will not be accepted.Date of Original Release: March 1, 2017. Credit may be obtained for these courses until February 28, 2018.Copyright Statement: Copyright © 2017-2019. All rights reserved.Overall Purpose/Goal: To provide excellent reviews on key aspects of allergic disease to those who research, treat, or manage allergic disease.Target Audience: Physicians and researchers within the field of allergic disease.Accreditation/Provider Statements and Credit Designation: The American Academy of Allergy, Asthma & Immunology (AAAAI) is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. The AAAAI designates this journal-based CME activity for 1.0 AMA PRA Category 1 Credit™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.List of Design Committee Members: Alexandra F. Santos, MD, PhD, and Helen A. Brough, MBBS, PhDLearning objectives:1.in vitro tests for diagnosing food allergy and their diagnostic performance.To describe the different 2.To analyze the results of allergy tests to determine the likelihood of clinical allergy.3.To explain the factors that influence the decision to perform an oral food challenge.Recognition of Commercial Support: This CME has not received external commercial support.Disclosure of Significant Relationships with Relevant Commercial Companies/Organizations: A. F. Santos has received research support from the Medical Research Council , Immune Tolerance Network and National Institutes of Health, and the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust; has received lecture fees from Thermo Scientific, Nutricia and Infomed; has received travel support from European Academy of Allergy and Clinical Immunology (EAACI), British Society of Allergy and Clinical Immunology (BSACI), Academy of Medical Sciences, Portuguese Society of Allergy and Clinical Immunology (SPAIC), Spanish Society of Allergy and Clinical Immunology (SEAIC), and French Meeting of Molecular Allergology. H. A. Brough has received consultancy fees from MEAD Johnson Nutrition and Nutricia; has received research support from the Food Allergy Research and Education Charity grant, Immune Tolerance Network, and National Institutes of Health; has received lecture fees from MEDA Pharmaceuticals, Thermo Scientific, and Nutricia; receives royalties from Wiley Blackwell Ltd; has received travel support from British Society of Allergy and Clinical Immunology, MEDA Pharmaceuticals, Thermo Scientific, and Nutricia; and has received research support from Thermo Scientific, Stallergenes, and Meridien Foods.Food allergy (FA) is an adverse reaction caused by an abnormal response of the immune system to food allergens. Food allergies are classified based on the involvement of IgE antibodies in their pathogenesis.de novo synthesis of leukotrienes and cytokines, which contribute to the symptoms that patients experience during allergic reactions.in vitro assays reflect different aspects of the immunologic mechanisms of IgE-mediated FA. For instance, the amount of circulating allergen-specific IgE antibodies can be determined using immunoenzymatic assays, and basophil activation and T-cell proliferation in response to allergen can be assessed using flow cytometry . An OFC is the most accurate test to diagnose FA but requires expensive resources, highly trained personnel, and carries the risk of causing an acute allergic reaction. Therefore, in clinical practice, the diagnosis of FA is based on a combination of the clinical history and the results of allergy tests when possible. The clinical history, including the allergic reaction(s) to the culprit food and the dietary history, is the cornerstone of the diagnosis of FA; it guides the selection of allergens to be tested and the interpretation of allergy test results. In this review, we discuss the main in vitro tests for FA and how to make the most of these tests to decide whether an OFC is required to reach an accurate diagnosis of FA.Various ytometry . Some ofSpecific IgE (sIgE) testing has been used to diagnose FA for many years. Automated systems permit the use of enzymatic immunoassays for a large number of samples in a standardized way; however, levels determined with different methodology may not be comparable.Using the cutoff of 0.35 kU/L, sIgE testing has high sensitivity but poor specificity to diagnose FA. For example, in the case of peanut allergy (PA), sIgE to peanut has a sensitivity of 75% to 100% and a specificity of 17% to 63%.Diagnostic cutoff values can vary widely in different studies. For instance, the 95% PPV cutoff to diagnose PA was 15 kU/L in USConventional IgE testing uses natural extracts containing a complex mixture of proteins. Allergen sIgE to component allergen tests for IgE binding to single allergens, allowing more precise profiling of the allergen-sIgE repertoire. The list of allergenic molecules available for testing is not complete; thus IgE assays using extracts are likely to be useful for some time. sIgE testing to components is available for single allergens and for multiple allergens in microarrays. Multiplex assays may introduce concerns where they reveal sensitization to molecules with potentially no clinical relevance as they are all tested independent of the patient's history. However, multiplex assays can be useful in identifying patterns of sensitization in complex polysensitized patients  and in identifying the culprit allergen in patients with recurrent anaphylaxis.23The food that has received the most research into component allergens and their validation in terms of clinical relevance is peanut. The number of identified peanut allergens is extensive although not all of these are available for testing in clinical practice . The immOf the tree nuts, hazelnut has received the most extensive evaluation leading to the identification of seed storage proteins  as well as cross-reactive proteins  as allergens. sIgE to whole hazelnut has a poor predictive value for clinical reactivity due to cross-reactivity with birch pollen. Birch pollen-associated hazelnut allergy is the dominant phenotype, although Cor a 9 and 14 are the allergens more commonly associated with systemic reactions .27, 28, Casein (Bos d 8), beta-lactoglobulin (Bos d 5), and alpha-lactoglobulin (Bos d 4) are the major allergens in cow's milk. Sensitivity to various cow's milk proteins is widely distributed; thus generally no single allergen is considered to be immunodominant.The main hen's egg allergens are ovomucoid , ovalbumin , conalbumin , and lysozyme .in vitro test or other component allergens.Component-resolved diagnosis (CRD) of wheat allergy has gained interest as wheat extract IgE testing has a poor predictive value. The major wheat allergens relevant for FA (rather than Baker's asthma) are glutens that can be subdivided into gliadins  and glutenins (high molecular and low molecular weight). The role of omega-5-gliadin (Tri a 19) in wheat-dependent exercise-induced anaphylaxis has been shown in several studiesAllergens predictive of systemic reactions to soya include the seed storage proteins Gly m 5, 6, and 8. Gly m 5 and 6 predicted systemic allergic reactions to soy (with both positive Gly m 5 and 6 giving an odds ratio of 12 for severe reactions) more than the Bet v 1 homolog Gly m 4.To try to improve the diagnostic performance of food sIgE, the added value of total IgE and food-specific IgG4 has been tested in food-specific/total IgE ratios or food-specific IgG4/IgE ratios. Some studies showed an improvement in the prediction of OFC outcome with specific/total IgE ratios compared with sIgE alone,Food-specific IgG4/IgE ratios have been determined in various studies, but their diagnostic utility has not been established. Sensitized-tolerant children tend to have higher allergen-specific IgG4/IgE ratios than allergic children. For instance, peanut-sensitized tolerant patients have a higher peanut-specific as well as Ara h 1-, Ara h 2-, and Ara h 3-specific IgG4/IgE ratios compared with peanut allergics.The basophil activation test (BAT) is a functional assay that uses live basophils in whole blood to detect the ability of IgE to mediate activation of basophils after stimulation with allergen. It goes beyond the detection of IgE binding to allergen to test IgE function, which depends not only on the allergen-sIgE levels but also on IgE epitope specificity, affinity, and clonality.The main added value of the BAT in the diagnosis of FA compared with tests routinely used in clinical practice, such as SPT and sIgE to allergen extracts, is its enhanced specificity with often conserved sensitivity. For instance, the BAT to peanut showed 98% sensitivity and 96% specificity to diagnose PA, with the specificity reaching 100% in a subsequent validation. The specificity of the BAT ranged between 77% and 100% in other studies .11, 67, The BAT requires fresh blood and uses flow cytometry for which appropriate equipment and trained personnel are needed. It is anticipated that the BAT is reserved for selected cases where the results of routinely used tests do not allow a precise diagnosis. Indeed, in the previously mentioned study,As any other diagnostic test, the BAT cannot be used in isolation to diagnose FA. The results of the BAT need to be considered in light of the clinical history. In addition to patients with a negative BAT or nonresponder basophils, patients with BAT results that are discordant with the clinical history require an OFC to confirm or refute the diagnosis of FA.IgE specificity can be refined further by determining the allergen epitopes to which IgE binds. This has been evaluated using short linear allergen peptides of 15 to 20 amino acids bound to a solid phase  using immunofluorescence. Beyer et alSimilar methods have tested the utility of IgE to allergen peptides in diagnosing and in predicting the resolution of other food allergies.T-cell responses are central to the development of oral tolerance in nonallergic individuals and to the development of the allergic immune response in allergic individuals. Peanut allergic individuals have been shown to have greater proliferation of their T cells when their PBMCs were stimulated with whole peanut or individual major peanut allergens.in vitro after in vivo allergen exposure during the OFC. This is consistent with studies in mouse models.Food allergic patients may also have impaired regulatory T-cell function in response to specific food allergens. Dang et alThe tests available for routine use in the clinic can vary, with some practices using mainly SPT, others mainly sIgE, and others both. sIgE to allergen components is used in most specialist centers and the BAT is becoming increasingly used clinically. The other tests described in the previous section are reserved for use in the research setting, namely, peptide microarrays and T-cell assays.The ultimate goal of the allergy test result is to determine the probability of clinical allergy; this is then used to decide whether an OFC is warranted.The clinical relevance of a given allergen sIgE result can vary depending on the age of the patient, with lower levels of sIgE having increased clinical relevance in younger patients.95Diagnostic decision levels may be affected by the patients' ethnicity. Black race is associated with a higher prevalence of sensitization to foodsConcomitant atopic diseases can also modulate the clinical relevance of a given allergy test result. Patients with atopic eczema tend to have a polyclonal IgE response to allergens that often lacks clinical expression. This underscores the importance of a judicious selection of allergens to be tested. Grabenhenrich et alGeographical location is another factor that may influence the clinical relevance of a given sIgE level. A study by Vereda et alFinally, the clinical setting influences the predictive value of sIgE levels, with increasing likelihood of clinical allergy going from the general population to secondary care and then to specialist centers. In studies performed in the general population, the prevalence of sensitization to foods such as cow's milk and egg was much lower than in a population recruited from specialist centers. For example, approximately 8% and 3%-4% of children in National Health and Nutrition Examination Survey 2005-2006The main reason to perform an OFC is to identify the food that caused the allergic reaction for the initial diagnosis and for monitoring resolution of FA. Other reasons for an OFC include assessing the status of tolerance to cross-reactive foods  and expanding the diet in foods not yet introduced but with positive allergy tests. This occurs more frequently because of the increased use of anticipatory testing and has important resource implications.Many factors affect the decision as to whether to perform an OFC . The mosWhen monitoring a patient for resolution of FA, it is generally recommended that children with a 50% chance of experiencing a negative challenge be good candidates for an OFC.When considering performing an OFC it is vital that the patient or parents of the child undergoing the OFC understand the rationale for this and the importance of introducing the food into the diet after a negative challenge. Several studies have shown that 18% to 32% of patients do not introduce the food after passing an OFC.in vitro studies of basophil activation and mediator release assays where Ara h 2 and Ara h 6 have been shown to be the most potent elicitors of effector cell response.121Identifying patients at high risk of a severe reaction to foods is important for the management of patients diagnosed with FA. Previous studies have shown contradictory results about the utility of food-specific IgE levels in assessing severity of FA.The above data need to be applied with caution to the assessment of individual patients. For example, patients with raised Ara h 2 sIgE do not necessarily have severe PA and can actually pass a peanut OFCIn vitro allergy tests are useful in diagnosing IgE-mediated FA and support the decision of whether an OFC is necessary to reach an accurate diagnosis. Validated cutoffs are reliable when applied to a similar patient population to the one where they were developed. Patient-specific factors can modulate the probability of clinical allergy of a given sIgE result. IgE to allergen components can provide more precise information about IgE specificity. The BAT assesses the function of IgE in its ability to mediate allergen-induced effector cell activation. Further research is needed to improve our understanding about how the information of various tests can be combined for optimal diagnostic accuracy to reduce the need to perform OFCs to a minimum.


Reconstructing the dynamic response of the Antarctic ice sheets to warming during the Last Glacial Termination  allows us to disentangle ice-climate feedbacks that are key to improving future projections. Whilst the sequence of events during this period is reasonably well-known, relatively poor chronological control has precluded precise alignment of ice, atmospheric and marine records, making it difficult to assess relationships between Antarctic ice-sheet (AIS) dynamics, climate change and sea level. Here we present results from a highly-resolved ‘horizontal ice core’ from the Weddell Sea Embayment, which records millennial-scale AIS dynamics across this extensive region. Counterintuitively, we find AIS mass-loss across the full duration of the Antarctic Cold Reversal , with stabilisation during the subsequent millennia of atmospheric warming. Earth-system and ice-sheet modelling suggests these contrasting trends were likely Antarctic-wide, sustained by feedbacks amplified by the delivery of Circumpolar Deep Water onto the continental shelf. Given the anti-phase relationship between inter-hemispheric climate trends across the LGT our findings demonstrate that Southern Ocean-AIS feedbacks were controlled by global atmospheric teleconnections. With increasing stratification of the Southern Ocean and intensification of mid-latitude westerly winds today, such teleconnections could amplify AIS mass loss and accelerate global sea-level rise. Understanding centennial to millennial-scale variability of the Earth’s ice sheets is key to gaining insights into ice sheet-climate feedbacks132During the LGT, long-term warming was interrupted by the ~2,000-year duration cold event across the mid to high latitude Southern Hemisphere, known as the Antarctic Cold Reversal1113214161132Here we take a novel approach that investigates a new 800 m long ‘horizontal ice core’ that captures a unique record of ice-sheet dynamics and climate across the Weddell Sea Embayment (WSE)2, CH4 and N2O) provide key age tie points across the profile (We report results from an exposed ancient blue ice area (BIA) outcropping alongside Patriot Hills in Horseshoe Valley , a local profile , and dem profile  that mar profile .18O profile  from the Patriot Hills BIA identifies a two-stepped change in values during the LGT, with a ~39‰ increase recorded across the ACR between ~14.7–12.7 ka, followed by a millennial-duration isotopic plateau (~12.7–11.7 ka) . Using aofile SI . The deuofile SI . These aofile SI . Given tile SI 19, we inteThe marked change in δD captured in the record across the ACR implies an ice-sheet surface elevation decrease of ~615 m across Horseshoe Valley (at a rate of ~0.4 m/a), assuming an atmospheric lapse rate of 10 °C/kmThe regional climate and sea-level fingerprint of the ACR across the mid- to high-latitude Southern Hemisphere is difficult to reconcile. Recent modelling studies have demonstrated that it is possible to reconstruct the spatial pattern without substantial fresh water forcing in the Southern Ocean1121314CO2–depleted abyssal waters from 12.7 kaThe coincidence between changes in AIS elevation from the Patriot Hills, enhanced iceberg flux22The Patriot Hills preserves a record of significant AIS ice-sheet drawdown, mass loss and meltwater discharge during the ACR and across the LGT, contrasting markedly with previous interpretations of the configuration in the Weddell Sea sector of the AIS, which predict limited ice sheet drawdown since the local Last Glacial Maximum (LGM)We suggest these contrasts may reflect two factors: firstly, there is a question over the effectiveness of terrestrial cosmogenic isotope studies to truly reflect the former elevation of the LGM ice-sheet surface in areas of cold based non-erosive polythermalice sheet settings2725292930Supported by marine geological evidence of enhanced iceberg calving246The characteristics of the Patriot Hills BIA are rare in Antarctic terms, with Horseshoe Valley being a slow flowing (<5 m/a) compound glacier system situated within an over-deepened catchment, that coalesces with the Institute Ice Stream at the periphery of the WSE , making 19High-resolution analysis of the Patriot Hills BIA using ground-penetrating radar (GPR) demonstrates a remarkably consistent pattern of layering along the 800 m transect out from Patriot Hills with two distinct unconformities at 247 m (D1) and 360 m (D2) along the profile . These u19202), methane (CH4) and nitrous oxides (N2O) – taken from depth (>3 m) along the BIA transect (34196) as an indicator of contamination by modern air. The average concentration of 8 samples analysed for SF6 was about 5% of modern day atmospheric concentrations and less than 2% for two of the samples selected to develop the chronology. The available constraints indicate the complete 800-m long Patriot Hills BIA transect spans ~50 to ~2.3 ka. Here we focus on the central section of the record, captured between the unconformities at 247 and 360 m, which, with multiple age ties together with the presence of the volcanic horizons dated to 17.8 ka and 18.2 ka, provides a robust chronology across the Patriot Hills BIA sequence of uninterrupted isochrons , 279 m (18.2 ka) and 190 m (36.4 ka) , and a ctransect . The traansect 34, providiansect 34 that cansochrons . Further18O isotopic measurements at 5 m resolution at James Cook University (JCU), and the University of New South Wales (UNSW) ICELAB. At the AAD an on-line chromium reduction method on a EuroVector EuroPyrOH-HT system interfaced in continuous flow mode to an Isoprime isotope ratio mass spectrometer. Analytical precision is <0.5‰ and δD values are expressed relative to the Vienna Standard Mean Ocean Water (VSMOW) scale. To confirm δD values, particularly the rapid transitions across the periods defined by the LGT, δD and δ18O were measured independently at JCU using Diffusion Sampling - Cavity Ring-down Spectrometry (DS-CRDS). This system continuously converts liquid water into water vapour for real-time stable isotope analysis by laser spectroscopy . Each analytical run consisted of 12 standards interspersed with 44 unknown samples. Data processing was performed using a customised Excel™ template and included correction for between-sample memory, instrumental drift and normalisation to the VSMOW scale. Further details are available in the 18O using a Las Gatos Research Liquid Water Isotope Analyzer 24d . Reported overall analytical precision on long term ice core standards are <0.32‰ for δD, and <0.13‰ for δ18O values are expressed relative to the .δD isotopic measurements at 1 m resolution were performed across the Patriot Hills BIA transect at the Australian Antarctic Division (AAD). These results were confirmed and augmented by δD and δHow to cite this article: Fogwill, C.J. et al. Antarctic ice sheet discharge driven by atmosphere-ocean feedbacks at the Last Glacial Termination. Sci. Rep.7, 39979; doi: 10.1038/srep39979 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


GTPase protein promoting oxidative stress, in human endothelial dysfunction.Endothelial dysfunction contributes significantly to the development of vascular diseases. However, a therapy able to reduce this derangement still needs to be identified. We evaluated the effects of pharmacological inhibition of Rac1, a small NSC23766, a Rac1 inhibitor, on endothelium‐dependent vasorelaxation of saphenous vein segments collected from 85 subjects who had undergone surgery for venous insufficiency and from 11 patients who had undergone peripheral vascular surgery. The endothelium‐dependent vasorelaxation of the varicose segments of saphenous veins collected from patients with venous insufficiency was markedly impaired and was also significantly lower than that observed in control nonvaricose vein tracts from the same veins. Rac1 activity, reactive oxygen species levels, and reduced nicotine adenine dinucleotide phosphate (NADPH) oxidase activity were significantly increased in varicose veins, and NSC23766 was able to significantly improve endothelium‐dependent vasorelaxation of dysfunctional saphenous vein portions in a nitric oxide–dependent manner. These effects were paralleled by a significant reduction of NADPH oxidase activity and activation of endothelial nitric oxide synthase. Finally, we further corroborated this data by demonstrating that Rac1 inhibition significantly improves venous endothelial function and reduces NADPH oxidase activity in saphenous vein grafts harvested from patients with vascular diseases undergoing peripheral bypass surgery.We performed vascular reactivity studies to test the effects of Rac1 pharmacological inhibition rescues endothelial function and reduces oxidative stress in dysfunctional veins. Rac1 inhibition may represent a potential therapeutic intervention to reduce human endothelial dysfunction and subsequently vascular diseases in various clinical settings. Endothelial dysfunction leads to the development of arterial vessel inflammation, remodeling, and atherosclerosis and, by this means, is significantly associated with an increased incidence of ischemic heart disease, peripheral artery disease, and stroke.Oxidative stress is considered to be the main cause of endothelial dysfunction, mostly through the reduction of nitric oxide bioavailability,The small GTP‐binding protein Rac1 might represent a suitable target to inhibit maladaptive NOX activity.Thus, the aim of this study was to evaluate for the first time the effects of pharmacological Rac1 inhibition in human endothelial dysfunction. For this purpose, we conducted ex vivo studies with dysfunctional saphenous veins collected from patients with chronic vein insufficiency and saphenous veins harvested from patients who had undergone bypass surgery for peripheral chronic ischemia.We conducted our experiments on intact saphenous vein portions removed from 2 groups of subjects: (1) 85 subjects who underwent surgery for chronic venous insufficiency and varicose veins (group 1) and (2) 11 subjects who underwent bypass surgery for peripheral chronic ischemia (group 2). Inclusion criteria were (1) severe saphenous vein insufficiency and/or varicose veins (group 1),or chronic peripheral ischemia suitable for surgical revascularization by means of saphenous vein graft (bypass) (group 2); (2) age ≥18 and ≤85 years; (3) elective surgery. Exclusion criteria were (1) acute and chronic inflammatory diseases; (2) immunological disorders; (3) active infections; (4) previous organ transplantation; (5) any pharmacological therapy able to modulate endothelial function and/or either Rac1 or NOX activity within the previous month; or (6) any surgical complication.Vessels were collected immediately after surgical harvesting and transported to the laboratory in ice‐cold Krebs‐HEPES buffer to perform vascular reactivity studies.The study protocol was approved by all local ethics committees of IRCCS Neuromed and done in accordance with the Declaration of Helsinki. All participants gave written informed consent. Institutional review board approval was obtained from IRCCS Neuromed (No. 20160106‐1006).2PO4 1.2, NaHCO3 25, MgSO4·d7H2O 1.2, CaCl2 2.5, glucose 11.1, and disodium EDTA 0.026. The vessels were cleaned of adherent connective tissues and cut into rings 3 to 4 mm in length. Rings were suspended between 2 stainless steel L‐shaped hooks in a 10‐mL jacketed organ bath containing Krebs Ringer solution at 37°C and aerated with 95% O2 and 5% CO2. One hook was fixed to a micrometric manipulator allowing adjustments in resting tension of the rings, and the other was connected to a force displacement transducer  for the measurement of isometric force. The optimal point of the length‐tension relation had been previously described.Hypercholesterolemia was considered as total plasma cholesterol, <240 mg/dL smoking was considered as current or within the last 6 months, and hypertension as current treatment with antihypertensive agents.−9 to 10−6 mol/L) and contraction to 40 mmol/L KCl, which had to be reproducible at least 2 times. To study the endothelium‐dependent vasorelaxation, saphenous veins were precontracted with 10−9 to 10−6 mol/L of phenylephrine to obtain a contraction that corresponds to 70% to 90% of maximal contractions observed with KCl. When the constriction reached a stable plateau, increasing doses of acethylcoline (10−9 to 10−6 mol/L) were added.At the end of an equilibration period of 2 hours, the viability of the vessel segments was checked through the evaluation of the cumulative dose‐response curves to norepinephrine  or polyethylene glycol superoxide dismutase , which were added to the bathing medium 30 minutes before acetylcholine administration. Responses to acetylcholine were assessed in each vessel before and after administration of NSC23766. In time‐matched control experiments, we determined whether the precontraction induced by phenylephrine is stable enough for the period required to construct the cumulative relaxation curves to acetylcholine.Cumulative dose‐response curves to acetylcholine in saphenous veins were repeated in the presence of NSC23766 (30 μmol/L), LAll drugs used were purchased from Sigma  or Tocris Bioscience . A stock solution of phenylephrine or acetylcholine was freshly prepared to prevent oxidation. NSC23766 and L‐NAME were dissolved in distilled water.g for 20 minutes, and the supernatants were used to perform immunoblot analysis. Total protein levels were determined using the Bradford method. Rac1 activity was determined using a commercially available kit  as described below. Forty micrograms of proteins were resolved on 10% SDS‐PAGE, transferred to a nitrocellulose membrane, and immunoblotted with anti‐Rac1‐GTP  or anti‐Rac1 ; with anti–RhoA‐associated kinase 1 (ROCK1) (1:1000 (abcam); with anti‐p–endothelial nitric oxide synthase (eNOS) phosphorylated on serine 1177  or anti‐total‐eNOS ; or with anti‐β‐actin . Horseradish‐peroxidase–conjugated secondary antibodies were used at 1:3000 dilution . Protein bands were detected by ECL Prime , and densitometry analysis was performed using Quantity One software (Bio‐Rad Laboratories).Vessels were solubilized in lysis buffer containing 20 mmol/L Tris‐HCl, 150 mmol/L NaCl, 20 mmol/L NaF, 2 mmol/L sodium orthovanadate, 1% Nonidet, 100 μg/mL leupeptin, 100 μg/mL aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride. Then, samples were left on ice for 30 minutes and centrifuged at 10 621Low‐temperature SDS‐PAGE (LT‐PAGE) was performed for detection of SDS‐resistant endothelial nitric oxide synthase (eNOS) dimer and monomer, as described previously.Tissues were lysed in a buffer containing NP‐40 equipped by kit STA‐401‐1 . The p21‐binding domain of p21‐activated protein kinase bound to agarose beads was added, and active Rac1, binding PAK1, was separated by repetitive centrifugation and washing. Then, the specimens were boiled in Laemmli buffer and subjected to SDS‐PAGE, and Rac1 was quantified by Western blot analysis. In detail, Rac1‐GTP was detected with the monoclonal antibodies anti‐Rac1‐GTPγ  and total Rac1 with monoclonal anti‐Rac1 . Densitometry analysis was performed using Quantity One software (Bio‐Rad Laboratories). The amount of Rac1‐GTP was normalized to the total amount of Rac1 in tissue lysates for the comparison of Rac1 activity (GTP‐bound Rac1) among different samples.Dihydroethidium (DHE) was used to evaluate the levels of oxidative stress in saphenous veins as previously described.2 1.87, MgSO4 1.20, K2HPO4 1.03, NaHCO3 25.0, sodium HEPES 20.0, and glucose 11.1, pH 7.4. Periadventitial tissue was carefully removed, and the vessels were repeatedly washed to remove adherent blood cells. A 10% vessel homogenate was prepared in a 50 mmol/L phosphate buffer containing 0.01 mmol/L EDTA. The homogenate was then subjected to low‐speed centrifugation (1000g) for 10 minutes to remove unbroken cells and debris. Supernatants (20 μL) were added to glass scintillation vials containing 5 μmol/L lucigenin in 2 mL phosphate buffer. The chemiluminescence that occurred over the ensuing 5 minutes in response to addition of 100 μmol/L NADPH was recorded . In preliminary experiments, homogenates alone without addition of NADPH gave only minimal signals. Furthermore, NADPH did not evoke lucigenin chemiluminescence in the absence of homogenate. In some experiments, 30 U/mL of heparin‐binding SOD was added to determine the SOD‐inhibitable activity. This is a recombinant form of SOD that contains a heparin‐binding domain that permits a close association of SOD with cell membranes, obviating electrostatic repulsion of SOD. The subcellular fraction of cytosol and membranes were obtained as previously described.NOX activity in saphenous veins was measured before and after NSC23766 treatment. Vein segments (3 cm) were placed in a chilled modified Krebs/HEPES buffer containing (mmol/L) NaCl 99.01, KCl 4.69, CaClAll data are presented as mean±SEM. For continuous variables, we used a t test to compare 2 independent groups. When 2 samples from the same individual were compared, a paired t test was used. When more than 2 independent groups were compared, we used a 1‐way ANOVA analysis followed by Bonferroni post hoc test. Finally, in order to analyze the effects of our treatments on endothelium‐dependent vasorelaxation in response to increasing doses of acetylcholine, we performed a 2‐way repeated‐measures ANOVA with Bonferroni post hoc test for multiple comparisons.P value of less than 0.05 was considered statistically significant. All statistical analyses were conducted with Prism statistical software .A In order to study the role of Rac1 in the development of human endothelial dysfunction, we conducted ex vivo experiments on saphenous vein portions obtained from 85 subjects who underwent saphenectomy due to chronic venous insufficiency and/or varicose veins. The clinical characteristics of the study subjects are reported in We tested whether Rac1 inhibition could restore the endothelial function of dysfunctional saphenous veins. NSC23766, a pharmacological inhibitor of Rac1,Remarkably, we corroborated these data on the saphenous veins harvested from the entire study population of patients with chronic venous insufficiency and/or varicose veins and confirmed that NSC23766 could restore the endothelial function of altered saphenous vein segments. Interestingly, pharmacological Rac1 inhibition improved vasorelaxation of veins collected from patients without cardiovascular risk factors and from subjects with risk factors in a similar manner Figure , we founThen we conducted biochemical analyses on dysfunctional vein portions treated or not treated with NSC23766. First, we found that NSC23766 inhibits Rac1 activity in vein segments from patients both without and with cardiovascular risk factors, as indicated by the reduction of Rac1‐GTP content Figure .Overall, these data indicate that Rac1 inhibition reduces NOX activity and ROS production, coherently with the established activating function of Rac1 toward this ROS‐generating enzyme. Interestingly, we found that PEG‐SOD modestly but significantly improved acetylcholine‐induced vasorelaxation Figure C. These Finally, we tested whether Rac1 inhibition is able to improve endothelial function of portions of saphenous veins used as conduits for surgical revascularization (bypass) of patients with peripheral ischemia due to severe artery disease . In factIn the present study we explored the effects of pharmacological Rac1 inhibition on human endothelial dysfunction. We found that Rac1 inhibition significantly improves endothelial function in dysfunctional saphenous veins collected from subjects with chronic vein insufficiency. Such beneficial effects were nitric oxide–dependent. Mechanistically, Rac1 inhibition reduced NOX activity and superoxide production. In addition, it downregulated ROCK1 and promoted eNOS upregulation. We also found that Rac1 inhibition improves endothelial function of saphenous vein grafts collected from patients who underwent bypass surgery for peripheral ischemia due to severe vascular disease.Rac1 belongs to the Rho family of GTPases.We believe that our study significantly extends this evidence by demonstrating for the first time that that Rac1 also plays an important role in the development of human endothelial dysfunction. We found that Rac1 inhibition restores the endothelial function of dysfunctional saphenous vein segments in a nitric oxide–dependent manner. In fact, the vasorelaxant effects of NSC23766 were abrogated by L‐NAME. These data suggest that Rac1 pharmacological inhibition may increase nitric oxide bioavailability. Mechanistically, Rac1 inhibition may reduce nitric oxide degradation by reducing NOX activity and production of superoxide, which rapidly reacts with nitric oxide to form peroxynitrite.Rac1 inhibition may directly increase nitric oxide production.Our study suggests that Rac1 activation may play a role in vein‐related diseases. Recent evidence indicates that endothelial dysfunction contributes to vein‐related diseases. A procoagulative state, which is usually associated with endothelial dysfunction, may favor deep vein thrombosis.Our study also has some limitations that should be acknowledged. First, Rac1 pharmacological inhibitor may have some nonspecific effects, although we confirmed that this compound significantly reduces Rac1 activation and NOX activity. We only had a small group of healthy vein portions. However, we were able to make a direct comparison of healthy versus diseased vein tracts from the same saphenous veins, which allowed us to demonstrate that varicose veins have endothelial dysfunction and significant Rac1 activation. Finally, DHE‐ and lucigenin‐based assays may not be completely specific for superoxide anion detection, although these are widely used and validated methods, and both of them provided similar results, which were also consistent with the Rac1‐GTP and eNOS uncoupling data.In conclusion, our study demonstrates that Rac1 pharmacological inhibition rescues endothelial function and reduces oxidative stress in dysfunctional saphenous veins. Accordingly, these emerging data may have applicative/translational implications because Rac1 inhibition may represent a potential therapeutic intervention to reduce human endothelial dysfunction and subsequently vascular diseases in several clinical scenarios, particularly vein‐related diseases. Of course, future studies are encouraged to test this hypothesis and to confirm our results, testing the efficacy of Rac1 inhibition in other models of human endothelial dysfunction, particularly in human arteries.This study was financially supported by University of Rome Sapienza to Giacomo Frati, from Ministero della Salute to Sebastiano Sciarretta and Roberto Carnevale (GR‐2013‐02355401). The funding source had no involvement in the research design and preparation, the writing, or the decision to submit this article for publication.None.Data S1. Vascular Reactivity StudiesFigure S1. A, Acetylcholine (ACh) vasorelaxation in preconstricted mice mesenteric arteries in basal condition  and after treatment with Rac‐1 inhibitor at 30 μmol/L for 30 minutes  (n=4 for each group). B, Nitroglycerin (Nitro) vasorelaxation in preconstricted mouse mesenteric arteries in basal condition  and after treatment with Rac‐1 inhibitor at 30 μmol/L for 30 minutes  (n=4 for each group).Figure S2. In situ detection of superoxide generation in segments of mouse mesenteric arteries treated with H2O2 (5 μmol/L); with H2O2 plus NSC23766 (30 μmol/L); with angiotensin II ; or with angiotensin II plus NSC23766.Click here for additional data file.


Pseudomonas aeruginosa and Pseudomonas putida. In this study, we show the role of S/T phosphorylation of one motility protein, FliC, in regulating multiple surface-associated phenomena of P. aeruginosa PAO1. This is the first report of occurrence of phosphorylation in the flagellar protein, flagellin FliC in its highly conserved N-terminal NDO domain across several Gram negative bacteria. This phosphorylation is likely a well-regulated phenomenon as it is growth phase dependent in planktonic cells. The absence of phosphorylation in the conserved T27 and S28 residues of FliC, interestingly, did not affect swimming motility, but affected the secretome of type 2 secretion system (T2SS) and biofilm formation of PAO1. FliC phosphomutants had increased levels and activities of type 2 secretome proteins. The secretion efficiency of T2SS machinery is associated with flagellin phosphorylation. FliC phosphomutants also formed reduced biofilms at 24 h under static conditions and had delayed biofilm dispersal under dynamic flow conditions, respectively. The levels of type 2 secretome and biofilm formation under static conditions had an inverse correlation. Hence, increase in type 2 secretome levels was accompanied by reduced biofilm formation in the FliC phosphomutants. As T2SS is involved in nutrient acquisition and biofilm dispersal during survival and spread of P. aeruginosa, we propose that FliC phosphorylation has a role in ecological adaptation of this opportunistic environmental pathogen. Altogether, we found a system of phosphorylation that affects key surface related processes such as proteases secretion by T2SS, biofilm formation and dispersal.Protein phosphorylation has a major role in controlling the life-cycle and infection stages of bacteria. Proteome-wide occurrence of S/T/Y phosphorylation has been reported for many prokaryotic systems. Previously, we reported the phosphoproteome of  Successful survival of environmental microbes in diverse niches requires highly co-ordinated switching of life strategies. The two pre-dominant lifestyles of bacteria are the free-swimming planktonic state and the surface-associated biofilm state , 2. WhilSurface appendages such as flagella and pili can facilitate these lifestyle switches –12. AmonThe second group of surface-associated processes that impact the bacterial lifestyle, especially during interaction with hosts are the secretion systems that affect the immediate environment. Six independent secretion systems have been classified in Gram negative bacteria, namely, Type 1–6 secretion systems , 18. SomBacillus subtilis, Lactococcus lactis and Escherichia coli were some of the first to be estabilished [Post-translational modifications such as phosphorylation, glycosylation, acetylation, methylation and lipidation signal for lifestyle transition by causing structural or functional changes on fully mature proteins . In recebilished –26. Thesbilished , 27, 28.P. aeruginosa PAO1, a successful environmental pathogen capable of colonizing diverse niches [P. aeruginosa. Hence, our study was directed towards the roles of FliC phosphorylation in the processes of motility, secretion and biofilm formation. Subsequently, we have uncovered effects of S/T phosphorylation of FliC on protease secretion by T2SS and biofilm dispersal. Till date, there are no other reports showing the importance of phosphorylation of a motility protein in affecting the virulence phenomena of bacteria. We have attempted to provide a novel perspective to the widespread functions of S/T/Y phosphorylation.We have previously reported the phosphoproteome of e niches . Among te niches , 30. The600.The bacterial strains used in this study are listed in fliC-FL T27A and ΔfliC-FL S28A were generated by site directed mutation of phosphosites T27 and S28 to alanine, denoted by T27A and S28A, while the phosphomimic ΔfliC-FL S28D was generated by site directed mutation of phosphosite S28 to aspartate, denoted by S28D. The site-directed mutation for fliC T27A was created by fusion PCR approach, whereas, fliC S28A and fliC S28D were created by cloning full length fliC gene into pJET vector , followed by mutagenesis using the Quik Change II XL Site-Directed Mutagenesis Kit . Primers used in this study are listed in fliC gene were cloned into pUC18-miniTn7T-GM vectors and electroporated into PAO1 ΔfliC. The clones were verified by genomic DNA isolation and sequencing of mTn7 insertion site. The following names were used for the resulting strains: ΔfliC- FL T27A (complemented threonine phosphomutant), ΔfliC- FL (complemented full length FliC), ΔfliC- FL S28A (complemented serine phosphomutant) and ΔfliC-FL S28D (complemented serine phosphomimic).The phosphomutants ΔPlate motility assay was carried out by stabbing overnight diluted cultures in semisolid 0.3% w/v agar  and determination of swim zones after 9 h and 16 h at 37°C . Bacteri600 levels after 13 h growth. Differences in growth were normalized using OD600 values, both for starting the cultures and at harvesting stage. Secretion starts from 9 h onwards. The supernatants were filtered through 0.20μm PES filter (Sartorius) and precipitated with Trichloroacetic acid (TCA) to get the extracellular protein profile as previously described [http://rsbweb.nih.gov/ij/) developed by Wayne Rasband, National Institutes of Health, Bethesda, MD.Overnight grown cultures were diluted 1:100 in 100 ml LB broth and 5 ml was harvested at approximately same ODescribed . The preescribed . DensitoP. aeruginosa elastase  was used as standard to calculate the units of active elastase per μg of total secreted protein.The assay was adapted from the method described by . ElastinIntracellular proteins were extracted from cell pellets after separating the supernatants for extra-cellular protein extraction. The pellets were washed once and resuspended in extraction buffer  containing complete Mini Protease Inhibitor Cocktail (Roche). Homogenization of cells was carried out in a Micro Smash MS-100  with 0.1mm glass beads in screw cap tubes at a pulse of 4,000rpm for 20 seconds in 8 cycles. The cells were then centrifuged to get the intracellular proteins in the supernatant.Overnight grown cultures were diluted 1:100 in 100 ml LB broth and 50 ml was harvested at 13 h. The cell pellets were washed once with 50mM sodium phosphate buffer (pH 8) and resuspended in the same buffer containing Complete Mini Protease Inhibitor Cocktail (Roche). The cells were homogenized in a sonicator for 4 min with 40 second on and 20 second off cycle. Cell suspensions were centrifuged to remove cell debris. The supernatants were centrifuged again at 125,000g at 4°C for 30–45 minutes to separate the membrane protein fraction (pellet) and cytoplasmic and periplasmic fractions (supernatant). The pellets were dissolved in the extraction buffer with protease inhibitor and stored in—80°C until further use .st base (USA) after sending them the expressed elastase B and FliC proteins as gel strips. All secondary antibodies were anti-rabbit/mouse IgG conjugated with alkaline phosphatase (Sigma). All incubations with primary and secondary antibodies were done at room temperature. The substrate used for detection was ImmobilonTM Western chemiluminescent AP substrate (Millipore).Proteins run in 12% SDS-PAGE gel were transferred onto ECL nitrocellulose membrane to probe with respective antibodies. The XcpP, XcpY and XcpZ rabbit antibodies were kind gifts from Dr. Gerard Michel, CNRS, France , 37. Mou1/2 gauge needle and centrifuged to remove cell debris. The supernatant was centrifuged at 50,000xg for 2 h. The resultant pellets were then dissolved in acidified water (pH 2.0) adjusted with Triflouroacetic acid (TFA) and centrifuged again for 2h at 50,000xg. All buffers used contained Complete Mini Protease Inhibitor Cocktail (Roche) and Phosphatase Inhibitor (Roche) tablets. The extracted FliC was subjected to phosphorylation analysis as described below. FliC was reduced (10mM dithiothreitol) and alkylated (50 mM iodoacetamide) at room temperature. It was then digested with Lys-C  (Wako) for 3 h and then diluted 4 times with 50 mM ammonium bicarbonate for overnight trypsin (Promega) digestion at room temperature. The resulting peptide mixture was acidified with TFA and desalted with a SDB-XC solid-phase extraction cartridge. The phosphopeptides were enriched using titanium dioxide beads as described previously [m/z 300–1500) for 250 ms, followed by high-sensitivity MS/MS scans from the top 7 most abundant precursor ions, each with a 100-ms accumulation time.Overnight cultures of one litre P. aeruginosa at early (6 h) and late (13 h) log growth phases grown at 37°C were harvested, sheared through a 23eviously , 39. TheP. aeruginosa PAO1. The search criteria used in this study were as follows: trypsin specificity allowing up to 2 missed cleavages; fixed modification of carbamidomethyl (C); and variable modifications of oxidation (M), acetylation (protein N-term), and phosphorylation (ST) and (Y). The precursor mass tolerance was set at 20 ppm, and the fragment ion tolerance was set at 0.1 Da. Peptides were considered identified if the Mascot score yielded a confidence limit above 95% based on the significance threshold (p < 0.05) and if at least three successive y- or b-ions with an additional two and more y-, b-, and/or precursor-origin neutral loss ions were observed, based on the error-tolerant peptide sequence tag concept [The peak list of each raw MS spectrum was generated as previously described . Peptide concept . The pho595.The biofilm formation assay was adapted from O’ Toole and Kolter . Briefly® BacLight™Bacterial Viability Kit . Imaging was performed in Confocal Laser Scanning Microscope  using the multi-track mode to minimize fluorescent bleed-through. Secretome was extracted as described previously from biofilms grown in 6-well plates under static conditions.The imaging methods were adapted from Meritt et al. . Briefly3) were used for growing biofilms [8 cfu/ml. Biofilms were allowed to develop for 7 days and monitored every 24 h. In total, 14 flow cell setups were used for PAO1 WT, ΔfliC, ΔfliC-FL T27A, ΔfliC-FL and ΔfliC-FL S28A strains. Each day, one flow cell was sacrificed and visualized by staining with 100–200μl of live/dead stain from LIVE/DEAD® BacLight™ Bacterial Viability Kit .The flow cell experiment was adapted from . Three-cbiofilms . The floAll microscopic observations and image acquisitions were performed by a Confocal Laser Scaning Microscope, CLSM  using the multi-track mode to minimize fluorescent bleed-through. The excitation wavelengths for GFP and Ds-Red were 488 and 561 nm respectively. The emission wavelengths were 509 and 584 nm respectively. For each flow cell channel, five images near the inlet (5–10 mm from the inlet) and two images near the outlet were taken. The resultant stacks obtained from images were focussed in the centre of the channel . For image analysis, the image stacks were quantified for each biofilm type using IMARIS . The average volume of live, dead and total cells were then calculated for estimating the attachment / detachment patterns of biofilm cells . The imaIn order to confirm the presence of T27 and S28 phosphorylation in FliC, we carried out its phosphopeptide enrichment and nano LC-MS/MS from early and late log phase grown PAO1 WT. Analysis of phosphorylation profile showed T27 residue to be phosphorylated. This phosphorylation was growth-stage specific as it was present in the FliC of late log grown WT cells and not in the early log grown WT cells . S28 wasfliC, ΔfliC-FL T27A, ΔfliC-FL and ΔfliC-FL S28A strains by both end-point motility assay for populations and video microscopy analysis for observing speed at single cell level. The swim zones and swimming motility speeds did not have any significant differences between the phosphomutant strains and WT strains , a phosphomimic. S28 was chosen because aspartate is a closer phosphomimic to serine and also to test whether it undergoes S28 phosphorylation. Secretome profile of ΔfliC—FL S28D was similar to that of ΔfliC–FL, thereby demonstrating the role of FliC phosphorylation at S28 in affecting the secretome levels of T2SS (fliC- FL T27A (39%) and ΔfliC—FL S28A (33%) strains when compared to ΔfliC–FL 27, (T/S/A)28 28 . This coP. aeruginosa [In the context of non-motility related functions of flagella, export machineries of flagella and secretion systems of Gram-negative bacteria are evolutionarily conserved , 51–53. ruginosa . Some evruginosa . Hence, Three lines of evidence establish the effect of S/T phosphorylation on T2SS. First, levels of extracellular proteases secreted by T2SS are increased in the FliC phosphomutants and are restored in the FliC phosphomimic mutants. This highlights the role of FliC phosphomutants, especially S28 residue in T2SS functions. Second, the increase in extracellular protease levels of phosphomutants was not due to increased production of proteases, as there was no increase in intracellular levels of proteases in phosphomutants, even under increased extracellular proteases. These findings raise the possibility of a feedback mechanism that maintains the level of intracellular proteases in cells. Third, the regulation of T2SS by FliC phosphorylation did not lead to increase in secretion machinery as the levels of T2SS membrane components were unaffected by FliC phosphorylation.Our study on the effects of FliC phosphorylation on biofilm attachment and dispersal led to two conclusions. Both initial attachment and detachment during dispersal stage were delayed by the loss of FliC phosphorylation in static and dynamic flow biofilms. As each of these processes still proceeded in the lack of phosphorylation, it suggested that FliC phosphorylation regulates the timing and rate of these processes without affecting biofilm architecture. Interestingly, in static biofilms, FliC phosphorylation seemed to be required for rapid attachment and maintaining low levels of T2SS proteases. It is possible that the association of T2SS factors in the biofilm matrix leads to decreased effectors in the culture filtrates. Hence, the FliC-T2SS interaction is also evident during biofilm growth. Overall, it is probable that FliC phosphorylation here could act to integrate environmental cues with the signals for attachment or dispersal of biofilms. FliC phosphorylation also seems to affect the impact of bacterial cells on the immediate environment through the modulation of protease secretion, hence playing a role in this two-way integration of cellular processes with the environment.P. aeruginosa upon FliC phosphorylation.This study adds a new facet of serine and threonine phosphorylation in bacterial systems. FliC phosphorylation seems to affect both secretome levels and biofilm-related phenomena, which brings up the level of importance of FliC phosphorylation in these processes. Altogether, our study shows the presence of a phenomenon which affects at least two surface-associated processes of secretion and biofilm formation in S1 FigfliC-FL T27A vs. ΔfliC-FL and ΔfliC-FL S28A vs. ΔfliC-FL. at both 9h and 16h. (B) Motility speeds determined by video microscopy analysis represented as fraction of live speeds of cells falling within the different percentile categories. Error bars indicate mean ± SD computed from four biological replicates with three technical replicates each. Bonferroni multiple comparison analysis not significant, p-values > 0.05 for ΔfliC-FL T27A vs. ΔfliC-FL and ΔfliC-FL S28A vs. ΔfliC-FL.(A) Quantification of motility zones formed in semisolid agar (0.3%) at 9h and16h respectively. Error bars indicate mean ± SD computed from four biological replicates with three technical replicates each. Student’s t-test p-values > 0.05 for Δ(TIF)Click here for additional data file.S2 FigfliC-FL T27A, ΔfliC-FL and ΔfliC-FL S28A strains. Proteins were loaded based on equal number of cells as shown by RNA Pol α-subunit levels (bottom panel).Immunoblot of extracellular FliC (top panel), and intracellular RNA polymerase (RNA Pol) α-subunit (bottom panel) at 13 h for PAO1 WT, Δ(TIF)Click here for additional data file.S3 FigfliC, ΔfliC-FL T27A, ΔfliC-FL and ΔfliC-FL S28A strains. Error bars indicate mean ± SD computed from four biological replicates with five technical replicates each. Student’s t-test p-values <0.05 for ΔfliC-FL T27A vs. ΔfliC-FL and ΔfliC-FL S28A vs. ΔfliC-FL.Quantification of biofilms formed in polystyrene round-bottom tubes by crystal violet staining at 5h for PAO1 WT, Δ(TIF)Click here for additional data file.S4 FigfliC, ΔfliC-FL T27A, ΔfliC-FL and ΔfliC-FL S28A strains across all 7 days. Live and dead cells are represented in green and red, respectively. Panels are represented as a-WT, b-ΔfliC, c-ΔfliC- FL T27A, d-ΔfliC-FL and e-ΔfliC-FL S28A, respectively. Magnification is under 40X oil lens. Scale bars indicate a distance of 50 μm.Comparison of biofilm architecture in confocal-ortho view for PAO1 WT, Δ(TIF)Click here for additional data file.S5 FigP. aeruginosa PAO1, 2- P. putida GB-1, 3- P. putida KT2440, 4- P. putida W619, 5- P. putida F1, 6- P. protegens Pf-5, 7- P. flectens Pf101, 8- E. coli K12, 9- E.coli K12 W3110, 10- E.coli CFT073, 11- E.coli EDL93 and 12- S. paratyphi A SARB42 respectively. Identical, strongly similar and weakly similar residue positions are indicated as (*), (:) and (.) respectively.Clustal Omega alignment shows the extent of conservation of threonine 27 and serine 28 residues across different bacterial species. Species are indicated as 1- (TIF)Click here for additional data file.