Escherichia coli survival in sterile physiological saline (SPS) was influenced by the treatment conditions: unfrozen, frozen-thawed and fully frozen (phase transition). In order to probe the enhanced phase transition microbial destruction, vibration effects of phase transition position were created and discussed. Test samples were placed in HP chamber for treatment  at room temperature and a special cooling device was used to maintain the phase transition conditions. Results showed that the phase transition from ice I to ice III of frozen SPS could be realized based on the cooling of a 20% sodium chloride solution. HP treatment under fully frozen conditions produced the best lethal effect compared to unfrozen and freeze-thaw samples. Vibration tests were carried out by using model solutions and apple juice to explore the behavior of phase transition. A synchronous and advance phase transition of internal apple juice was realized, respectively, by using pure water and 5% sodium chloride solution as external vibration sources, and the advance phase transitions of external pure water were realized by using 5% sodium chloride solution and 5% glucose solution as internal vibration sources.It was demonstrated that the inactivation of high pressure (HP) treatment on  Escherichia coli O157:H7 in carambola juice to a safe level, a pressure of up to 600 MPa needed to be applied. From the perspective of economic cost and safety guarantee demand, the pressure of HP processing in commercial application should not be so great. Therefore, researchers have begun to shift the focus of HP applications using the traditional and other variables in other ways.High pressure (HP) processing is becoming a high-tech pasteurization/sterilization technology which is attracting much attention all over the world because it overcomes the thermal damage to quality and nutrients in food that characterizes conventional thermal processing ,2,3,4. IAt present, the synergistic treatment of HP and low temperature is considered to be effective for pasteurization methods ,11. TheoUnder high pressure and low temperatures, water can form different ice crystals and undergo different solid-solid phase transitions . Under tA custom developed laboratory-designed self-cooling device was used in this study. This cooling system achieves automatic cooling in the HP chamber set-up without the necessity for any external temperature control system on the traditional high-pressure apparatus. Thus, it can be employed in any existing high pressure equipment operating at room temperature. Compared with the previous complex and expensive HPLT equipment, this way to realize high-pressure and low temperature is both convenient and economical. Li et al.  tested tSodium chloride and glucose exist in many real foods, including fruits and vegetables . Their pEscherichia coli in sterile physiological saline by HP treatment given to samples in three different forms: unfrozen, frozen-thawed and fully frozen (phase transition); (2) carry out vibration tests by using model systems as possible link for the inactivation and the law of phase transition.Therefore, the purpose of this study was to use a laboratory-designed self-cooling device to: (1) obtain the temperature and phase transition information for establishing an HPLT process and demonstrate the inactivation of A laboratory-designed self-cooling device  was usedE. coli strains ATCC 25922 (CGMCC 1.2385) were used and purchased from China General Microbiological Culture Collection Center . For the revival of freeze-dried strains, sterile nutrient broth  was used as liquid medium for initial activation at 37 °C for 24 h, and then the resuscitated cultures were inoculated to a sterile nutrient agar  slant and incubated at 37 °C for 24 h before being stored at 4 °C. In order to maintain the viability of strains, we made new sterile nutrient agar slant every month, and then used inoculant ring to pick out several bacterial strains from the old nutrient agar slant and transferred them to the new nutrient agar slant in the form of a scribed curve. They were cultured at 37 °C for 24 h and then stored at 4 °C. To prepare the working cultures, several loops of the strain were scraped from the slant of the nutrient agar, and then inoculated into 50 mL nutrient broth amd placed in a 37 °C thermostatic oscillator bath  for 24 h at a shake speed of 150 rpm. On the second day, several loops of E. coli suspension in the above nutrient broth were re-inoculated in to 150 mL nutrient broth and incubated at 37 °C for 24 h to obtain the inoculum for testing.Freeze dried cultures of E. coli pellets were obtained, and the supernatant was discarded. After washing the pellets several times with SPS, the E. coli cells suspended in SPS were divided into five parts after shaking evenly by an oscillator. The first part used for plating on brain-heart infusion agar  and the initial population was counted as about 108 CFU/mL. The other four parts were packed in 9 mL sample containers, one of which was used as an untreated frozen sample, and the other three were used for HP treatment.To prepare the sterile physiological saline (SPS), 8.5 g sodium chloride crystals were dissolved in 1000 mL distilled water and then autoclaved at 121 °C for 15 min. The inoculum obtained in E. coli in three different formats: unfrozen state, frozen-thawed state and the fully frozen (phase transition) state. The three prepared samples reserved for HP experiment in As shown in The unfrozen-state experiment meant the samples obtained in E. coli. The untreated frozen samples and pressure-treated samples were transferred to a sterile ultra-clean platform and homogenized for 10 s with a stomacher to ensure the uniform distribution of E. coli. Sterile physiological saline (0.85% w/v NaCl) was used for serial dilution of samples. After the BHIA was cooled and solidified, the plates were turned over and incubated at 37 °C for 48 h. Colony count was taken after two days.Brain-heart infusion agar  was selected to evaluate the lethal effect of HP treatment on In order to further explore the factors affecting the phase transition position of food, the influence of vibration factors on the phase transition position of ice I to ice III of water in apple juice and model solutions was investigated. In the vibration test, the composition and operation steps of the cooling system were somewhat different from that described in For internal vibration experiments, the cooling medium container with the 50 mL centrifugal tube was frozen for 48 h, and the centrifuge tube was not pulled out but left as the sample container. Then the sample was poured into the 50 mL centrifuge tube, and the cap was twisted on, and the sample was frozen at −30 °C for 24 h. For external vibration experiment, a 4 mL sample container was placed into the cavity structure, and cold pure water was poured to fill the remaining gap. The reason for pouring cold pure water was to increase the volume change during phase transformation and increase vibration intensity. The mechanism can be explained as follows: when the phase transition from ice I to ice III occurs in the solution, it is water as a solvent that causes the volume change. Compared with 5% glucose solution and 5% sodium chloride solution, there is no solute in pure water. Therefore, when the phase transition from ice I to ice III occurs between the same volume of solution and pure water, the content of solvent water in the solution is less than that of pure water due to the existence of solute. Thus, the volume change of pure water is also the largest due to the largest volume during phase transition. After screwing on the cap, the sample was put into the −30 °C freezer for 24 h. The rest of the cooling system composition and operation procedures were exactly as described in p < 0.05).SPSS  software was used for one-way analysis of variance (ANOVA) and the differences in average values were compared  and liquid water (1 g/cm3), the phase transformation of metastable ice I and metastable water to ice III was a process of volume reduction on the whole. Consequently, the cooling medium with a volume of nearly 440 mL produced a volume change during the phase transition, resulting in a pressure drop of ~6 MPa. For the frozen sample, the frozen SSPS entered the metastable zone of ice III and ice II successively after the pressure exceeded 210 MPa. As reported in earlier studies, metastable ice I was observed in the ice II region, and the nucleation of ice III could be realized in the ice II region [Surprisingly, when the pressure increased to the triple point of liquid-ice I to ice III (210 MPa), neither the sample nor the cooling medium underwent the phase transition from ice I to ice III. Based on the phase diagram of pure water , the phaI region . Hence, Escherichia coli cells. As the extension of the holding time did not significantly improve the destruction effect of phase change [The existence of metastable ice I and metastable water in the thermodynamically stable regions of ice III and ice II made the transition from ice I to ice III very unstable, and the phase transition range may differ greatly (more than 100 MPa) . Therefoe change , experimE. coli cells in unfrozen, freeze-thawed and frozen SPS conditions after different HP treatments. The initial concentration of E. coli before freezing was 1.4 × 108 CFU/mL. In the process of atmospheric freezing, the formation and growth of ice crystals led to a small destruction of bacterial cell membrane, resulting from damage to microbial cells [E. coli caused by freezing observed in this experiment was relatively very small, only 0.14 log, which may be due to the rapid cooling rate which offers better protection against cellular damage due to the associated small size ice crystals. Similar results were obtained previously by Zhu et al. [E. coli in frozen carrot juice after freezing. When the freezing speed was slow, the damage of bacterial cells could be higher [al cells . The inau et al.  who obsee higher .E. coli reduction was only the pressure level. The inactivation effect was very small at 150 MPa and 240 MPa: only 0.01 log and 0.25 log, respectively, which also indicated the strong pressure resistance of E. coli in SPS. When the pressure was 330 MPa, the inactivation E. coli increased to 1.14 log, a small step increase. Obviously, for unfrozen samples, 300 MPa HP treatment did not offer a significant advantage in inactivation of E. coli. The lethal effect of high-pressure treatment on E. coli cells in unfrozen SPS were similar to some previous studies [For unfrozen samples, the factor affecting  studies ,16,30,31E. coli destruction was not only dependent on the pressure, but also by the freezing process before pressurization. Although freezing alone resulted in only 0.14 log reduction in E. coli, after 24 h of freezing, some of the surviving E. coli were likely injured by the freezing treatment due to ice crystal formation but could recover in the brain heart medium, so an overall decrease in population was not shown. These bacteria injured cells could possibly be inactivated under pressure. As a result, high pressure treatment of frozen-thawed samples resulted in a much higher cell destruction than that of unfrozen samples. The destruction of E. coli at 150, 240 and 330 MPa were 1.31, 1.54 and 2.92 log, respectively. The microbial reduction at 150 MPa was similar to the lethal effect caused at 330 MPa treatment of unfrozen samples, implying that to achieve the same inactivation effect, the pressure required for frozen-thawed samples would be much lower than that of unfrozen samples.For the frozen thawed samples after treatment with high pressure, E. coli should be considered from three aspects: (1) the freezing process before compression, (2) the synergistic effect of high pressure and freezing during high pressure treatment and (3) phase transition from ice I to ice III. The freezing process before pressurization made some E. coli cells injured, and the injured cells were more likely to be destroyed during pressurization. The synergistic effect of high pressure and freezing can be understood as follows: during the HP treatment of frozen samples, E. coli cells in SPS remained in the frozen state, and the pressure resistance was significantly lower than that of E. coli in a liquid state; at the same time, the samples located within the cooling system were subjected to additional pressure created by the solid cooling medium, and hence E. coli cells were subjected to a greater mechanical stress. Under the combined action of the two, the lethal effect of high-pressure treatment of frozen E. coli would be higher than that of freezing-thawed E. coli and unfrozen E. coli samples. The destruction effect achieved at 150 MPa was a little more than the lethal effect of frozen-thawed samples treated at 240 MPa, which would result in reduced operating pressure and costs from the point of view of commercial application. From 240 MPa to 330 MPa, the bactericidal effect was significantly improved by 1.39 log. According to the temperature and pressure curves in E. coli before and after phase transition had a significant difference (p < 0.05), indicating that the phase transition from ice I to ice III was an important factor affecting the inactivation of E. coli. Under the condition of freezing, the rigidity of bacterial cell membrane and cell wall can increase [For the pre-frozen samples, the inactivation effect of HP treatment on increase , thus deE. coli under the same pressure and different states, there was no doubt that the inactivation level of E. coli in frozen treated samples was the highest, followed by the inactivation of E. coli HP treated frozen-thawed sample, and the lethality of E. coli in unfrozen samples was the least. With the increase of pressure, the E. coli survivors in various states all gradually decreased, and high-pressure treatment of frozen samples had the best bactericidal effect. Although the data related with effect of phase transition of ice under high pressure condition on inactivation of E. coli was smaller than those in many other studies [E. coli was killed by about 3 logs at 400 MPa. Similarly, Li et al. [E. coli. In our experiment, the HPLT phase transition at 330 MPa killed 3.68 log of E. coli cells, which was relatively small compared with the data of phase transition microbial inactivation in high-acid medium, but relatively reasonable compared with the data of phase transition microbial destruction in food medium with a pH value close to neutral.Comparing the destruction results of  studies ,29, this studies  selectedi et al.  chose miAccording to the temperature and pressure profiles discussed in As the name suggests, the metastable phenomenon is unstable and can be sensitive to external disturbances. Therefore, the vibration source might affect the metastable state, making the metastable limits easier to be broken/altered, and change the phase transition position  accordingly. Vibration experiments can be divided into internal and external vibration experiments according to the relative location of vibration source.In the external vibration, the cooling medium located around the sample was used as the vibration source, and the sample located at the core served as the target object. The shock vibration generated by the instantaneous volume reduction of water in the vibration source during the phase transition of ice I to ice III was used to realize the synchronous or advance phase transition of ice I to ice III in the target object. In the external vibration test, frozen pure water, frozen 5% sodium chloride solution and frozen 5% glucose solution were used as vibration sources, and frozen apple juice was used as the core target.Taking the temperature and pressure curves of frozen pure water vibrating frozen apple juice as an example , when thBecause the freezing point of pure water is higher than that of any other solution, when the pure water reached the phase transition curve of ice I-liquid, its temperature curve was placed above the temperature curve of apple juice, which was different from the temperature curve placed below the sample temperature curve when 20% sodium chloride solution was used as a cooling medium in In the study of pure water as a vibration source and apple juice as the target object, it was observed that shock vibration can affect the phase transition position of ice crystals. In order to further evaluate the impact of the magnitude of the shock vibration (instantaneous reduction of pressure during phase change of vibration source) and the position of the shock vibration (position of the pressure during phase change of vibration source) on the phase change position of the target object, further research was carried out with 5% sodium chloride solution and 5% glucose solution as the vibration source and apple juice as the target object. There were two reasons for choosing these solutions as vibration sources: (1) In the preliminary experiments, it was observed that the phase transition pressures of metastable ice I and metastable water to ice III in 5% sodium chloride solution and 5% glucose solution were 246 MPa and 229 MPa, respectively, which happened earlier than that of pure water, so it can be used as the difference in shock vibration position; (2) During the phase transition of 5% sodium chloride solution and 5% glucose solution, the instantaneous reduction of pressure was 23.8 MPa and 17.8 MPa, respectively, which can be used as the difference of the magnitude of the shock vibration. The results are shown in p > 0.05) in phase transition pressure. On the contrary, although the phase transition pressure (246 MPa) of 5% sodium chloride solution was relatively backward, the shock vibration generated during phase change was relatively large (23.8 MPa), which gave enough energy to the apple juice in metastable state to break through the energy barrier of phase transformation; thus, the phase transition took place in advance, and the phase transition pressure was significantly (p < 0.05) advanced by 40 MPa.By comparing the results in To summarize, the impact of the magnitude of the shock vibration of the vibration source on the phase change position of the target object cannot be ignored. Although the position of the shock vibration of the vibration source in front of the target object in this experiment did not necessarily lead to the early phase change of the target object, the impact of the shock vibration position cannot be completely ruled out. Perhaps the phase transition pressure of 5% glucose solution in this experiment was too close to the triple point of liquid-ice I-ice III (210 MPa), and the phase transition reaction near the triple point was more complex, which affected the transmission of shock vibration.In the internal vibration experiments, the sample located inside the cooling system was used as the vibration source, and the cooling medium around the sample was used as the target object. The instantaneous reduction of volume of water in the vibration source during the phase transition of ice I to ice III was used to produce instantaneous collapse vibration in the target object and promoted the ice I to ice III phase transformation in the target object to occur in advance.p > 0.05) between the phase transition pressure of pure water obtained by glucose solution and sodium chloride solution as a vibration source. According to the standard deviation of phase transition pressure of pure water, it can be found that although the phase transformation of pure water can be realized in advance through an internal vibration test, the position of phase transition was still unstable, which may be caused by the random occurrence of phase transition of water in the vibration source. This phenomenon also existed in external vibration experiments.In the internal vibration experiment, 5% sodium chloride solution and 5% glucose solution were used as vibration sources, and pure water was used as the target object. The experimental results are shown in The principle of internal vibration experiment can be described as follows: (1) when metastable ice I and metastable water to ice III phase transitions occurred in the vibration source (5% sodium chloride solution and 5% glucose solution), the volume decreased instantaneously; (2) a gap was created between the vibration source at the center of the cooling system and the surrounding target object (pure water). Under the action of high pressure, the pure water collapsed and was squeezed together instantly; (3) at this time, the pure water in metastable state gained energy due to collapse vibration, broke through the energy barrier of phase transition, and completed the phase transition of metastable ice I and metastable water to ice III. The external vibration method has proved that the instantaneous reduction of pressure (the magnitude of shock vibration) during the phase change of vibration source had an impact on the early occurrence of phase change of target object. Hence, in the internal vibration experiment, the sample volume was changed from 4 mL to 50 mL in order to produce greater collapse vibration to realize the early phase transition. The advantage of the internal vibration method is that it improves the processing capacity of the target object from 4 mL of the external vibration method to 400 mL of the internal vibration method, and the amount of vibration source (50 mL) in the internal vibration method is much less than that in the external vibration method (446 mL), which can be of great significance in the cost-savings of a practical application.These vibration experiments are evaluated as possible contributors for microbial destruction enhancement achieved in the first part of this study when the samples were subjected to HP treatment under fully frozen conditions. Under these conditions, the samples experience phase transition effects and vibrational effects can predominate. This type of detailed analysis of metastable state kinetics and positional effects on microbial destruction has never been analyzed before, and such research is only the beginning. In the future, more detailed experimental evaluation can be expected to be carried out in order to more deeply analyze the law and mechanism of ice crystal phase transition, as well as to carefully evaluate the effect of these vibrations on microbial destruction kinetics.E. coli in SPSS was demonstrated to be different when HP treated in unfrozen, frozen-thawed and fully frozen states. The freezing process before compression, the synergistic effect of pressure and freezing during HP treatment and phase transition from ice I to ice III contributed the best inactivation effect, especially the phase transition process. In addition to microbial kinetics, experiments were also designed to understand the vibration behavior of test samples undergoing phase transitions. By selecting the appropriate vibration source, the target object can break through the metastable state in advance and realize the phase transition from ice I to ice III, which was the first attempt in the research field of HPLT phase transition. Factors affecting the phase transition position need to be further explored in the future, and relevant research is also underway. Because the phase change pressure is an important working parameter in the process of phase change microbial, breaking the metastable state and advancing the phase change position means that it can greatly reduce the working pressure during industrialization. Considering the economic cost and equipment safety, this is the best of both worlds.The self-cooling equipment developed in our laboratory can maintain test samples in a frozen state even when the pressure chamber is held at room temperature processing conditions, thus enabling the phase transition studies to be carried out without sophisticated equipment. The inactivation of Current experiments have demonstrated the effect of phase change microbial destruction. The vibration studies have opened up new avenues of research in the phase transition inactivation of microorganisms.


Analysis shows the impact of the SO sea ice concentration on northward swell peaks in September–November, coinciding with the maximum sea ice extent in the Antarctic region of the Indian Ocean. W3no_ice simulations are biased more by ~ 60% and ~ 37% in significant wave height and period, respectively, against W3with_ice when compared with NIO mooring data. W3no_ice simulates low-frequency swells and travels fast towards NIO, with implications for operational oceanography. We have shown that the forecasts of the timing of high swell events along NIO coasts can be erroneous by ~ 12 h if the SO sea ice concentration is not included in the model. Further, W3no_ice could potentially produce false swell alerts along southeastern Australian coasts. In summary, our study highlights the importance of the SO sea ice concentration inclusion in the wave models to accurately simulate NIO waves.The present study analyzes the impact of the Southern Ocean (SO) sea ice concentration on the north Indian Ocean (NIO) wave fields through swells using 6 years (2016–2021) of WAVEWATCH III (WWIII) simulations. We did two experimental runs of WWIII, one with sea ice concentration and winds as the forcing (W3 They also serve as a crucial connection between the Antarctic regions and the tropical climate and provide a buffer protecting the Antarctic coastline from the SO's intense storms and ocean swells5.The sea ice coverage of the Southern Ocean (SO) undergoes a complete seasonal cycle, with minimum ice cover during February and maximum during September–October. The Marginal Ice Zones (MIZ), the transitional zone between the open sea and dense, thick ice packs further south, is an important region where intense coupling between waves, sea ice concentration, ocean, and atmosphere occurs. MIZ is usually not a sharply demarcated edge but a belt comprising ice floes with patches of open water in between them and plays a vital role in regulating global weather and climate patterns. The sea ice in the MIZ regulates the global ocean's carbon and heat uptake6, while the attenuation and scattering of waves under the ice ensure its final decay8. Efforts to integrate the wave-sea ice interactions into the ice/ocean model started long back9, but the non-availability of in situ observations for validating the parameterizations hinders success. Major approaches to modelling the sea ice concentration of MIZ vary from simple models with dependence on floe size to direct numerical simulations (DNS) using granular models with either a single floe diameter10 or flow diameters from a sample11. These studies  underscored the importance of two-way wave-ice interaction, ways of parameterizing it in sea ice models, and its effects. While the role of sea ice concentration in MIZ in regulating the Antarctic polar climate received much scientific attention, its role in modulating wave climate of remote places, like the north Indian Ocean (NIO), through swell generation did not receive the required focus.The variations of sea ice concentration in the Antarctic MIZ impart local and remote changes. Locally generated wind waves travel through the loosely packed sea ice concentration of MIZ and further contribute to more ice breakage13. These waves travel outside this wave-generating area and northward as long-period swells, modifying the wave climate of the northern hemisphere. Studies in the Indian Ocean (IO) show that the region bounded by 30–50° E and 70–50° S is a strong swell-generating area14. Several studies use modelling tools and satellite observations to focus on the IO's swell generation mechanism, propagation route, and energy spread. The swell wave power density of the IO increases from west to east and tends to pile up along the west coast of Australia and then spreads northward of IO15. Another modelling study14 found that the swell wave height contours in the IO have a pronounced northward feature, and the dominant swell direction is from the south year-round. Generally, there is a convergence of opinion that the SO swells play an important role in determining the wave characteristics of NIO16. But, to date, there has not been any study determining the role of sea ice concentration of the SO MIZ in regulating the swell generation and, subsequently, the wave fields of NIO.Northward of the MIZ, strong year-round westerlies blow over practically an unlimited fetch, generating some of the fiercest waves on the planet17. Hence any modifications to wave climate have an important societal impact on their livelihood. For example, the long period swells from the SO cause flash flooding along the Indian coastline, called the Kallakkadal events18, creating much distress in the coastal community. The SO swells cause coastal inundations, hindrance to maritime operations, and possible damage to agriculture by contaminating freshwater reservoirs along coasts. Therefore a thorough knowledge of the modulation of NIO swell characteristics by the SO sea ice cover is significant, especially in the wave projection and forecasting context.The regions in and around the IO are home to roughly 2.6 billion people, which is 40% of the global population, out of which one-third are located within 20 km of the coastlineThis article is an effort in that direction to estimate and discuss the impact of the SO sea ice concentration on the NIO swell wave characteristics using WAVEWATCH III (WWIII) modelling tools. The rest of the article is organized as follows. Data description and the methodology are described in section two. The differences between the two WWIII simulations we used in this study are discussed in section three and four. We end this article with a summary and discussion in section five.19 based on an algorithm designed to meld the high-resolution bathymetry with the shoreline database to develop the optimum grid. WWIII integrates the spectral wave energy balance equations in space and time with discretized wave numbers and directions. The present study uses a spectral resolution of 29 frequencies ranging from 0.035 to 0.5 Hz at an increment of 10% in 36 directions. The WWIII package includes many dissipation/input parameterizations, of which switch ST420 is used in this study. As we plan to quantify the impact of the SO sea ice concentration on the swells in NIO, we used the wave-ice parameterization scheme21 which provides simple energy flux blocking depending on the local ice concentration.The numerical wave model used in the present study is the WAVEWATCH III- version 6.07 (WWIII) . INCOIS-WWIII setup has four mosaic grids in nested pattern  with spatial resolution varying from coarser 0.5° global to the finer 0.04° coastal grids. ETOPO1 bathymetry  from National Geophysical Data Centre and global shoreline database  is used with an automated grid generation package V2.2The wind data used is the 3-hourly European Centre for Medium-range Weather Forecast (ECMWF) winds with a spatial resolution of 0.25° × 0.25°. The daily sea-ice concentration data product from National Snow and Ice Data Centre's Soil Moisture Active Passive (SMAP) L1-L3 Ancillary NOAA Data, Version 1, for 6 years (2016–2021) is used. This dataset, having global coverage and spatial resolution of 0.03° (4 km polar gridded), is used as an input field in WWIII for analyzing the impact of sea ice concentration in the SO on NIO swells.22 under the National Data Buoy Program (NDBP). These moorings are named as AD06 , AD07 , AD09 , all located in the Arabian Sea; BD08 , BD11 , and BD14  all in Bay of Bengal basin. Statistical comparison of model results with observation data is made using Pearson's linear correlation coefficient (r), Bias, Root Mean Square Error (RMSE), and Scatter Index (SI). Altimeter data from Jason-2 has been used for the validation of Hs in the SO. Jason-2 covers a swath width of 6–7 km with repeat cycles at about 10 days.The model results are then validated against data from six offshore moored buoys from NIO Fig. , deployeno_ice), and in the second, WWIII was run with both winds and the SO sea ice concentration as the input fields (W3with_ice). The monthly averaged difference between W3no_ice and W3with_ice for a period of 6 years (2016–2021) in significant wave height (Hs), swell height (HsS), mean wave period (Tm), and swell period (TmS) at AD07 (blue bars) and BD08 (red bars) locations are shown in Fig. no_ice and W3with_ice is observed in the SON period. The simulations without sea ice concentration generate low-frequency swells with more wave height in the NIO basins than simulations with sea ice concentration. The maximum monthly mean difference in Hs (HsS) between the two simulations (W3no_ice − W3with_ice) in the NIO was observed in October, approximately 6.5 cm (6–7 cm), respectively. In the September–October period, a similar trend is noted in Tm and TmS in the NIO basins. The impact of sea ice concentration on the SO swell waves is maximum during the September–October–November (SON) season and minimum during March–April–May (MAM), coinciding positively with the spatial variation of sea ice concentration in the SO  propagating northwards. The total agreement of the model with the buoy cannot be seen in any of the wave parameters, which is expected due to the discrepancies involved in the forcing wind, ice concentration fields, the model parameterization scheme, etc.The difference between W3with_ice and W3no_ice simulations against the six NIO mooring observations during the SON period of 6 years (2016–2021). The statistics shown are 95% significant (p = 0.05) using Student’s t-test. In all the NIO mooring locations we considered in this study, W3no_ice HsS simulation biases are 8–10 cm more than W3with_ice simulations compared to observations. We found that W3no_ice is ~ 60% (37%) more biased than W3with_ice simulations in HsS (TmS) averaged over the NIO mooring locations. A comparison of Tm and TmS suggests that W3no_ice produces more low frequency (peak shift towards lower frequencies) swell waves compared to the swells in W3with_ice. The spectral energy data available for September 2017 at the BD08 location is used for the energy comparison between the two model simulations , W3no_ice and Wwith_ice3 behave almost similarly. Overall, W3with_ice is better at predicting low-frequency swells than the other simulation in the NIO basins.Table with_ice and W3no_ice simulations for the ice-free regions of the SO  for SON of 2017. Both W3with_ice and W3no_ice simulations are correlated well with satellite Hs, but W3with_ice Hs exhibit more scatter than W3no_ice Hs. One noticeable feature is the overestimation (~ 20 cm) of mean Hs by W3no_ice in the lower ranges, which are mainly the developing seas. Hence, similar to the wave field comparisons in the NIO with mooring data, satellite Hs comparison in the ice-free regions of the SO suggests that W3with_ice simulates Hs better than W3no_ice.In-situ observations are unavailable from the SO for wave field comparison between the two simulations. One possible option to ascertain the quality of wave simulations in the SO is to compare the Hs from the satellite with the model simulations. Figure with_ice and W3no_ice simulations and found that W3with_ice simulates accurate wave parameters. This section shows the local and remote implications of the SO sea ice concentration inclusion as an input field in the wave simulations. Figure with_ice simulation. Around 1,00,00 km2 area is covered with sea ice concentration in the southern sector of IO during SON, which acts as a large fetch area for wave generation in W3no_ice simulation. The area around 30–50° E and 70–50° S is considered a strong swell generating area14 with a fetch size of ~ 44,000 km2. In W3no_ice, the entire 44,000 km2 is available as fetch for swell generation, while in W3with_ice, ~ 46% of the area is ice-covered during SON  and the other with only winds as forcing (W3no_ice), to explore the effect of the SO sea ice concentration on wave simulations. Our analysis suggests a non-negligible difference in HsS and TmS in the SO  sea ice concentration on the Indian Ocean (IO) wave conditions using WAVEWATCHIII (WWIII) numerical wave model simulations. The impact of the SO sea ice concentration on IO swells is not explicitly addressed in scientific literature. However, past studies on IO wave climate proved the effect of SO swells on IO coastal areas. In the present study, we did two WWIII model simulations, one with winds and the SO sea ice concentration as forcing fields  we used here.23. We did not focus on validating different wave-ice parameterization schemes here but selected the simple IC0 scheme, which provides energy flux blocking depending on the local ice concentration and does not consider the thickness of the ice. Studies suggest that the simulation of wave fields under ice-covered conditions relies on the accuracy of sea ice concentration data used as a model forcing and the accuracy of the wave-ice parameterization23. Future studies may require validating the sea ice concentration data and the wave-ice parameterizations.WWIII employs several wave-ice parameterization schemes  to account for the effect of sea ice concentration in wave simulations2 in DJF to a maximum of 1,08,308.75 km2 in SON, and a wave model without this large sea ice extent will grossly overestimate the wave growth. Hence our arguments in this article will be valid irrespective of the above caveats, and any change in the ice extent in the SO will substantially impact NIO through the SO swells. Thus our study recognizes sea ice concentration in the SO as a critical factor in modifying wave characteristics in the NIO and underscores the need to include it in wave modeling for forecasting and climate simulations.The sea ice extent in different seasons in the Indian sector of the SO varies largely, from a minimum of 74,208.5 km


Background and Objectives: Using an appropriate torque to tighten set screws ensures the long-term stability of spinal posterior fixation devices. However, the recommended torque often varies between different devices and some devices do not state a recommended torque level. The purpose of this study is to evaluate the effect of set screw torque on the overall construct stability and fatigue life. Materials and Methods: Two commercial pedicle screw systems with different designs for the contact interface between the set screw and rod  were assembled using torque wrenches provided with the devices to insert the set screws and tighten to the device specifications. The axial gipping capacity and dynamic mechanical stability of each bilateral construct were assessed in accordance with ASTM F1798 and ASTM F1717. Results: Increasing or decreasing the torque on the set screw by 1 Nm from the recommended level did not have a significant effect on the axial gripping capacity or fatigue strength of Group A (p > 0.05). For Group B, over-tightening the set screw by 1 Nm did cause a significant reduction in the fatigue strength. Conclusions: Excessive torque can damage the rod surface and cause premature failure. When insertion using a manual driver is preferred, a plane contact interface between the set screw and rod can reduce damage to the rod surface when the set screw is over-torqued. Pedicle screws are commonly used to treat disorders of the spine through instrumented spinal fusion. The aim is to correct deformities and stabilize the spine to safely achieve arthrodesis. Stiff and stable constructs have been shown to improve fusion rates and increase the strength of the fusion mass ,2.Spinal surgical techniques have evolved considerably over the past 10 years, particularly with the use of minimally invasive surgery where pedicle screws can now be inserted through a 2–3 cm incision .Standard top-loading pedicle screws have a tulip-type screw head with a U-shaped slot to accommodate the rod, which is held in place by a set screw. The clamping force of the system is mainly determined by the ability of the set screw to maintain a tight clamp on the rod when the rod is subjected to a radial force. The set screw can be torqued manually using screwdrivers, but this is prone to error with variation in the torque applied. Inadequate torque may lead to set screw loosening and failure, while excessive torque may lead to fracture of the rod ,6,7. TheImplant disassembly due to loosening the set screw is uncommon, but insufficient tightening of the inner nut into the pedicle screw head can predispose the system to rod migration ,11. Few Some rod and screw systems are supplied with a torque-limiting driver or breakaway set screw design to allow for consistent tightening of all set screws. However, this is not standard with all pedicle screw systems, and the recommended driving torque is often not detailed in the instructions supplied with the product. The effect of set screw torque on construct stability and screw loosening has not been investigated to date. The aim of this study is to evaluate the stability of commercially available pedicle screw constructs tested according to ASTM F1798 and ASTM F1717 using different torques for the set screws. Insufficient preloading or inadequate tightening of set screws can create stress concentrations and compromise implant stability. Hence, this study will investigate the relationship between the tightening torque and overall construct stability to improve understanding for clinical applications.Two commercially available pedicle screw and rod systems were assessed in this study, both using 4.0 mm-diameter screws that accommodate a 5.5 mm-diameter rod . Both syt-test was used to detect significant differences in AGC for different torque values .The axial gripping capacity (AGC) was assessed using 15 pedicle screws and 15 spinal rods for each device (Group A and B). One end of the spinal rod was clamped to the actuator using a collet. A poly-axial screw assembly was then placed on the load cell and rested on a metal tube with a recess to allow the rod to advance a. A pedix-axis and 20 mm on the y-axis. Set screws were tightened at three different torque values to simulate standard insertion (manufacturer recommended), over-tightening by 1 Nm and under-tightening by 1 Nm. Dynamic compression bending tests were performed on both constructs according to the methods described in ASTM F1717 [Dynamic mechanical testing was performed on 11 bilateral constructs of each device, in total consisting of 44 pedicle screws and 22 rods. Blocks of ultra-high molecular weight polyethylene (UHMWPE) were formed according to ASTM F1717. As shown in TM F1717 . All tesp > 0.05) between the AGC values recorded for Group A. Group B had an average AGC of 1775.07 N using the standard insertion toque, and over-tightening the set screw did not significantly affect the gripping capacity (p > 0.05). However, there was a significant decrease in AGC when the set screw was under-tightened (p < 0.05).Group A had an average AGC of 2019.65 N when using the manufacturer’s recommended torque to tighten the set screw, while the AGC increased or decreased, respectively, when the torque value was higher or lower than the standard insertion . There wInternal fixation with top-loading pedicle screw systems requires the set screws to be sufficiently tightened to hold the rod in place and achieve a secure fixation and symmetric load condition. It has been reported that operators using hand-held manual drivers can show considerable variation in the torque applied ,15. The The function of set screws is to secure the rod to the poly-axial mechanism. Previous reports indicated that inadequate tightening of the set screws could result in rod disengagement from the screw head . In the Even in cases where a torque-limiting driver is supplied with the implant, an orthopedic surgeon still has the option to tighten the set screw manually. Loosening set screws after implantation has been reported with an incidence rate of 2–12% at the long-term follow up ,22. To mThis study has some limitations. First, only two commercially available poly-axial screws were evaluated using three torque values for the set screws. The methods used are adequate for this study because the intent was to investigate how over- and under-tightening of set screws affects the performance of the construct. In addition, testing was performed in accordance with ASTM F1798 and ASTM F1717 rather than aiming to replicate physiological conditions, so rod bending and variations in the poly-axial tilt angle were not evaluated. ASTM standards are typically used to compare different component designs or surgical techniques in terms of relative mechanical parameters ,25. FinaWithin the limitations of this study, the results demonstrated the importance of achieving the target torque values when tightening the set screw. Insufficient torque can lead to rod slippage or failure of the poly mechanism, while excessive torque can damage the rod surface and reduce the fatigue life. A line contact surface between the set screw and rod can damage the rod surface when the screw torque exceeds the recommended level. The optimal torque range for the set screw often depends on the design and properties of the construct, and, as such, there is no universal torque suitable for all devices. The authors recommend that the target torque range be stated with all devices, and a torque-limiting driver be provided as standard instrumentation for non-breakaway set screw systems. In pre-surgery planning, surgeons should confirm the torque limit handles with manufacturer-recommended torque or set screws with the breakaway head feature have been provided in instrument and implant sets, respectively.


Economic sustainability involves the development of an organisation that meets its future needs through an integrated policy, planning, and social learning process. The purpose of this study was to investigate the mediating role of competitive advantage in the relationship between strategic orientation and economic sustainability under unpredictable circumstances. This study collected quantitative data from a total of 284 halal small and medium enterprises (SMEs) from Indonesia through structured interviews. Data were analysed using partial least squares structural equation modelling (PLS-SEM). Moreover, this study adopted artificial neural network (ANN) analysis for a model-free estimation using non-linear, multilayer, and parallel regression. The results revealed statistically significant and positive effect of strategic orientation on economic sustainability. Additionally, this study found that competitive advantage expanded the effect of strategic orientation on economic sustainability. Findings of ANN analysis confirm high prediction accuracy of the model. Findings of the sensitivity analysis highlighted the importance of innovation, network and technological orientation, and the positive effect of competitive advantage on halal SMEs economic sustainability. In order to achieve long-term economic sustainability, halal SMEs should therefore focus on innovation capacity, vertical and horizontal networking and adoption of the latest technologies. The uniqueness of this study focused on the strategic orientation and value of competitive advantage of halal SMEs towards economic sustainability. Additionally, this study was the first to develop hybrid SEM–neural network analysis to apply sensitivity analysis for the evaluation of the contribution of each exogenous predictor towards the endogenous construct. Indonesia Maju) is one of the government’s initiatives to support sustainability development. Shariah economy and finance  have been identified as key contributors considering that the vast global opportunity of $1.9 trillion in 2020 and domestic opportunity of $184 billion in the related consumer spending market have not been fully tapped. Indonesia is the largest domestic halal economy market, by 87.0% of the total population or about 236.53 million people. This amount is equivalent to 12.7% of the entire Muslim population in the world. The halal economy has remained more unscathed by the effects of the COVID-19 pandemic than the national economy, with a contraction of − 1.70%, as compared to − 2.07% for the national economy in 2020 .The environmental system of an economy is the central factor to gain business opportunities for sustainable development. The concept of sustainability and the interdependence of the economy and environment are increasingly important for policymakers. Small and medium enterprises (SMEs) play essential roles of contributing to the economic growth of developing countries and creating income and job creation; however, numerous small business entrepreneurs face failure , which was proposed by Penrose , is the This study argued that strategic orientation enhances halal SMEs’ competitive advantage in a hyper-competitive environment. Thus, this study attempted to explore and develop a framework of developing hybrid SEM–neural network analysis on how customer orientation, competitor orientation, technology orientation, network orientation, and innovation orientation affect the economic sustainability of halal SMEs through competitive advantage in Indonesia. The above problem statements and the lack of sufficient findings on the related areas prompted the current study to contribute to the existing body of knowledge and address other gaps, such as the inconsistent findings on the relationship between strategic orientation and economic sustainability in related literature.In this study, RBV was captured with respect to strategic decision-making orientation and competitive advantage towards the economic sustainability of halal SMEs in Indonesia. This study designed, developed, and validated an instrument to measure the dimensions of strategic orientation, competitive advantage, and economic sustainability using the hybrid SEM–neural network analysis.RBV (a bona fide theory), which was introduced by Penrose , emphasiBarney  observedMoreover, with respect to emerging markets, studies on RBV have suggested that local firms are interested in using foreign alliances to acquire advantages over their domestic rivals, in emphasising the importance of network ties as an intangible resource for entrepreneurial start-ups, and in understanding the changing benefits of unrelated diversification as economic institutions develop Barney, . The gloBarney  stressedThe concept of the triple helix model was originally proposed by Etzkowitz and Leydesdorff . TheoretFurthermore, Carayannis and Campbell  added a The current study focused on balancing development and sustainability by addressing both innovation and sustainability by modifying the original triple helix model. This study considered the triple helix concept within the context of halal SMEs, specifically in terms of their economic sustainability, towards achieving innovation and sustainability in a hyper-competition setting. There is an increasing awareness that a knowledge-based society operates according to a different set of dynamics, as compared to an industrial society, with an emphasis on SMEs. Knowledge-based innovations are more closely linked to sources of new knowledge and human resources. Fostering a continuous process of the formation of halal SMEs based on advanced technologies moves to the heart of strategy orientation.Additionally, this study supported the quadruple and quintuple innovation helix to change its spin as the production of new knowledge and technology, which has become an increasingly important element for the performance of halal SMEs. This can also drive the sustainability of halal SMEs towards meeting the global and local knowledge economy and society through strategy, democracy of knowledge, and innovation. At this level of the quadruple and quintuple innovation helix, the enhancement of the performance of SMEs and other knowledge-producing institutions often becomes the key issue. The concept of democracy of knowledge, ecology, and society is crucial for halal SMEs to obtain innovation. Sustainable development, progress, and performance across different dimensions of halal SMEs explain the success of quadruple helix.Competitive advantage can be sustained by generating innovative ideas, quick response to customers’ needs, and development of new beneficial products for customers  was employed in this study to assess the complex cause–effect relationships  . Thus, H1a was supported. The present study supported the study by Lee et al. , which supported H1b. Besides that, competitor orientation was found to exhibit significant and positive influence on competitive advantage (H2a)  and economic sustainability (H2b) . These findings were found to be consistent with the findings reported by Tseng et al.  and economic sustainability (H3b) . In other words, H3a and H3b were accepted. Park and Zhang  and economic sustainability (H4b) . These results supported both H4a and H4b. This study also demonstrated the significant and positive influence of innovation orientation on both competitive advantage (H5a)  and economic sustainability (H5b) . This present study supported the study of Haffar et al.  . Thus, H6 was accepted. Desiana et al.  was calculated according to the criterion suggested by Cohen  of all factors at the individual level (single factor). Referring to Table Next, effect size . Moreover, competitive advantage mediated the relationship between competitor orientation and economic sustainability . Similarly, competitive advantage mediated the relationship between technology orientation and economic sustainability  and the relationship between network orientation and economic sustainability . Finally, this study also demonstrated the mediation effect of competitive advantage on the relationship between innovation orientation and economic sustainability . Table The obtained results of this study revealed the mediation effect of competitive advantage on the relationship between customer orientation and economic sustainability  values for training and testing  and economic sustainability of halal SMEs. This study proposed RBV to explain the roles of halal SME owners’ capabilities of implementing strategic orientation and competitive advantage towards economic sustainability in this highly competitive market Barney, . The resThis study demonstrated the significant influence of strategic orientation on economic sustainability of halal SMEs. The results of this study confirmed the significant mediating role of competitive advantage in increasing the performance and promoting economic sustainability among halal SMEs. Similarly, Deyganto  found thBesides that, this study found the positive and significant influence of technology orientation on competitive advantage and sustainability. Technological and innovative activities and the potential for both radical and incremental changes are central for the sustainability of halal SMEs. This study also found the significant influence of technology orientation on economic sustainability, such as technological superiority to differentiate new products. This study supported the findings of Park and Zhang  on technThis study demonstrated the significant and positive influence of innovation orientation on competitive advantage and economic sustainability. Desiana et al.  showed tFurthermore, this study argued that competitive advantage is a crucial factor in the relationship between strategic orientation and economic sustainability. Theoretically, the integration of competitive advantage as a mediator in this study extended the RBV theory and literature on dynamic capability. This study concluded the significant role of competitive advantage in mediating the relationship between strategic orientation and economic sustainability. Additionally, the current study extended the halal entrepreneurship literature through human network, network capability, and competitive advantage in the digitalisation era. Besides that, this study demonstrated the mediating role of competitive advantage. Halal SMEs need to recognise the importance of competitive advantage by transforming new systems in manufacturing, product delivery system, and online sales platform of halal products in order to accomplish strategic diversification and innovation . The quadruple and quintuple innovation helix concepts  are two important basic theories that explain strategic orientation, competitive advantage, and economic sustainability of halal SMEs. This study developed RBV through competitive advantage as a mediator, and this study found that all constructs increase the economy sustainability of halal entrepreneurship. Competitive orientation, customer orientation, competitor orientation, network orientation, innovation orientation, and technological turbulence can adequately improve the survival and sustenance of halal enterprises into an unforeseeable future. Resource capability and competitive advantage can improve the efficiency of enterprises. Strategic orientation is an attribute that can adequately improve the competitive advantage of halal enterprises. Moreover, the quadruple and quintuple innovation helix concepts explain the significant roles of environment, policy, and knowledge for SMEs to gain sustainability. The democracy of innovation, knowledge, and strategy help halal SMEs to expand their market and position in hyper-competitive market.This study offered several recommendations for halal small businesses to remain sustainable or competitive through strategy orientation. The study also presented relevant recommendations for policymakers and other concerned bodies. Based on the findings of this study, innovation, customer, competitor, network, innovation, and competitive intensity are critical in determining the robustness of resource capability. These aspects adequately improve the growth of sales and the survival and sustenance of halal enterprises into an unforeseeable future, as well as the efficiency of enterprises and competitive advantage of halal enterprises, particularly in trying to be different from other competitors. Additionally, decisions on the survival of enterprises into an unforeseeable future, resource capability of competitive advantage and environmental turbulence should be prioritised. Finally, when it comes to the decisions on economic sustainability, resource capability of strategic orientation should be the main focus.This present study’s findings also contribute to the social environment. Halal SMEs should focus on strategic orientation by combining internal resources (innovation and network) and external resources (customer orientation and competitor orientation) to gain competitiveness and sustainability. Secondly, customers have been increasingly aware with healthy consumption and habit of purchasing halal products. Consuming halal products can create an eco-friendly system that creates positive life circumstances.Additionally, this study suggested a few policies. Firstly, the regulation maker of halal certification (Islamic Council of Indonesia) serves as an important strategic institution for sustainability by proposing and educating customers to consume healthy and halal products. Secondly, education is a central plot for new start-ups to obtain entrepreneurial knowledge and training. Thirdly, the government should provide infrastructure development and policies of halal products, which focus on innovation, competition, and growth of SMEs, for halal enterprises to make significant contributions towards economic sustainability. Additionally, the government can support halal SMEs to invest in modern production facilities  in order to have capabilities and capacities of local industries that meet international quality standards, resulting in more halal SMEs that are able to enter new export markets. Moreover, the government, society, and SMEs should increase knowledge, innovation, and culture change to gain their market share locally and globally and expand the performance and economic sustainability of SMEs.This study also recommended that science and halal SMEs focus on implementing strategies, as these strategies facilitate SME managers or owners to design, develop, and enhance resource-based approach and capabilities. With respect to the quadruple and quintuple innovation helix concepts, establishing science and knowledge in universities can be helpful for quicker exchanges or interactions of strategies and capabilities among universities, industries, the government, and actors of SMEs. Last but not the least, the developed questionnaire in this study served as a tool to discover shortcomings and deficiencies in the path of promoting sustainability in science and SMEs.Overall, policy, education and training, and infrastructure development should focus on halal SME innovation, competition, and sustainability for halal enterprises to make significant contributions towards social implementation.This study encountered a few limitations. Cross-sectional data were obtained from 284 halal SMEs in Indonesia. Thus, a larger sample is recommended for future research to generalise and better understand the implementation of strategic orientation and policy decision-making for halal SMEs. Secondly, competitive orientation, customer orientation, competitor orientation, network orientation, innovation orientation, and technological orientation were employed in this study to explore the role of strategic orientation as well as the mediating role of competitive advantage. Additionally, the profitability and cost reduction were employed in this study to represent economic sustainability. Therefore, other indicators of economic sustainability can be implemented to achieve various outcomes. Lastly, the dynamic capability approach can be explored in relation to the economic sustainability of halal enterprises in developing and developed countries.


This study aimed to investigate the inhibition of human important phase II metabolic enzyme sulfotransferases (SULTs) by phthalate monoesters, which are important metabolites of phthalate esters (PAEs).in vitro incubation system was utilized for preliminary screening, and 100 μM of phthalate monoesters was used. Inhibition kinetics were carried out to determine the inhibition of SULTs by phthalate monoesters.Recombinant SULT-catalyzed metabolism of p-nitrophenol (PNP) was employed as the probe reactions of SULTs to investigate the inhibition of 8 kinds of phthalate monoesters towards SULT isoforms. An Ki) towards SULT1B1 and SULT1E1. The inhibition kinetic parameters (Ki) were calculated to be 2.23 μM for MOP-SULT1B1 and 5.54 μM for MOP-SULT1E1. In silico docking method was utilized to understand the inhibition mechanism of SULT1B1 by phthalate monoesters.Multiple phthalate monoesters have been demonstrated to exert strong inhibition potential towards SULT1A1, SULT1B1, and SULT1E1, and no significant inhibition of phthalate monoesters towards SULT1A3 was found. The activity of SULT1A1 was strongly inhibited by mono-hexyl phthalate (MHP), mono-octyl phthalate (MOP), mono-benzyl phthalate (MBZP), and mono-ethylhexyl phthalate (MEHP). Monobutyl phthalate (MBP), MHP, MOP, mono-cyclohexyl phthalate (MCHP), and MEHP significantly inhibited the activity of SULT1B1. MHP, MOP, and MEHP significantly inhibited the activity of SULT1E1. MOP was chosen as the representative phthalate monoester to determine the inhibition kinetic parameters (All these information will be beneficial for understanding the risk of phthalate monoester exposure from a new perspective. Phthalate esters (PAEs) are softening chemicals that are widely used in homes and industries as plasticizers . PAEs arSulfotransferases (SULTs) are an enzyme that catalyzes the sulfuration of various endogenous and exogenous substrates. Sulfuryl transfer reaction has important basic biological significance . SULT cain silico docking and in vitro–in vivo extrapolation (IVIVE) were carried out.The purpose of this study was to investigate the inhibitory effect of phthalate monoesters on the activities of human SULTs. Eight phthalate monoesters commonly used in industry were selected as inhibitors, and four SULT isoforms  were selected for the experiments. In addition, preliminary inhibition screening, inhibition kinetic type, and parameters were determined, and 2 were purchased from Sigma-Aldrich . Ultra-performance liquid chromatography (UPLC) grade acetonitrile was purchased from Tianjin Saifurui Technology Ltd. Millipore Elix 5 UV and Milli-Q Gradient Ultra-Pure Water System were used for the preparation of ultra-pure water. Other reagents were of ultra-performance liquid chromatography (UPLC) grade or of the highest grade commercially available.Eight phthalate monoesters -UV instrument. The column used for UPLC separation is a C18 column  with a flow rate of 0.4 ml/min and a column temperature of 25°C. The mobile phase is 0.5% formic acid aqueous solution for phase A and acetonitrile for phase B. The gradient condition was used as follows: 0–6 min, 5% B; 6–8 min, 95% B; 8–14 min, 95% B; 14–17 min, 5% B. The detection wavelength was 280 nm. The incubation system  containing 100 μM of phthalate monoesters, Tris-HCl buffer , MgCl2 (5 mM), PAPS (40 μM), SULTs, and PNP was used to determine the Michaelis constant (Km) of PNP metabolites. The used concentration range was 1–400 μM for SULT1A1, 0.1–7 mM for SULT1A3, 0.1–1 mM for SULT1B1, and 0.1–1.2 mM for SULT1E1. The kinetic parameters (Vmax and Km) were determined through fitting the data to the Michaelis–Menten equation or the substrate inhibition equation using Prism 4 software.The incubation system  containing 100 mM of Tris-HCl buffer (pH 7.4), 5 mM of MgCl2, and 40 μM of PAPS, and the concentrations of 4-nitrophenol and SULTs are different. The concentrations of 4-nitrophenol were 40, 1,200, 40, and 120 μM for SULT1A1, SULT1A3, SULT1B1, and SULT1E1, respectively. In addition, the concentrations of SULT isoforms were 10, 10, 20, and 10 μg/ml for SULT1A1, SULT1A3, SULT1B1, and SULT1E1, respectively. The absence of phthalate monoesters was set as the negative control. After pre-incubation at 37°C for 3 min, 40 μM PAPS were added for the reaction. The metabolic reaction is terminated by the addition of acetonitrile of the same volume and ensures that the incubation time can reach 30–60 min according to different SULTs. The optimized microsome protein concentration and incubation time were used to ensure linear velocity (v). The final mixture was centrifuged at 12,000 rpm for 10 min, and the supernatant of 10 μl was then used for ultra-performance liquid chromatography (UPLC)-UV instrument. All experiments were repeated in two independent experiments. Screening of phthalate monoesters was carried out with a SULT inhibition rate greater than 80% for subsequent experiments.4-Nitrophenol was employed as a nonselective probe substrate for human SULT isoforms to investigate the inhibition of phthalate monoesters on SULT isoforms. The incubation system  containing 100 μM of phthalate monoesters,100 mM of Tris-HCl buffer (pH 7.4), 5 mM of MgCl50) was calculated. Based on different SULT isoforms, multiple concentrations of 4-Nitrophenol  and phthalate monoesters  were used to determine the inhibition kinetics. Lineweaver–Burk plots are used to determine the type of inhibition kinetics. The second plot was used to calculate the inhibition kinetic parameters (Ki), using the linear slope of the Lineweaver–Burk double reciprocal plot and the concentration of the inhibitor phthalate monoesters.The concentration-dependent inhibition effect of phthalate monoesters on SULTs was investigated using different concentrations of phthalate monoesters (ranging from 0 μM to 100 μM), and the half inhibition concentration  structure of SULT isoforms was established using a homology modeling method. Autodock Version 4.2 was employed to dock the flexible small molecule of phthalate monoesters into the rigid protein of SULTs. The non-polar hydrogen atoms of SULT enzymes were merged. The gridbox was generated with 60 × 60 × 60 in X, Y, and Z coordinates, covering the entire ligand-binding site. Lamarckian Genetic Algorithm (LGA) method was selected to possess molecular docking study for the binding of phthalate monoesters towards SULTs. The interactions between phthalate monoesters and SULTs were analyzed, including hydrogen bonds and hydrophobic contacts.In order to better clarify the molecular interaction between phthalate monoesters and SULTs, an t-test. Multiple groups were compared using the one-way ANOVA, and p < 0.05 was considered to be significant.Experimental data were expressed as the mean value plus standard deviation (S.D.). Statistical analysis between two groups using a two-tailed unpaired Student’s The inhibition of phthalate monoesters on SULT1A1, SULT1B1, and SULT1E1 is shown in 50 was further determined. The concentration-dependent inhibition of MHP and MOP towards SULT1A1, SULT1B1, and SULT1E1 was exhibited . As shown in Ki). According to Ki) were calculated to be 2.2 μM and 5.5 μM for the inhibition of MOP on SULT1B1 and SULT1E1, respectively.Some phthalate monoesters significantly inhibited SULT1A1, SULT1B1, and SULT1E1, and ICxhibited Figure 3in silico docking method to analyze the mechanism of its inhibition of SULT1B1. The in silico docking method was used to dock the chemical structure of phthalate monoesters into the activity cavities of SULT1B1, and the representative docking results of MHP and MOP were given. The active site where SULT1B1 binds to MHP consists of amino acid residues ARG-131, SER-49, TRP-53, GLY-50, THR-52, THR-51, PHE-256, LYS-48, PRO-255, SER-139, ARG-258, HIS-142, and PHE-143, as shown in Since phthalate monoesters showed broad inhibition on SULT1B1, SULT1B1 was selected as the representative SULT isoform. We used the in vitro determination system was used to investigate the inhibition behavior of phthalate monoesters on the activity of various isoforms of SULTs. The results showed that SULT1A1, SULT1B1, and SULT1E1 were strongly inhibited by some kinds of phthalate monoesters, but no significant inhibition on SULT1A3. In addition, the results also showed that short-chain phthalate monoesters, such as MMP and MEP, had no significant inhibition on all SULTs isomers, while SULT1B1 was relatively significantly inhibited.In this study, an IVIVE was used to calculate the in vivo potential inhibition of SULTs isoforms by phthalate monoesters. MOP was chosen as the representative phthalate monoesters, and the determined Ki values were 2.23 μM and 5.54 μM for SULT1B1 and SULT1E1, respectively. According to the [I]/Ki ratio ([I]/Ki > 0.1) evaluation standard, the threshold values were calculated to be 0.223 and 0.554μM for the inhibition of MOP towards SULT1B1 and SULT1E1, respectively. Therefore, when the in vivo exposure concentration of MOP is greater than 0.223μM, the metabolism of endogenous substances mediated by SULT1B1 may be inhibited. When the in vivo exposure concentration of MOP is greater than 0.554μM, the metabolism of endogenous substances mediated by SULT1E1 may be inhibited. SULT1A1 and SULT1B1 are generally considered to be the main enzymes involved in the detoxification of exogenous drugs in the human body , the National Natural Science Foundation of China (81970351 and 81822005), the Key Research and Development Program of Shaanxi (No. 2020SF-253), the Central University Basic Science Foundation of China (119132971000056), and the Clinical Research Award of the First Affiliated Hospital of Xi’an Jiaotong University, China (No. XJTU1AF-CRF-2017-006).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The handling editor [FZ] declared a shared affiliation with the author [CH] at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The gold standard to diagnose food allergy (FA) is a double-blind, placebo-controlled food challenge (OFC), even if it shows potential risk of severe allergic reactions for the patient and is time-consuming. Therefore, easier, and less invasive methods are needed to diagnose FA and predict the tolerance, changing the clinical practice.The main aim of this study was to assess whether the total IgE values at the diagnosis of FA were associated with the duration of the tolerance acquisition and thus of the food elimination diet.We retrospectively analyzed the medical records of 40 patients allergic to milk or egg who performed an OFC for the reintroduction of the causal food at the Pediatric Allergy and Respiratory Unit of the University of Chieti from January 2018 to December 2020.β = 0.152; CI, 95% 0.04–0.27) after adjusting for age, sex, and type of allergy (milk or egg). We also showed a significant correlation  between the total IgE values and the duration of the elimination diet and a significant correlation between the casein specific IgE values at diagnosis of FA and the severity of the clinical presentation .We found a positive association of total serum IgE with the elimination diet duration , may be useful in the prognostication of natural tolerance acquisition. Cow’s milk is the most common and clinically important food allergen in children, likely because it is an important nutrient source in early childhood . Howeverin vitro  is considered the gold standard to diagnose FA and to assess the attained tolerance , but it in vitro , affectiin vitro , 14. TheThe main aim of this pilot study was to assess the association between total IgE levels at the diagnosis of FA and the duration of the causal food elimination diet in order to establish the best timing to perform an OFC without severe allergic reactions.•children diagnosed with IgE-mediated allergy to milk (group 1) or egg (group 2) who were on a specific food elimination diet; the diagnosis was based on clinical history and positive food-specific IgE values;•OFC test performed to assess for tolerance acquisition, in the Pediatric Allergy and Respiratory Unit of the University of Chieti from January 2018 to December 2020;•available total IgE values at the diagnosis (time 0), specific IgE for egg and milk and recombinant milk allergens  at the diagnosis (time 0) and at the time of OFC (time 1).We carried out a retrospective study to investigate the clinical and laboratory parameters in milk and egg allergic children. The inclusion criteria were:(1)Mild (grade 1): isolated local reactions of the skin or mucous membranes in the site of contact with the allergen.(2)Moderate (grade 2): allergic reactions involving skin areas away from the site of contact with the allergen, the upper airways, and/or the gastrointestinal tract.(3)Severe (grade 3): severe allergic reactions, potentially at risk of life, which include signs which include cardiovascular, neurological, bronchial and/or laryngeal signs and symptoms.An accurate family and personal medical history was collected by the parental interview and by consulting medical records. Symptoms of allergy at the onset and children age at the onset of FA, at the beginning of food elimination diet and of food reintroduction were obtained. To better describe our study population, symptoms at the allergy onset were classified as localized  or systemic (anaphylaxis) and characterized according to the severity of symptoms . The defAll the patients involved in the study were selected to perform OFC when their food-specific IgE results fell below 15 KUA/L that we usually use as a cut-off; furthermore, none of them presented recent clinical reactions to accidentally administered causal food in the previous twelve months. In our center, we usually visit allergic patients annually and perform food-specific IgE dosages each time .Primary outcome was to investigate the association between total IgE levels at baseline and the duration of elimination diet (years).- to assess the difference between food-specific IgE levels (milk or egg) at the diagnosis of FA and at the time of food re-introduction;- to assess the association of different recombinant milk allergens with the severity of the clinical manifestations at the diagnosis.Secondary outcomes were:Written informed consent was obtained from parents or legal representatives of all children. The study was approved by the local Ethical Committee of the University of Chieti, and it was conducted in compliance with ethical principles based on the Declaration of Helsinki.χ2 test for categorical variables. Pearson's correlation was used to assess the correlation between total IgE levels and duration of elimination diet.Continuous data were presented as mean and standard deviation (SD) or median and range 5%–95%. Categorical data were presented as absolute numbers and percentages. Shapiro-Wilk test was used to investigate the normality of the distribution of all variables. Given the non-normal distribution of total IgE, the logarithmic transformation has been applied to normalize this variable. Nonparametric tests, such as Wilcoxon test or Mann-Whitney test, were used for continuous variables, while the P-value <0.05 was considered for the significance of our statistical reports.In addition, a multivariate linear regression model was performed to investigate the association between total IgE and duration of elimination diet after adjusting for potential confounding factors, such as sex, age at the diagnosis and type of allergy. Spearman's correlation was performed to assess the correlation of different recombinant milk allergens  with the severity of symptoms at the diagnosis. Statistical analysis was conducted using Stata 15 Software for Statistics and Data Science for WindowsWe enrolled 40 patients: 14 (35.0%) were on milk elimination diet and underwent milk OFC, while 26 (65.0%) were on egg elimination diet and performed egg OFC. Clinical and laboratory characteristics of our population were reported in Clinical manifestations at the onset occurred at the median age of 9 months in the group 1. Symptoms described by parents were skin reactions in 7/14 (50.0%) patients, gastrointestinal disorders in 7/14 (50.0%) patients, upper respiratory involvement in 1/14 (7.1%) patients, anaphylaxis in 2/14 (14.3%) patients. Symptoms were mild in 2/14 (14.3%), moderate in 8/14 (57.1%) and severe in 4/14 (28.6%) children.Clinical manifestations at the onset occurred at the median age of 12 months in the group 2. Symptoms reported by parents were skin reactions in 12/26 (46.2%) patients, gastrointestinal disorders in 8/26 (30.8%) patients, bronchial involvement 3/26 (11.5%) patients, anaphylaxis in 3/26 (11.5%) patients. Symptoms were mild in 10/26 (38.5%), moderate in 12/26 (46.15%) and severe in 4/26 (15.4%) children.One of 14 (7.1%) milk OFC resulted positive; therefore, 13 children in the group 1 (92.9%) reached the tolerance and started a free diet. Three of 26 (11.5%) egg OFCs were positive; therefore, 23 children in the group 2 (88.5%) reached the tolerance and started a free diet.β = 0.153; CI, 95% 0.04–0.27).The median duration of food allergy (from onset of symptoms to reintroduction of food) was 4.3 years in patients with milk allergy and 5.7 years in patients with egg allergy. We obtained a positive association between total IgE levels and duration of the elimination diet, after adjusting for age, sex, and type of allergy as well   and the duration of the elimination diet in the study population by using Pearson's correlation (= 0.007) .p = 0.008; p = 0.02; p = 0.03; respectively). We also found a significant difference among specific IgE values for egg white and yolk at the diagnosis and those obtained at the time of OFC . As regards the secondary outcomes, we also found a significant difference among specific IgE values for casein, α-lactalbumin and β- lactoglobulin at the diagnosis and those obtained at the time of OFC . On the contrary, no correlation of specific IgE for α-lactalbumin and β-lactoglobulin with severity of symptoms at the onset of allergy was found.In addition, we observed a significant correlation between specific IgE values for casein at the diagnosis and the severity of symptoms at the onset of FA (Rho = 0.66; In the literature, cow's milk allergy appears during early infancy since the first two months of life , 19. EggIn group 1, 50.0% of milk allergic patients presented cutaneous manifestation, whereas the percentage of cutaneous manifestation reported in the literature is higher. For example, Martorell et al.  found thThe median duration of milk allergy in our sample was 4.3 years. This finding differs slightly from the data in the literature, which reported an allergy resolution rate of 45%–50% within the first year of life, 60%–75% within 2 years, and 85%–90% within the third year of life . The medTo date, OFC remains the gold standard to diagnose FA as indicated by EAACI food allergy and anaphylaxis guidelines . HoweverCurrently, the evaluation of total IgE is only considered a supplemental diagnostic measure for allergic diseases, because of the relatively low sensitivity and specificity and the undefined cost-effectiveness . In the Conversely, Beigelman et al.  found thWe also found a substantial reduction of specific IgE values from diagnosis to the execution of OFC. It is well known that the greater the serum IgEs level, the greater the probability of allergic reactions , 7. In 2As a last point, we found a significant correlation between specific IgE for casein and the severity of the symptoms at the onset of FA. Casein is the main protein allergen in cow's milk  and seveThis is the first study which examined the association between total serum IgE at baseline, and the duration of elimination diet in milk and egg allergic children. However, some methodological limitations should be discussed. Firstly, a retrospective study design could lead to bias related to the type of study. However, we considered several measures to minimize bias and to increase the validity of our findings. To minimize potential selection bias, we fixed clearly defined inclusion/exclusion criteria identifying the population of interest. To minimize potential measurement bias, data were collected by a trained research pediatrician using a predefined questionnaire. Secondly, the small sample size could have affected the power of the study. Lastly, although we adjusted for several confounders, we might not have had information on all possible confounding factors. Therefore, our findings should be confirmed in observational prospective studies with a large sample of allergic children rightly selected who underwent double-blind placebo controlled OFCs.In conclusion, we would suggest that total IgE values could be useful in managing children with FA. Particularly, this simple and cheap test should be considered to guide the decision of the duration of the elimination diet and the timing for food reintroduction. We also confirmed the utility of specific IgE for casein to predict the severity of the clinical manifestations. In clinical practice, the allergy tests commonly used to support food allergy diagnosis are skin prick test and specific IgE levels, with total IgE levels, which can also be used to monitor the spontaneous resolution of food allergy over time.


Urbanization is leading to biodiversity loss through habitat homogenization. The smooth, featureless surfaces of many marine urban structures support ecological communities, often of lower biodiversity, distinct from the complex natural habitats they replace. Eco-engineering  seeks to enhance biodiversity and ecological functions on urban structures. We assessed the benefits to biodiversity of retrofitting four types of complex habitat panels to an intertidal seawall at patch (versus flat control panels) and site  scales. Two years after installation, patch-scale effects of complex panels on biodiversity ranged from neutral to positive, depending on the protective features they provided, though all but one design (honeycomb) supported unique species. Water-retaining features (rockpools) and crevices, which provided moisture retention and cooling, increased biodiversity and supported algae and invertebrates otherwise absent. At the site scale, biodiversity benefits ranged from neutral at the high- and mid-intertidal to positive at the low-intertidal elevation. The results highlight the importance of matching eco-engineering interventions to the niche of target species, and environmental conditions. While species richness was greatest on rockpool and crevice panels, the unique species supported by other panel designs highlights that to maximize biodiversity, habitat heterogeneity is essential.This article is part of the theme issue ‘Ecological complexity and the biosphere: the next 30 years’. ComplIn the sea, as on land, urbanization is simplifying habitats and reducing their complexity ,10. ThisThe low complexity of marine urban structures has the potential to drive marked and pervasive changes in marine benthic communities . BiofilmEnhancement of the complexity of marine urban structures is ideally done at the time of structure design , but forWhether complexity facilitates or inhibits a particular group of species will be dependent on the match between the species' niche and the microhabitats provided . For exaAdditionally, ecological benefits of complexity may depend on its efficacy in mitigating key environmental stressors ,21. On iHere, we assess how retrofitting complex habitat to seawalls influences biodiversity at the patch and site scales. Specifically, we attached complex habitat panels of four different designs and flat control panels across the tidal elevation gradient of a seawall in Sydney Harbour . We then compared ecological communities at the eco-engineered seawall and at unmodified (control) seawalls and natural (reference) rocky shores. We hypothesized that: (i) over time complex panels would acquire a greater number of species (i.e. species richness) and abundance of mobile and sessile invertebrates and algae than flat (control) panels; (ii) complex panels of differing designs would support communities distinct from one another; (iii) differences in community structure among panel designs would be driven by the availability of microhabitats that mitigate heat and desiccation stress; and (iv) at the site scale, eco-engineered seawalls would develop greater species richness and distinct assemblages compared with unmodified control seawalls, and similar richness and assemblages to reference rocky shores.. 2 (a)The study was conducted at five sites on the northern side of Sydney Harbour, New South Wales, Australia: one vertical seawall site installed with complex panels (hereafter ‘Eco-engineered site'), two otherwise similar vertical seawall sites unmodified by panels (hereafter ‘Control sites') and two reference rocky shore sites with a mixture of vertical and sloping horizontal surfaces (hereafter ‘Reference sites'). The Eco-engineered and Control sites were located at Sawmillers Reserve , and the Reference sites at Balls Head Reserve  . The sitca 25 mm pitting across the panel surface), (iii) rockpools (15 cup-like structures per panel designed to hold water as the tide retreats), and (iv) swimthrough . Panels were attached to seawalls using three stainless steel rods drilled into the seawall such that the panels sat approximately 10 cm off the surface of the wall, eliminating the need to clear existing marine life on the seawall or level the seawall prior to panel installation.The Eco-engineered and two Control sites were situated on the seawall at Sawmillers Reserve. This vertical intertidal seawall was approximately 320 m long and had approximately 2 m of intertidal zone. At the Eco-engineered site, seventy-two 550 × 520 × 100 mm concrete ‘Living Seawalls' panels designed by Reef Design Lab  were installed in November 2018 along a 12 m length stretch of shoreline . Panels  (b)Panel-scale sampling tested the hypotheses that within the Eco-engineered site: (i) complex panels would support greater richnesses and abundances of species than flat, control panels and that these differences would strengthen through time as more species colonized; (ii) complex panels of differing designs would support distinct benthic communities; and (iii) differences in communities among complex panels would reflect differences in the microhabitats present, and not be explained by differences in surface area alone. Four replicate panels of each of the five designs  were rein situ using a 25 × 25 cm quadrat with 25 intersecting points, which was placed over their centre. Within each quadrat, the per cent cover of sessile biota was assessed using the point-intercept method and mobile biota were counted. To represent the full sessile community, both primary  and secondary (i.e. growing on the primary species) cover were recorded separately and summed to give a total cover that could exceed 100%. Sessile biota that were present within the quadrat but did not fall beneath a point intercept were given a count value of 0.1 (0.4%) to note their presence. All organisms were identified to species, or where this was not possible, morphospecies.First, to test for differences in communities among panel designs, four panels of each design were sampled Second, to test whether differences in sessile and mobile communities among complex panel designs reflected differences in microhabitats, we subsampled inner  and outer surfaces  of complex panels. Microhabitats were subsampled on four replicate panels of each of the complex designs  at 2, 4, 6, 8, 12 and 24 months following panel installation. For the first five time points (i.e. until 12 months) six replicate mini-quadrats were haphazardly sampled within each of the two microhabitats per panel. At 24 months, this was reduced to three replicates per microhabitat per panel, owing to the substantial growth that slowed sampling. Microhabitats were sampled using 4 × 4 cm quadrats, constructed of transparent film that was marked with 3 × 3 evenly spaced lines to create nine intersection points. The quadrats could be moulded to fit the contour of microhabitats. Sessile and mobile species were quantified within quadrats using the methods described above. For each time point, we averaged mini-quadrats across each microhabitat of a panel to obtain a single estimate of community structure for the inside versus outside microhabitat of each panel.To test for differences in the thermal environment between the inner and outer microhabitats of each complex panel design and the surface of the flat panel, a single iButton data logger  was randomly positioned in each microhabitat of each of three replicate panels per design, at the mid-intertidal elevation. The loggers, waterproofed with Plastidip rubber coating , were programmed to record temperatures with 0.5°C accuracy at 20 min intervals during December 2018–February 2019 and increased to 60 min intervals from February 2019. Loggers were attached to panels using Fisher FIS EM Plus 390 S epoxy mortar and were interchanged approximately every 4–10 weeks to provide near-continuous monitoring of temperature throughout the experiment. (c)Site-scale sampling was conducted to test the hypotheses that: (i) prior to panel installation, the Control and Eco-engineered sites would support fouling communities distinct from those at Reference sites, and (ii) after panel installation, the Eco-engineered site would initially support communities distinct from those at either Control or Reference sites, but through time acquire communities more similar to those at the Reference sites. To test these hypotheses, each of the five sites  was sampled twice before (6 and 1 month prior) and four times after  installation of habitat panels. The exception was Control Site 2 and Reference Site 2, which were only sampled from 6 months onwards (i.e. the four ‘after' times). At the Eco-engineered site, the sampling was done on the unmodified seawall before and on the panels after installation.Owing to strong vertical gradients in communities, sampling of sessile and mobile communities was stratified by tidal elevation. Three intertidal elevations were sampled at each site: high (MLWS + 0.9 m); mid (MLWS + 0.6 m); and low (MLWS + 0.3 m). At the two before-installation times, five replicate 25 × 25 cm quadrats were haphazardly sampled per elevation of each site, at low tide. For the four sampling times after installation of panels, this was increased to 10 replicate quadrats to better document biodiversity. Quantification of sessile and mobile species within quadrats was as described for the panel-scale sampling. (d)Multivariate and univariate permutational analyses of variance (PERMANOVA)  tested hThe development through time of panel-scale effects of complexity on mid-intertidal communities was assessed using three-way analyses, with the factors: Panel design (fixed); Time (fixed) and Panel identity . Panel identity was included as a factor in analyses as the same panels were repeatedly sampled through time. Time was considered a fixed factor as we hypothesized that differences among panels would strengthen through time as additional colonization occurred. These analyses were run on all multivariate and univariate metrics, with the exception of functional group covers, which were instead run separately on 12 and 24 month data and included only the factor Panel design. This approach was taken because of the low abundances of some taxa at earlier sampling times.To assess the extent to which panel-scale differences were driven by differences in protective microhabitats, we conducted a second set of analyses that compared the inner and outer microhabitats of complex panel designs (as sampled using mini-quadrats). These analyses had four factors: Panel design , Microhabitat , Time (fixed) and Panel identity . Similar analyses, but also including the Control microhabitat, were run on the mean, maximum and minimum temperatures recorded by loggers each calendar month. Analyses of temperature variables excluded the factor Panel identity, as loggers were rotated among panels and time points were hence temporally independent.Finally, site-scale effects of complexity were assessed using three-way permutational analyses of variance (PERMANOVAs) with the factors Treatment , Site (nested within Treatment) and Time (fixed). Before versus After was not included as a factor in our analysis owing to predictions of temporal change at the Eco-engineered site following panel installation as well as lack of replication of the Control and Reference sites prior to panel installation (i.e. only one Control and one Reference site were sampled before). A separate analysis was run on data from each of the three tidal elevations, as differences in communities among these otherwise dominated analyses.a posteriori pairwise PERMANOVAs. In instances where too few permutations (i.e. fewer than 100) were available for a given test, p-values were obtained through Monte Carlo simulations )