Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-β-lactamase-1 (NDM-1) coexpression in members of the K. pneumoniae complex  isolated from human and animal hosts, based on inhibition of ceftazidime-avibactam (CZA) and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, or avibactam (AVI). While the presence of blaKPC-2 and blaNDM-1 genes was confirmed by whole-genome sequencing, PCR, and/or GeneXpert, coexpression was successfully detected based on the following: (i) a ≥5-mm increase in the zone diameter of ATM (30 µg) disks plus AVI (4 or 20 µg) and ≥4-mm and ≥10-mm increases in the zone diameters for “CZA 50” (30 µg ceftazidime [CAZ] and 20 µg AVI) and “CZA 14” (10 µg CAZ and 4 µg AVI) disks, respectively, when we added DPA (1 mg/disk) or EDTA (5 mM) in a combined disk test (CDT); (ii) a positive ghost zone (synergism) between ATM (30 µg) and CZA 50 disks and between CZA 50 and DPA (1 mg) disks, using the double-disk synergy test (DDST) at a disk-disk distance of 2.5 cm; (iii) ≥3-fold MIC reductions of ATM and CZA in the presence of AVI (4 µg/mL), DPA (500 µg/mL), or EDTA (320 µg/mL); and (iv) immunochromatography. Although our results demonstrated that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex, additional studies are necessary to confirm the accuracy of these methodologies by testing other Gram-negative bacterial species and other KPC and NDM variants coexpressed by WHO critical priority pathogens detected worldwide.Reports of Gram-negative bacteria harboring multiple carbapenemase genes have increased in South America, leading to an urgent need for appropriate microbiological diagnosis. We evaluated phenotypic methods for detecting IMPORTANCE Alerts regarding the emergence and increase of combinations of carbapenemases in Enterobacterales in Latin America and the Caribbean have recently been issued by PAHO and WHO, emphasizing the importance of appropriate microbiological diagnosis and the effective and articulated implementation of infection prevention and control programs. In this study, we evaluated methods based on inhibition of ceftazidime (CAZ), ceftazidime-avibactam (CZA), and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, and avibactam (AVI) inhibitors for the identification of KPC-2- and NDM-1-coexpression in members of the K. pneumoniae complex recovered from human and animal hosts. Our results demonstrate that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex. Enterobacterales (CRE) has increased in South America and the Caribbean (Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase-1 (NDM-1) seems to be the major threat to public health .KPC belongs to class A carbapenemases, which share a serine residue at their active site that confers hydrolytic properties  that cantic site  and can tic site . Strikintic site . As a rePhenotypic methods to detect carbapenemases have been based on the use of inhibitors, where an increase in size of the inhibition zone of carbapenem-containing disks is observed by using combined disk (CDT) methods , 13, wheK. pneumoniae complex members  recovered from human and animal hosts in South America.In this study, we evaluated methods based on inhibition of ceftazidime (CAZ), ceftazidime-avibactam (CZA), and ATM by DPA, EDTA, and AVI inhibitors for the identification of KPC and NDM coexpression by K. pneumoniae-related species, including K. pneumoniae, K. quasipneumoniae, and K. variicola, displaying resistance to broad-spectrum cephalosporins and ceftazidime-avibactam were identified in human and animal hosts (K. pneumoniae strains 331 (susceptible to imipenem and meropenem) and MV940851 (susceptible to imipenem).Fifteen al hosts . RegardiKlebsiella spp. showed mCIM+ eCIM− results, 2 K. pneumoniae strains displayed an indeterminate result . It is important to emphasize that eCIM is not an accurate method to detect suspected coproduction of class A and class B carbapenemases, as it only detects MβLs if both the mCIM and eCIM are positive, whereas mCIM+ eCIM+ results may be caused by NDM or MβLs plus AmpC.Initially, metallo-β-lactamase and serine carbapenemase production was screened by using mCIM and EDTA-modified carbapenem inactivation (eCIM) . In thisK. pneumoniae complex, whereas 2 of 4 CZA-resistant K. pneumoniae strains displayed positive bands for KPC production alone . Coproduction of NDM and KPC was confirmed by PCR and/or GeneXpert, and further genomic analysis predicted blaKPC-2 and blaNDM-1 genes. On the other hand, genomic analysis of four CZA-resistant K. pneumoniae strains confirmed the presence of blaKPC-3, blaKPC-31, blaKPC-113, and blaKPC-114 variants. Expression of KPC-31 and KPC-114 was not detected by immunochromatography.Strikingly, immunochromatography revealed coproduction of NDM- and KPC-type carbapenemases in 11 members of the For the CDT, with different EDTA and DPA concentrations tested, 5 mM EDTA/disk and 1,000 µg DPA/disk were chosen for inhibition activity of MβL, since these concentrations showed no inhibitory activity across the bacterial growth of all screened isolates when sterile blank disks impregnated with 10 μL of 0.1 M EDTA and 10 mg/mL DPA were tested. On the other hand, in order to detect expression of NDM enzymes, ceftazidime-avibactam at 14 μg/disk  and 50 μg/disk  were used, in accordance with guidelines for disk-diffusion antimicrobial susceptibility tests of EUCAST and CLSI, respectively.K. pneumoniae complex isolates, an increase of ≥10 mm in the size of inhibition zones were observed around CZA 14 disks containing 10 µL of 0.1 M EDTA or 10 µL of 10-mg/mL DPA, in comparison to the inhibition zones of CZA disks without EDTA or DPA. Otherwise, for the same KPC-2+ NDM-1+ isolates, increases of ≥4 mm in the size of inhibition zones were observed around CZA 50 disks containing 10 µL of 0.1 M EDTA or 10 µL of 10-mg/mL DPA, in comparison to the inhibition zones of CZA disks without EDTA or DPA. Additionally, for KPC-2 and NDM-1 coproducers, increases of ≥5 mm in the size of inhibition zones were observed around the ATM 30-μg AVI 4-μg disks, in comparison to the inhibition zones of ATM disks without AVI (K. pneumoniae isolates (strains 330 and 331), which were NDM-1 negative and carried blaKPC-113 or blaKPC-114 gene variants, exhibited an increase of ≥10 mm in the inhibition zones around CZA-DPA and displayed no increase in the inhibition zones around CZA-EDTA disks. On the other hand, the blaKPC-31-positive K. pneumoniae strain MUV940851 displayed an increase of ≥10 mm in the inhibition zones around CZA-DPA and CZA-EDTA disks, while no increase around the ATM-AVI disk was detected, supporting the prediction that coproduction of KPC-2 and NDM-1 must be based on a positive synergistic effect shown using both CZA-EDTA and ATM-AVI disk combinations and not a single combination in the CDT test.For KPC-2-positive NDM-1-positive hout AVI  and B. EK. pneumoniae strains suggested that combinations of monobactams and AVI produced inhibitory effects on some KPC variants, in a similar way as for NDM-type carbapenemases. In this respect, since AVI is able to covalently bind to some bacterial penicillin-binding proteins (PBPs), synergistic activity of ATM-AVI against KPC-113 and KPC-114 may be related with different activities on multiple PBP targets , a positive ghost zone (synergism) was observed between CZA 50 and ATM disks and between CZA 50 and DPA disks, with disks positioned at a disk-disk distance of 2.5 cm  ATM and CZA resistance is observed; (ii) KPC-2 and NDM-1 coproducers are mCIM+ and eCIM−, and (iii) KPC variants conferring resistance to CZA could be susceptible to meropenem, displaying an indeterminate mCIM and eCIM result, as interpreted by CLSI guidelines. All CDT and DDST results are summarized in Although NDM-1For MIC reduction assays, the final concentrations of EDTA, DPA, and AVI were fixed at 320, 500, and 4 µg/mL, respectively, since these concentrations produced no antibacterial activity against any screened isolates, allowing us to observe a ≥3-fold decrease in ATM and CZA MICs among NDM-1 and KPC-2 coproducers in the presence of inhibitors. In Enterobacterales. Thus, the combination of aztreonam plus avibactam appears to be a promising option against Enterobacterales isolates coproducing class A and class B β-lactamases while awaiting development of new antimicrobials  are susceptible to aztreonam and are inhibited by EDTA or DPA  with an Illumina NextSeq platform and/or by GeneXpert , or by PK. pneumoniae IBL2.4), NDM-1 and CMY-48 (Citrobacter freundii PG4), NDM-1 and CTX-M-15 (K. pneumoniae Kp183), NDM-1 and PER-2 (Escherichia coli 2ECMBL), CTX-M-15 and SHV-28 (K. pneumoniae PRETA), and CMY-2 (E. coli Em1cro) producers were used as controls (Both the CDT and DDST were adapted from methods previously described for the detection of carbapenemases . ATM 30,controls . All assE. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as susceptible controls (For MIC determinations, ATM, CAZ, EDTA, and DPA were purchased from Sigma-Aldrich, and avibactam was purchased from Selleckchem. All MICs were determined by the broth microdilution methodology outlined in ISO 20776 , 51. In controls . All asscontrols , 51.


As a result, these k values decreased gradually as the pH of the sample increased from 5.0 to 7.0. The Arrhenius model was applied to describe the heat dependence of the k-values. The activation energy (aE) was calculated for each case of GF2, GF3, and GF4 degradation at pH values of 5.0, 6.0, and 7.0. The result showed that rice bran FOS is very thermostable at neutral pH while more labile at acidic pH.Thermal degradation kinetics of fructooligosaccharides (FOS) in defatted rice bran were studied at temperatures of 90, 100, and 110 °C. FOS extracted from rice bran and dissolved in buffers at pH values of 5.0, 6.0, and 7.0 were prepared for the thermal treatments. The residual FOS (including 1-kestose (GF2), nystose (GF3), and 1F-fructofuranosylnystose (GF4)) contents were determined using the ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method. The results showed that the thermal degradation kinetics of GF2, GF3, and GF4 followed a first-order kinetic model. Thermal degradation rate constants ( Bifidobacterium spp. and Lactobacillus spp., while reducing or maintaining pathogenic populations  at low levels [‘Fructooligosaccharides’ (FOS) is the common name for fructose oligomers, including three major representatives known as 1-kestose GF2), nystose GF3), and 1F-fructofuranosylnystose GF4). FOS are widely present in a wide variety of foods and feedstuffs. They are naturally occurring sugars with potentially beneficial nutritional effects [, nystose, and 1F-. FOS arew levels ,7,8. Thuw levels ,14,15,16w levels ,18,19. Tw levels ,21,22,23w levels ,25.Oryza sativa) is a global crop that has a long history of safe usage as an indispensable food for humans [Rice . Cellulase preparation was provided by Novozymes  and delivered by Trung Son Technology . The cellulase preparation is in brown color, noted with an activity of 700 EGU/g and the best-storing temperature of 4 °C. Standard FOS set, including GF2, GF3, and GF4, with a purity 99% was purchased from Fujifilm Wako Pure Chemical Corporation . Acetonitril, water, and methanol for UPLC analysis were from Merck .®, Merck, Darmstadt, Germany), being added with 0.1 mL cellulase, and incubated in a water bath shaker at 50 °C for 1 h. The mixture was then blended for 2 min using the IKA T25 disperser (5000 rpm), added with an equal volume of ethanol, stirred, allowed to stand for 15 min, and centrifuged at 5252× g for 12 min . The supernatant was recovered and dried by lyophilization. The dried powder was stored at −20 °C for further use. The yield of fructooligosaccharides extracted from rice bran was about 0.7 g/kg rice bran.The extraction of FOS from defatted rice bran was performed according to the method of Patindol et al.  with min® culture tubes 16 × 100 mm with closed cap . Isothermal treatments were conducted in a temperature-controlled block heater , Tokyo, Japan). After isothemal treatment, the samples were immediately cooled down in ice water and measured for the residual contents of GF2, GF3, and GF4.Rice bran crude extracts (100 mg) and buffers  were added to Falcon 50 mL conical centrifuge tubes  for well mixing. The mixed samples of 5 mL were enclosed in Kimax2 column (150 mm × 2 mm × 5 µm)  [The rice bran-extracted samples thermally treated were analyzed for the residual GF2, GF3, and GF4 contents using an ultra-performance liquid chromatography system equipped with an ESI  coupled to an MS/MS system . The chromatographic separation was performed on a Luna amino—NHCA, USA) . The eluA0 and A are respectively initial- and remaining concentrations at time t (min); and k is the degradation rate constant (min−1).Degradation of GF2, GF3, and GF4 can be described by a first-order kinetic model [k can be determined from a linear regression analysis of ln(A/A0) versus time.Equation (1) is valid under isothermal conditions, whereby the degradation rate constant T is absolute temperature (K); T0 is reference absolute temperature (K); k0 is k at T0 (min−1); aE is activation energy (kJ mol−1), and TR (8.314 J mol−1 K−1) is the universal gas constant.The temperature dependence of the degradation rate constants can be estimated using the Arrhenius equation (Equation (2)):The activation energy can be estimated by linear regression analysis of the natural logarithm of the rate constant versus the inverse of absolute temperature.k value in the present case) and the independent variables  have been formulated [Y is predicted response; β0 is constant; β1, β2, β11, β22, and β12 are unknown parameters of variables for linear, quadratic, and interaction terms, respectively; X1 and X2 are independent variables.Many empirical polynomial models describing the relationship between the predicted response  were constructed using regression analysis.The UPLC-ESI-MS/MS analysis of the standard FOS solutions  was performed with analytical data presented in k values, estimated using linear regression analysis of ln(A/A0) versus t, are reported in The effect of combined temperature and pH on the thermal degradation of rice bran-extracted GF2, GF3, and GF4 dissolved in buffered solutions was studied at 90, 100, and 110 °C and pH values of 5.0, 6.0, and 7.0. As observed, the combined temperature-pH degradation of the rice bran GF2, GF3, and GF4 samples could be adequately described by a first-order model (Equation (1)) in the temperature range of 90–110 °C . Degradaln(k) versus 1/T formed a linear line with a good correlation coefficient , in the temperature range studied, could be estimated using the Arrhenius equation (Equation (2)), with an activation energy range of 61.8–77.7 kJ·mol−1 obtained for GF2, 67.5–92.3 kJ·mol−1 for GF3, and 49.2–72.6 kJ·mol−1 for GF4 , with aX1 as the temperature variable and X2 as the pH variable on the experimental data, the model parameters were estimated using nonlinear regression analysis . Based on the model parameters estimated, however, it was shown that the terms β0 and β22 were redundant, as indicated by the large standard error (~100%). As a consequence, these terms were omitted, and a reduced version of Equation (3) was used ). Model parameters estimated based on Equation (4) are shown in By fitting Equation (3) with k values, respectively) as a function of temperature, pH, experimental k value, and estimated k value (data not shown). In addition, the parity plots of the predicted k values based on Equation (4) versus the experimental k values were established for GF2, GF3, and GF4 ), no tendency was found by graphing residuals (differences between experimental and predicted  and GF4 . The dev version ,35.k of 0.016676, 0.021072, and 0.025567 min−1 corresponding to 8, 10, and 12% loss, respectively, of rice bran GF2, GF3, and GF4 for a total process time of 5 min were simulated and represented in isorate contour plots (By inserting all model parameters of ur plots .Rice bran-extracted GF2, GF3, and GF4 were rather thermally stable compounds at neutral pH while more labile at acidic pH. Among these, GF3 was more heat sensitive compared to GF2 and GF4. A mathematical equation was suggested for a description of the temperature-pH behavior of rice bran-extracted GF2, GF3, and GF4 during the processing of rice bran-based foods. This equation could be useful in designing alternative processing conditions for temperature-pH processing of rice bran-based products. Degradation kinetic studies of rice bran FOS in real food products would be interesting for food processors to evaluate the potential of temperature-pH processing of these products. Similar works can be applied for process stability studies of many other food quality attributes. This type of research is a good approach for the calculation and optimal design of processes for the food processing industry.


Allergies constitute an important public health problem, and epidemiological data is crucial to developing strategies for its prevention and therapy. Few population-based studies are available for data on allergies and sensitization. However, as these studies are expensive and time-consuming, novel approaches are searched for.A large monocentric IgE dataset was used to analyse quantitative sensitization data in different age and gender groups and compared the results to available epidemiological data.A total of 14,370 patients who sought medical care at the Department for Dermatology and Allergology at the Technical University of Munich, Germany was analysed. Total IgE and sensitization measured in specific IgE levels to common food allergens and aeroallergens were compared between females and males, age groups, and the year of testing (2003–2021).8283 females (57.6%) and 6087 males (42.4%) were tested. The average number of specific IgE tests per patient was 12.3 ± 11.4. Total IgE increased after birth with age and reached a peak between 4–6 years in males and 10–12 years in females. Males had higher specific IgE for all common aeroallergens  and food allergens  except for cat epithelia. Data closely reflected results of population-based studies in the literature.This study shows that, despite potential patient and test selection bias, the results of the quantitative IgE-dataset analysis closely reflect results of population-based data. Thus, as large cohorts can be examined with a minute amount of effort, this surrogate method appears promising to supplement epidemiology research.The online version contains supplementary material available at 10.1186/s13223-022-00717-8. Allergic diseases constitute an important public health problem in Western societies , 2. AccoClassic methods to generate this data include cohort and case-control studies as well as cross-sectional studies. These instruments have been successfully employed in the past to create much of the epidemiological backbone to allergy research . HoweverSpecific IgE, together with skin prick testing, indicates sensitization, which is a measure and prerequisite for a potential IgE-mediated allergy. In the course of routine testing of allergy patients, large sets of IgE data are generated, which offer the potential to be pooled and analysed. We wanted to explore the potentials and pitfalls of quantitative IgE analysis and created a dataset by extracting the results of 14,370 allergy patients who had undergone IgE-testing. We analysed sensitizations over patient age and gender in order to elucidate possible trends to then appraise their validity by comparing the results with data from German health surveys , 9. MoreIn this cross-sectional, retrospective, mono-centre study we used IgE sensitization data from 14,370 patients who sought medical care at the Department for Dermatology and Allergology, Technical University of Munich, Germany between January 2003 and February 2021. Most patients had symptoms suspected to be allergic . Like similar studies in this field , the pat® Phadia™, Laboratory Systems by Thermo Fisher Scientific were used to assess sensitization quantitatively measured in specific IgE antibodies. We analyzed the total IgE in kU/l and specific IgE levels in kUA/l for the following solutions: birch, mugwort and timothy grass pollen, house dust mite (Dermatophagoides pteronyssinus) and cat epithelia for aeroallergens as well as cow’s milk protein, chicken egg white, peanut, cod and wheat flour for food allergens. For statistical analysis, all values below the threshold [Dermatophagoides pteronyssinus is referred to as house dust mite in our analysis.Laboratory in-vitro tests from ImmunoCAPhreshold  were treThe data were analysed using Python 3.8.8 and the statistical libraries pandas 1.2.4, NumPy 1.20.1, and statsmodels 0.12.2 [The study population consisted of 8283 females (57.6%) and 6087 males (42.4%), altogether 12,834 adults (89.3%) and 1536 children (10.7%). The average age of the patients was 43.5 years (sd: 20.3) ranging between 0 and 106 years. The average number of specific IgE tests per patient was 12.3 , which did not differ between sex (p = 0.431) and age groups (p = 0.065). The top three tested allergens were timothy grass pollen , house dust mite  and birch pollen  of all patients, was 292.3 kU/l (sd: 805.5). Total IgE in male patients (373.3 ± 92.1) was on average higher  than in female patients , cow milk protein , peanut , wheat flour  and cod , wheat flour at the age group 31–33 years , while chicken egg white shows no differences in any age group. Cod sensitization differs in the age groups 4–6 years  and 46–48 years  and peanut sensitization three distinct groups: 19–21 years , 40–42 years  and 52–54 years  and after 2010 . Furthermore, in many cases, allergen panels created specifically for the hospital were taken . This leads to the fact that only a small part of all possible sensitizations was measured in each patient. For rare allergens, sensitizations only in a small number of cases were determined. Interestingly, despite these limitations, our results correspond surprisingly well to data from birth cohorts and may offer new insights, which can be confirmed in population-based studies , 29.On the other hand, mass quantitative IgE analysis has decisive advantages. First, population-based conclusions can be extracted from data about how sensitizations develop over long periods of time. In contrast to most studies, which mainly examine children for reasons of practicability, the present method makes it possible to easily examine a wide variety of age groups with a minute amount of effort involved.In summary, the present data demonstrates that our patient-based data results are largely congruent with already existing population-based sensitization data and therefore appear to be a valid surrogate in allergy research. To fully exploit the potential of quantitative IgE analysis available for allergy research, further studies are needed. Among other things, it would be interesting to apply the same methodology to a dataset that includes diagnostics of an even broader spectrum of sensitization of each patient, e.g. as determined in Thermo Fisher's ISAC diagnostics . SimultaAdditional file 1: Table S1. Comparison of total and specific IgE for food and aeroallergens by age group.Additional file 2: Table S2. Sex differences in sensitizations to food and aeroallergens by age group.


Leptonychotes weddellii and crabeater seals' (CESs) Lobodon carcinophaga breeding season distribution in the Weddell Sea, determined from satellite imagery. We contrast the theoretical climate impacts on both ice‐obligate predators who differ in life‐history characteristics: CESs are highly specialized Antarctic krill Euphausia superba predators and breed in the seasonal pack ice; Weddell seals (WESs) are generalist predators and breed on comparatively stable fast ice. We used presence–absence data and a suite of remotely sensed environmental variables to build habitat models. Each of the environmental predictors is multiplied by a ‘climate change score’ based on known responses to climate change to create a ‘change importance product’. Results show CESs are more sensitive to climate change than WESs. Crabeater seals prefer to breed close to krill, and the compounding effects of changing sea ice concentrations and sea surface temperatures, the proximity to krill and abundance of stable breeding ice, can influence their post‐breeding foraging success and ultimately their future breeding success. But in contrast to the Ross Sea, here WESs prefer to breed closer to larger colonies of emperor penguins (Aptenodytes forsteri). This suggests that the Weddell Sea may currently be prey‐abundant, allowing the only two air‐breathing Antarctic silverfish predators (Pleuragramma antarctica) (WESs and emperor penguins) to breed closer to each other. This is the first basin‐scale, region‐specific comparison of breeding season habitat in these two key Antarctic predators based on real‐world data to compare climate change responses. This work shows that broad‐brush, basin‐scale approaches to understanding species‐specific responses to climate change are not always appropriate, and regional models are needed—especially when designing marine protected areas.The impacts of climate change in Antarctica and the Southern Ocean are not uniform and ice‐obligate species with dissimilar life‐history characteristics will likely respond differently to their changing ecosystems. We use a unique data set of Weddell  We built habitat models for crabeater (CES) and Weddell (WES) seals using presence–absence data identified from satellite images. The models show that because the dietary specialist crabeater seals breed on pack ice close to where their preferred prey (Antarctic krill) is most abundant, they are at risk to lose breeding habitat. Weddell seals, being a dietary generalist who breed on more stable fast ice, are likely to buffer climate change effects. Antarctic toothfish [Dissostichus mawsoni]). How do we plan for possible compensatory or additive changes to an ecosystem such as fishing and climate change? A first step is that we must understand the habitat associations and life histories of those marine predators that indicate the health of ocean ecosystems.Climate change will impact animal populations differently based on a combination of the latitude, baseline ecosystem stability and the life‐history strategy of a particular species, with ultimate impacts on global distribution and abundance of populations  is a mixed capital‐income breeding, long‐lived pinniped  has a specialist strategy in many regards. Though, a capital breeder and likely one of the most abundant marine mammals in the world  that live and breed mostly under the pack ice and pack ice edges comprises >90% of diet , leopard seals (Hydrurga leptonyx), Adélie penguins , emperor penguins (Aptenodytes forsteri), Antarctic petrels , snow petrels (Pagodroma nivea), South polar skua  and humpback whales (Megaptera novaeangliae). The Weddell Sea has some of the highest concentrations of primary productivity because of its relatively large sea ice edge where phytoplankton blooms occur as the ice melts  and fast ice (WESs) habitats in the Weddell Sea , the suite of environmental variables also differed. For models of CES habitat, the 22 environmental variables broadly included variables of ocean depth, temperature, oceanography and ice conditions (see Appendix Table A1 for the full list). Models of WES habitat considered 19 environmental variables, which were categorically similar but differentiated slightly: for example, fast ice conditions, proximity to penguin colonies, bathymetry (meanbathy), distances to glacier (glacierdist) and to the continent Antarctica (distToShore) and distances to the 300 m (cont300dist) and 800 m (cont800dist) bathymetry contours (Appendix Table A2). Because most oceanographic and ice variables are inherently collinear, we filtered for the most informative set by calculating variable inflation factors for the entire Weddell Sea region using the R library ‘fmsb’  to evaluate model performance, while the remaining 80% of the data were used to train the models. The hold‐out data had equal numbers of seal presence and absence points  and were selected randomly across the entire study region. To compensate for the unbalanced number of seal presence versus seal absence records for both species  and a random sample of equal size  of seal absence records. Without balancing the data set, the models would optimize on predicting absences well at the expense of increased error in predicted presence. We ran 500 bootstraps for each of the three constituent models for each species. The RF and BRT models were tuned, respectively, in each bootstrap using the ‘tuneGrid’ function in ‘caret’ through compiling a range of candidate models and choosing the best candidate model based on the R2 value that was calculated from 10‐fold cross‐validation. Cross‐validation folds were created by randomly dividing bootstrap records into 10‐folds containing an approximately equal number of records. We calculated the R2 value as the goodness‐of‐fit measure for the RF and BRT models of each bootstrap using the 20% hold‐out test data.The full modelling procedure is detailed in the study by Wege et al. , so here2.4The main aim was to identify environmental variables that influence CES and WES breeding habitat. For each of the bootstrap samples, we calculated permutation variable importance for the RF and Maxent models and percentage contribution of each variable in the BRT models. These methods differ with respect to the way variable importance is calculated, but the form and magnitude of important explanatory variables are comparable among them. To enable us to compare permutation importance with percentage relative influence values produced by the different models, we calculated the relative proportion each of these variables contributed to seal breeding habitat.change importance product’, which is the climate change score multiplied by the proportional importance of the variable for each of the three model types (see above paragraph). This was done to come up with a relative index that considers the importance of the environmental variable for each species’ life history and the likelihood of change of that aspect of the species' environment. The aim here was to distinctly separate variables that are both important and also likely to change substantially, from less important or more stable environmental situations for each species.Returning to methods outlined in the studies by Laidre et al.  and Sini33.1The Variance inflation factors of the CES data indicated high collinearity between 10 variables and consequently, only 14 of the 24 variables were retained for habitat modelling: bathy, dist_canyon, dist_shelf, ice_edge_dist, ice_sd, oldice_cv, sal200_600, slope, vmix, vmix_sd, windmag, sst, shflux and shflux_sd. Variance inflation factors calculated for the WES data indicated high collinearity for two variables. Subsequently, 17 of the 19 variables were retained: ADPEabund, ADPEdist, cont300dist, cont800dist, DecemberIcePresence, distNearestIceEdge, distToShore, EMPEabund, EMPEdist, fastIceRatio, glacierdist, InTrough, meanbathy, meanslope, Persistence2Years, PredictabilityDec5Years and PredictabilityOct5Years.3.2As in the study by Wege et al. , the CES3.3The CES RF, BRT and Maxent top variables across all models were as follows: distance to ice edge, bathymetry, standard deviation of ice concentration and distance to the shelf Figure . For WES4More than 10 years ago, Siniff et al.  discusseWe found that the most influential variables for CESs, that are also likely to change the most in the future, are connected to their ability to forage effectively post‐breeding. Crabeater seals are capital breeders that do not forage during their breeding season , an important food item in all three species’ diets  is due to the Weddell Sea being prey‐abundant for these fast ice obligates. These community ecological effects, rather than physical properties of the ocean, are likely to be the most important for WESs in the future, which is a critical finding for the future of conservation. Simply put, really only two air‐breathing Antarctic silverfish predators (WESs and emperor penguins) that can dive to the same depths (Goetz et al., We note that although the error margins around the partial dependence plots Figures  and 7 seA primary objective in conservation biology is the maintenance of biodiversity (Niesenbaum, Supplementary MaterialClick here for additional data file.


The marginal ice zone is the dynamic interface between the open ocean and consolidated inner pack ice. Surface gravity waves regulate marginal ice zone extent and properties, and, hence, atmosphere-ocean fluxes and ice advance/retreat. Over the past decade, seminal experimental campaigns have generated much needed measurements of wave evolution in the marginal ice zone, which, notwithstanding the prominent knowledge gaps that remain, are underpinning major advances in understanding the region’s role in the climate system. Here, we report three-dimensional imaging of waves from a moving vessel and simultaneous imaging of floe sizes, with the potential to enhance the marginal ice zone database substantially. The images give the direction–frequency wave spectrum, which we combine with concurrent measurements of wind speeds and reanalysis products to reveal the complex multi-component wind-plus-swell nature of a cyclone-driven wave field, and quantify evolution of large-amplitude waves in sea ice. Unprecedented 3D imaging of waves and sea ice floes from a moving icebreaker in the Antarctic marginal ice zone during a polar cyclone reveals a complex wind-plus-swell sea state, where contrasting ice-driven attenuation and wind forcing coexist. Autonomous platforms that operate in harsh polar environments, such as autonomous underwater vehicles3 and drones4, are pushing the boundaries for in-situ observations, generating data for essential calibration and validation of satellite remote sensing, and measuring properties beyond the capabilities of contemporary satellites. The marginal ice zone (MIZ), which is characterised by dynamic interactions between large-amplitude surface waves and relatively small and thin ice floes, is difficult for satellites to capture6 and a major target for improved observations8. Wave evolution and ice properties in the MIZ are intimately coupled11, and, hence, there is demand for a technology capable of simultaneously monitoring both wave activity and ice cover properties, which can capture data during storms when wave–ice interactions are most intense.Improved observational capabilities are needed to understand the often paradoxical and baffling regional and inter-annual variabilities of Antarctic sea ice12 at a frequency-dependent rate that induces a downshift of the peak frequency13, as well as modifying the directional wave spectrum14. The attenuation rate has become a research focus, as it informs predictions of the width of the ice-covered region impacted by waves and, hence, the MIZ extent16. Major advances in measuring wave attenuation in the MIZ have been made over the past decade, including dedicated campaigns in the Arctic17 and Antarctic18. State-of-the-art in-situ measurements mostly come from arrays of wave buoys, where the buoys can be traditional open water buoys20 deployed between floes in regions of low ice concentration  or bespoke buoys deployed on ice floes20 large enough to support the buoys  but small enough that the floes follow the waves. Attenuation rates are generally calculated by applying an exponential decay ansatz to measurements provided by neighbouring buoys, in terms of the significant wave height20 or a more detailed analysis in which the ansatz is applied to each component of the one-dimensional (frequency) wave spectrum, under the assumption of a stationary wave field23.Historical in-situ measurements of waves in the MIZ show the ice cover attenuates wave energy exponentially over distance25) has generated a new understanding of wave attenuation in the MIZ, particularly on how the wave attenuation rate depends on frequency26. Certain theoretical models reproduce the observed frequency dependence, but the dominant sources of attenuation are still hotly debated27 and empirical models often rely upon28. Further, the measurements have revealed a large range of attenuation rates25, even at comparable frequencies, which is attributed to dependence on ice cover properties, such as ice thickness, areal ice concentration and floe sizes, as well as momentum transfer from winds over the ice cover. Satellite and model-hindcast data have been used to derive empirical relationships between measured attenuation rates and ice concentration23, ice thickness29 and winds23. In contrast, floe sizes in the MIZ are below satellite resolutions and have only recently been integrated into large-scale models30, so that coincident floe size data have been limited to visual observations during deployment. Overall, data on ice properties are too sparse or unreliable to validate theoretical models.The recent surge in measurements (including remote sensing31. Airborne synthetic aperture radar (SAR) is an alternative method to measure frequency–direction wave spectra and has been applied over 60–80km long transects of the MIZ33. However, stereo-imaging, being an in-situ technique, can be used to measure sea-ice geometrical properties simultaneously34 and can be combined with co-located meteorological measurements, e.g., wind velocities. Further, in contrast to SAR35, stereo-imaging resolves wind sea components of the wave spectrum , as well as swell (long wavelength systems no longer under the effect of winds).Stereo-imaging techniques are emerging as a tool for in-situ monitoring of waves and ice properties in the MIZ. In principle, the images can be used from a moving vessel, as in open waters, to reconstruct the sea surface elevation in time and space, thus enabling analysis of wave dynamics in two-dimensional physical space, the frequency–direction spectral domain, and wave statistics36 use a camera system on a fixed platform on the edge of a lake to quantify incoming and reflected energy fluxes of relatively small waves (<0.3m) in pancake and brash ice. Smith & Thomson37 use camera images from a moored vessel in the Arctic MIZ during calm conditions  to calculate bulk wave properties and pancake floe velocities. Alberello et al.34 use an autonomous stereo-camera system on a vessel moving through the winter Antarctic MIZ during a cyclone to measure pancake floe shapes and sizes.To date, the use of stereo-imaging techniques in the MIZ has been limited in scope. Campbell et al.34 combined with automated image reconstruction software. We report the extreme sea state created by the cyclone over a >40km transect into the Antarctic MIZ, and validate a subset of the results with co-located buoy measurements. The sea state deep into the MIZ during the cyclone is shown to be more complex than previously thought, and partitioning of the two-dimensional spectra is required to analyse wave evolution of the cyclone-driven wind sea. Further, evidence is given of momentum transfer from winds through 100% ice concentration, based on comparing attenuation of the significant wave height over distance with an empirical model18, which is considered to be a benchmark due to the large size of the underlying data set and that the measurements were made in the Antarctic MIZ during the sea-ice growth period.In this article, we demonstrate the potential to monitor the evolution of the frequency–direction wave spectrum from the images captured by Alberello et al.∘ South and 30∘ East during an explosive polar cyclone. It encountered the ice edge (the northernmost location where ice concentration exceeds 10% in a 1km radius around the vessel38) at 08:00 UTC, and reached 100% ice concentration at 09:00 UTC, approximately 10km from the ice edge. It continued South, whilst remaining in 100% ice concentration34. Over this time, strong winds  up to 10m North-East of the icebreaker and > 6m at the ice edge  when the icebreaker entered the MIZ . The mean directional spread, a measure of the breadth of the wave spectrum in direction42, was ≈60∘ and, like the mean wave direction, was steady during the day41. Wave height and period at the ice edge increased throughout the day, due to the intensification of the cyclone, thus creating a non-stationary incident wave field. A detailed analysis of the sea state indicates that ≈70% of the total significant wave height is due to waves generated locally (wind sea). Swell contributes the remaining 30%, which also comes from a north-easterly direction but with a slight offset of less than 20∘ from the wind sea.On the 4th July 2017, the South African icebreaker S.A. Agulhas II entered the Antarctic MIZ at 62d = 5–44km, where the distance is taken along the mean wave direction , the ice concentration was ic ≈50%44 and consisted of pancake ice floes  and the remaining 50% was water between the floes oes Fig. b. At allons Fig. e, and thons Fig. f. At allons Fig. e. Therefη(t), at each measurement location, from which the one-dimensional frequency spectra, E(f), and two-dimensional frequency–direction spectra, E, are derived (see Methods). At the two deepest measurement locations (d = 43–44km), wave buoys (waves-in-ice observation systems47 of the type used in the previous studies21), were deployed on ice floes48 and the frequency spectra they provide are used to validate the analysis of the stereo-camera images  and upper tails (f > 0.10Hz), which show over and under estimation of energy, respectively, relative to the buoys. These modes carry a small amount of energy and make only minor contributions to integrated parameters, such as the significant wave height and mean period, for which values derived from the images and buoys differ by ≈5% and ≈2.5%, respectively.The three-dimensional images Fig. a are useons d = 4–44km, waf < 0.16 Hz, or −1.4 < log10(f) < −0.8, corresponding to periods of ≈7–25s; see error bands in Fig. 49.The uncertainty associated with spectra derived from the stereo-camera images is primarily white noise . The noise level is negligible for the most energetic modes  to HS = 4.6m at d = 44km (bottom panel), and, hence, wave energy attenuates over distance. Statistical analysis of the individual waves indicates consistency with Gaussian (linear) theory. For instance, the kurtosis (fourth order moment of the probability density function of the surface elevation and a measure of wave nonlinearity50) is close to three, similar to Gaussian sea states51. The maximum individual wave heights at each location, which are part of energetic wave groups, tend to diminish with distance into the MIZ. Nevertheless, large waves are recorded tens of kilometres into 100% ice concentration, e.g., Hmax ≈ 8m at d = 43km (second to bottom panel), which corresponds to Hmax/HS ≈ 1.6, close to the maximum height expected in a linear sea state43. The steepness associated with the largest individual waves, εmax = πHmax/(2λ), where λ is the wavelength, is a measure of the strength of the wave42. It decreases from εmax ≈ 0.19 at the ice edge (d = 5km) to ≈ 0.10 at the deepest measurements locations (d = 43–44km). Despite the attenuation of the steepness over distance, these maximum values are expected to be large enough to have an impact on the ice cover, for example, by keeping the ice cover unconsolidated52.Surface elevation timeseries around the largest individual waves recorded (42), with the bandwidth at the two deepest measurement locations ≈80% of the bandwidth close to the ice edge. The frequency spectrum is unimodal at the first four measurement locations (d ≤ 36km) but becomes bimodal at the deepest two measurement locations (d = 43–44km) where a low-frequency peak appears (peak period ≈ 15s). The low-frequency peak indicates the swell initially north of the ice edge . The swell overlaps the wind sea system in frequency space, but the two systems are clearly separated in two-dimensional frequency–direction space . Note that the buoys do not return the directional spectrum18 and, hence, their measurements cannot be used to separate wind sea and swell systems, as they overlap in frequency space.Attenuation of wave energy with distance into the MIZ is also evident in the one-dimensional spectra Fig. b. As expcp/u1042, where cp is the phase velocity (ratio of wavelength to wave period) and u10 is the wind speed, is computed at each measurement location using wind speeds from the onboard met-station . The waves switch sharply to old (cp/u10 > 1.25) at the deepest two measurement locations. The sharp transition is partially due to the arrival of the swell system, but, as implied by the similar sharp transition in wave age for the wind sea , a sudden drop in wind speed from ≈25ms−1 to ≈17ms−1 is the primary cause.The wave age ion Fig. a. The va54 is negligible. The normalised peak period sharply increases when the swell system appears at the deepest two measurement locations (d = 43–44km), but the increase is relatively small for the wind sea. The normalised peak period values for the wind sea are consistent with the MBK spectral attenuation model21, where the ERA5 spectrum at the ice edge is used as the incident field for the model.Lengthening of the peak period with distance is evident in the frequency spectra Fig. b. The peσ0) is calculated from the two-dimensional spectra20 and normalised using the ERA5 spectra at the ice edge  but increases sharply at the final three locations , with the maximum spread  at d = 36km. The wind sea shows little variation in normalised directional spread over all locations . Thus, the peak in directional spread of the total sea at d = 36km is likely due to a combination of attenuation of the wind sea peak and emergence of the swell system. The directional spread of the total sea decreases at the deepest two locations as the swell dominates. The MBK attenuation model applied to the ERA5 two-dimensional incident spectra, with propagation distances of the spectral components dependent on their direction, predicts the normalised directional spread slightly decreases over distance from 50∘  at 5km to 43∘  at 43–44km, as the components travelling at oblique directions travel farther and, hence, experience greater attenuation leading to collimation14. This trend contrasts with the weak increasing trend in the observed directional spread of the wind sea by ≈ 1.22% per kilometre.The frequency averaged directional spread  further increases the attenuation rate to α = 29.4 × 10−6m−1. For comparison, the benchmark empirical model18 gives the rate 32.7 × 10−6m−1 . The uncertainty in the calculation of the distance from the ice edge  attenuates with distance , where wind speeds were generally < 10ms−1  and the wave spectra were most likely unimodal . Therefore, we argue that the fairest comparison is with the wind sea without wind input , although noting the subtracted wind input represents an upper bound as it does not consider ice cover. With these modifications to the wave field, which rely on analysis of the frequency–direction spectrum, the attenuation rate is less than the benchmark by ≈10% only. The agreement is remarkable considering the potential differences in conditions that may affect attenuation rates, such as the extreme wave heights and strong winds associated with the cyclone, and the ice properties. Therefore, the results provide support for the benchmark attenuation rate and evidence it holds for considerably larger waves than previously recorded in similar ice conditions. However, the results show the benchmark attenuation rate only applies to single-component seas, and does not describe the evolution of the complex sea observed deep into the MIZ during the cyclone.The benchmark significant wave height attenuation rate is greater than that derived for the total sea from the stereo-camera images by approximately a factor four. The benchmark rate is based primarily on measurements of low-energy sea states  of 4m. The stereo rig was installed on the monkey bridge of the icebreaker, ≈34m from the waterline and tilted 20S) was calculated based on pixels and approximated by a disk, from which a characteristic diameter 34.An automatic algorithm, developed using the MatLab Image Processing Toolbox, was applied to extract sea-ice metrics from the recorded images. To avoid sampling the same floe twice, images every 10s were analysed. Images were ortho-rectified and projected on a horizontal plane. A camera-dependent calibration was applied to convert pixel to metres. The images were processed to eliminate the vessel from the field of view, adjust the image contrast and convert the grey scales into a binary map, which isolates the solid ice shapes from background water or frazil ice. A morphological image processing was applied to improve the fidelity of the shape of identified pancakes. Floe area (60) was used to estimate a set of data points in space (a dense 3D point cloud) representing the 3D ocean surface. WASS analyses left and right stereo images to find photometrically distinctive corresponding points  that can be triangulated to recover their original 3D position in space. The operation is performed for each pixel of the stereo frames to produce a temporal sequence of 3D point clouds composed of millions of samples each. The data is also automatically filtered to remove possible outliers and the mean sea-plane is estimated independently for each frame.The Wave Acquisition Stereo System  as a discrete-time random variable. At each frame, the system state is updated with both the absolute IMU data (yaw-pitch-roll) and the mean sea-plane distance vector estimated from the point clouds. Both are modelled as Gaussian distributions in which the measurement covariance is given by the sensor manufacturer specifications, in the case of the IMU, or the empirical stereo estimation error for the cameras61, also referred to as quantisation noise, which depends on the object distance, the focal length and the cameras’ resolution, mutual position and declination. An estimate of this error was calculated as the difference between a known synthetic surface and its “back and forth” transformation, which consists of projecting the known surface onto the camera coordinate system and re-projecting it onto the original coordinate system by using the specific geometry of the stereo-camera setup61. This difference provides the spatial distribution of the error, the amplitude of which is uniformly distributed across wavenumbers/frequencies .A systematic source of uncertainty is the resolution errorα is estimated with respect to the ice edge and the dimensionless significant wave height according to the following equation:d is the distance of each measurement location from the ice edge calculated along the mean wave direction in the open ocean as provided by the ERA5 reanalysis, and HS/HS,ERA5 is the significant wave height normalised by the incident (open ocean) counterpart from the ERA5 reanalysis. To estimate the incident wave conditions , the delay between the time at which waves enter into the MIZ and the time at which waves are measured at a specific location was estimated through the wave group velocity. A standard least square fitting is applied to extrapolate an overall attenuation rate. Owing to the non-stationarity of the sea state conditions, each frequency components of the wave field is subjected to different forcing, besides ice-induced attenuation. Thereby, the estimate of a frequency-dependent attenuation rate18 is impractical herein as it would be subjected to significant uncertainty.The sea state conditions were non-stationary during the observation period, with wave height increasing ≈10% from 6.7m to 7.4m. Therefore, the attenuation rate d is ≈± 5%  with a Fourier expansion method62. The wavelet directional method resolve modes around the spectral peak  more accurately than the maximum likelihood method63. Thereby, it is the preferred approach to identify the complex multiple peak feature of the spectrum.With the stereo images, the strategy is to apply an array approach to extract timeseries of the surface elevation and a wavelet directional method to approximate the directional spectrum, noting that the estimate of the spectrum from timeseries allows resolving components with periods > 11.5s and, hence, wavelength > 200m . A virtual array with geometry comprising of a triangle inscribed in a circle of radius ≈1m inside a pentagon inscribed in a circle of radius ≈3m and with an additional probe in the central position (a co-array configuration) was used to allow a sufficient number of non-redundant spatial lags between elements and asymmetry, which ensure an accurate estimation of the directional spreading function64 and it is typically referred to as the encountered spectrum ES, where fS is the frequency detected by a moving object. To restore the original spectral shape, the encounter frequency is converted into the true frequency f through the linear wave dispersion relation as follows64:β is the angle between the ship heading and the (open water) mean wave direction from the ERA5 reanalysis. The true wave spectrum, E, is computed by performing a change of variable:dfS/df is the Jacobian of the transformation.The sea surface elevation extracted from the images is in a frame of reference that moves with the forward speed and heading of the ship. The directional spectrum is therefore distorted due to a Doppler shift65, which is a specific implementation of the inverse catchment scheme introduced by Hasselmann et al.66. The spectral peak that satisfies the conditionu10 is the wind speed, cp is the phase velocity, θ is the wave direction and ψ is the wind direction, is assumed to be associated with the wind sea. All other systems are swell and are ranked based on their energy contents as primary, secondary and tertiary swell. Only primary swell was considered.The partitioning of the wave spectrum is performed using the path of steepest ascent technique67 and the shape of the spectrum is limited by directional resolution (15∘), which makes it difficult to discriminate wind seas and swell that propagate in approximately the same direction.The incident sea state at the ice edge is obtained from ERA5 reanalysis, which provides hourly wave data at a resolution of ≈40km. The model performs well in the Southern Ocean but can miss the swell arrival timeThe ERA5 wave model uses a simplistic wave attenuation schemes in sea ice, which acts only at the outskirts of the MIZ (up to 15% ice concentration), and assumes waves are completely dissipated for ice concentration > 15%. To overcome uncertainties related to the model assumptions, we adopt, as a reference, the wave data in open ocean (0% ice concentration), in the proximity of the ice edge along the mean wave direction  and July 3rd (between 12:00 and 13:00 UTC at 56∘ South and 28∘ East) is shown in Table ∘.A more thorough evidence of the accuracy of the ERA5 reanalysis in the Southern Ocean through comparison against a large data set acquired during the Antarctic Circumnavigation Expedition is detailed by Derkani68.To provides evidence of consistency between the ERA5 reanalysis and the observations presented herein, and thus to allow using the former to produce incident sea states, a comparison against open ocean conditions recorded on July 221) proposed an  attenuation rate for each frequency component in the spectrum asBased on field measurements in the Antarctic MIZ, MBK , the frequency of the wave component and the distance from the ice edge on the direction of each component.The MBK attenuation rate is used to propagate the incident directional wave spectrum into sea ice up to the measurements locations using the expression42a1 = 2.88 × 10−3 and b1 = 0.4569. Eqn. (FERA5) from the known ERA5 wave height . The distance from the edge (d) is added to the fetchHS,wind) over the entire distance (F). The difference between the updated wave height and the one at the edge provides an estimate of the wave height attributed to wind input, i.e.In the open ocean, wind transfers momentum to the sea surface, forcing ocean waves to grow over distance (fetch). Wave growth is estimated with empirical models, which are expressed asPeer Review File


Preliminary testing on pure water ice yield elastic modulus and hardness measurements of 4.1 GPa and 176 MPa, respectively, which fall within the range of previously published values. Our approach demonstrates the potential of low temperature, in situ, instrumented nanoindentation of ice under controlled conditions in the SEM, opening the possibility for investigating individual structural elements and systematic studies across species and concentration of impurities to refine to constitutive equations for natural ice.Interest in the technique of low temperature environmental nanoindentation has gained momentum in recent years. Low temperature indentation apparatuses can, for instance, be used for systematic measurements of the mechanical properties of ice in the laboratory, in order to accurately determine the inputs for the constitutive equations describing the rheologic behaviour of natural ice . These properties are essential to predict the movement of glaciers and ice sheets over time as a response to a changing climate. Herein, we introduce a new experimental setup and protocol for electron microscope loading and  Glaciers and ice sheets presently cover ~10% of Earth’s land surface in alpine and polar regions, forming an integral part of the planet’s climate system, influencing regional- and global-scale climate as well as responding to climate change , 3. The Based on a compilation of deformation data and mechanical tests, impurity-rich glacial ice deforms on average 2.5× faster than impurity-poor Holocene ice in simple shear . While iStandardized testing methods for measuring the mechanical properties of ice at millimetre length-scales currently consist of laboratory creep experiments including uniaxial compression or tension experiments, flexural testing, extrusion experiments and fracture testing –15. SeveIn materials sciences, instrumented nanoindentation uses a nanometer-scale tip with known mechanical properties pressed into a material to probe its local mechanical properties . Hardnes2, 77 K) or liquid helium . Temperature control is challenging, and the constant formation of gas bubbles in the liquid cells result in turbulence that affect the load measurements during indentation. In another type of apparatus, the indenter can be retrofitted with refrigeration systems  and electric heaters to control the temperature of the specimen and indenter independently [in situ testing and observations under vacuum, together with precise control over the tip, sample and frame temperatures for minimised thermal drift.The last few decades have seen significant developments in low temperature nanoindentation. In one type of apparatus, the specimen and indenter can be fully immersed in a cryogenic liquid contained in an insulated vessel –27. For endently , 28–30. in situ instrumented nanoindentation of water ice using an Alemnis Low Temperature Module (LTM-CRYO) installed within a SEM. While the LMT-CRYO is a commercial product, we demonstrate a new procedure for preparing and loading ice onto the nanoindentation stage, cooling, testing and venting while preserving the specimen surface. We detail the setup and steps necessary to obtain preliminary measurements on the elastic modulus (E) and hardness (H) for pure water ice and offer key insights into how these measurements could be further optimised by researchers. Our approaches pave the way for complex nano- and micromechanical testing of ice and its individual structural elements, along with the possibility of high throughput, under controlled conditions in the SEM.Herein, we introduce an experimental protocol to conduct in situ instrumented nanoindentation of water ice in a controlled low temperature environment is outlined in in situ observations.The experimental setup for 2). We used an Aldrich® AtmosBag two-hand, non-sterile, size L, with zipper-lock . During the experiments, the glovebag is completely sealed therefore all the needed tools and materials must be placed inside the bag prior to sealing .To avoid frost contamination on the ice samples and on the indentation device during loading, we created an oxygen depleted atmosphere by attaching a glovebag to the outside of the SEM chamber and flowing in nitrogen gas  with circular cut-outs (3 mm diameter) along the edges at 120° angles to allow space for the sample holder screws  from an N2 line to the glovebag is initiated until the lowest possible dew point is reached. With this setup, a dew point of -35°C was achieved after approximately 30 minutes of purging. At this stage, the LTM-CRYO indentation system is cooled, including sample and indenter holders, to below 0°C but still above the dew point.To begin the experiments, the SEM chamber is vented and the door opened. After attaching the glove bag to the outside of the SEM chamber, N2. For the purpose of this study, we used type 1 (18 MΩ) deionized water for preparing our ice samples. Once the sample holder on the LTM-CRYO reaches a temperature of ~ -20°C, the ice sample is placed onto the holder by snapping the steel tablet into place on the magnet, with the ice droplet placed as close as possible to the holder center. Sample loading must be performed before the sample and indenter holders reach the dew point temperatures in order to avoid frost contamination. Before closing the SEM door, the indenter tip was aligned with the sample. After evacuating the chamber, the sample and indenter holder temperatures can be stabilized by regulating the LN2 flow to the indentation system and controlling the power on the electric heaters to the desired testing temperature. Secondary electron images of the ice sample prior to indentation are shown in In parallel, the ice sample is prepared by placing a water droplet onto the Cu tape on the tablet and submerging it into LN2 gas flow to the glovebag is once again initiated to prepare for sample unloading. Once the atmosphere stabilizes at the minimum dew point (~ -35°C), the LN2 flow to the sample and indenter holders is stopped to slowly warm up the indentation system, up to a temperature slightly above the dew point (> -35°C) of the glovebag atmosphere yet still at freezing conditions. Since the JEOL JSM-6490 vents using ambient air, we place a container connected to a direct N2 line near the intake valve to ensure a direct feed-in of N2 gas at atmospheric pressure into the SEM chamber to reduce the humidity during the venting procedure and prevent frost formation  and hardness (H) for the experiment was calculated from the load-displacement curve following the Oliver-Pharr analysis method [E of 1141 GPa and ν of 0.07 for the diamond Berkovich indenter were used for the calculations and the curve was optimized to fit 2–60% of the unload data. The maximum indentation depth was 3.56 μm yielding a E of 4.1 GPa and a H of 176 MPa for the ice sample. E agrees reasonably with previous measurements from laboratory experiments (E ~9–11 GPa) [E ~1 GPa) [E [E measurements and those reported from laboratory experiments. To avoid fractures, future ice samples could be slowly cooled by using an apparatus that allows for the precise control of the freezing kinetics [Nanoindentation was performed on one sample at a constant loading rate until reaching a peak load of 44 μN before unloading without a hold segment in order to minimize the effect of sublimation or melting of the ice surface. The loading and unloading experiments were conducted at a constant temperature of -90°C. A total of four indents were made, however, due to various complexities during the experiment, we report one single load-displacement curve to demonstrate the potential of our experimental setup and protocol ; S1 Files method . The geo–11 GPa) –33 and f ~1 GPa) –36. Disc GPa) [E , 37. Baskinetics .E. Although nanoindentation should not be as sensitive to surface interactions due to the much higher loads used for testing, it is still a surface sensitive technique therefore results could still be affected by plastification or interfacial pre-melting. Other factors that could contribute to underestimating E include the roughness and curvature of the frozen droplet surface. When indenting a curved surface, the asymmetry of the indent can introduce inaccuracies to the simple functional relationship used for estimating the contact area (A). The surface curvature of the ice sample could be minimized by substituting the Si wafer substrate with e.g. a porous material to increase the wettability of the surface  [In AFM studies, it has been demonstrated that the pressure exerted by the tip can either lead to the plastification or the interfacial pre-melting of the ice surface –44 once us gold) . While cH measurements also fall within the range of previously published measurements for water ice. For example, Pittenger et al. [H values in the range of 10–300 MPa. These values, however, were significantly higher than those measured by previous macroscopic indentation techniques on polycrystalline ice which measured H values of 10–50 MPa at temperatures of 0– -15°C [H∝P/A, where S is the measured stiffness and P is the peak load, H measurements are more sensitive to errors in the estimation of the contact area [H measurements. The significant variability between the reported values for E and H demonstrate the impetus to develop new methodologies for performing more systematic testing using established and robust techniques . Such techniques allow for a large number of experiments to be conducted at various conditions to deepen our understanding of ice mechanics.Although studies on ice hardness are currently very limited, our r et al.  measured0– -15°C , 48. In act area , which min situ instrumented nanoindentation of water ice within an SEM, yielding reasonable E and H measurements, further technical developments are required to make it routine and combine it within correlative analytical microscopy workflows to link structure-composition and properties. Such approaches require that the sample surface is stable, and the features created during the nanoindentation experiments be maintained for the duration of the analysis in the SEM, for e.g. electron backscatter diffraction (EBSD). The stability of ice is dependent on the pressure and temperature conditions in the SEM chamber, as these parameters control the thermodynamics and kinetics of sublimation and condensation, making the transfer between instruments critical to enable possible further analyses by cryogenic-transmission electron microscopy or cryo-atom probe tomography following preparation by focused-ion beam milling at cryogenic temperature [Although we have successfully performed the first perature –52.−6 hPa. At this pressure, the equilibrium temperature occurs at approximately -112°C. At equilibrium, as indicated in −6 hPa is estimated to be approximately 1 μm min-1 [E and H calculations. Nanoindentation experiments on water ice at the tested vacuum pressure should therefore be performed below temperatures of -112°C for correlative studies, or alternatively using SEMs with the possibility for low vacuum mode.Under high vacuum SEM conditions, the chamber pressure is typically on the order of 1×10μm min-1 –57. At t2. Therefore, the ice is quickly cooled to -195.8°C. After freezing, since the current set up can only reach a dew point of ~ -35°C, sample loading onto the indentation device must occur at slightly higher temperatures to avoid frost contamination. During the nanoindentation experiments, the ice sample can be cooled to the desired temperature, however, it must be warmed again to temperatures above -35°C for unloading. Since ice crystal structure varies with temperature [Additional limitations of the current approach include temperature variations during sample preparation and loading and unloading procedures. In this study, the samples were prepared by submerging a droplet of water into LNperature , such va2 gas keeping the humidity at a minimum. To avoid introducing humidity during sample unloading, the SEM also needs to be adapted to vent using N2. With this setup, the nanoindentation device could be cooled to lower temperatures while still preventing frost contamination and the temperature variations between the ice sample and the sample holder would be minimized. The addition of a port for a high vacuum cryo transfer system [These issues can be alleviated by using a new glovebox with a load lock and port for a high-vacuum cryo transfer system that can be attached to the SEM. A glovebox can be constantly purged with dry Nr system  would fain situ nanoindentation within an SEM is the ability to visualize the experiments and capture images of the sample surface prior and post indents, the resolution and the quality of the images are currently limited by the distortions generated by the electromagnetic interferences of the Sm-Co magnet on the sample holder  can be varied. However, as outlined, future technical developments are necessary to optimize this approach and link low temperature nanoindentation experiments to various correlative microscopy techniques.In this study, we have designed a simple experimental setup and protocol that allowed for the first S1 File(XLSX)Click here for additional data file.


Cicer arietinum L.) peptides have shown in vitro potential to inhibit the angiotensin I-converting enzyme (ACE-I). However, the potential molecular interactions between chickpea peptides (CP) and ACE-I as well as their ADMET (absorption/distribution/metabolism/excretion/toxicity) characteristics remain unknown. Thus, our aim was to study the in silico interactions of CP with ACE-I and the CP ADMET characteristics. Legumin and provicilin sequences were submitted to in silico analysis to search for ACE-I inhibitory peptides. Simulated enzymatic hydrolysis was performed using the BIOPEP-UWM database, and the ACE-I inhibitory peptides generated (EC50 ≤ 200 μM) were selected to perform molecular docking and ADMET analysis. After hydrolysis, 59 out of 381 peptides with ACE-I inhibitory potential were released. Based on A and B parameters, the legumin peptides showed better ACE-I inhibitory potential than the provicilin ones. CP mainly interact with residues from pocket S1 (Ala354/Glu384) and S2 (His353/His513) through hydrogen bonds (distances < 3.0 Å) and hydrophobic interactions . Through ADMET analysis, CP showed optimal values for inhibiting ACE-I in vivo. ACE-I inhibitory peptides from legumin and provicilin can bind strongly and tightly to the active site of ACE-I. Further studies to evaluate in vivo the antihypertensive effects of CP are warranted.Chickpea ( Hypertension is defined as systolic or diastolic blood pressure ≥ 130 or ≥ 80 mm Hg (stage 1), respectively, or systolic or diastolic blood pressure ≥140 or ≥ 90 mm Hg stage 2), respectively [, respectCicer arietinum L.) is the third most important pulse in the world based on total grain production, and it contains bioactive compounds with antidiabetic, antioxidant, antihypertensive, anti-inflammatory, and anticarcinogenic activity [Chickpea  were obtained from the UniProt database (http://www.uniprot.org (accessed on 6 December 2021)): legumin (UniProt ID: Q9SMJ4) and provicilin (UniProt ID: Q304D4). The BIOPEP-UWM™ database of bioactive peptides [The protein sequences of the main storage proteins in chickpea seeds (Chickpea seed protein sequences (legumin and provicilin) were evaluated for the prediction of ACE-I inhibitory peptides using the BIOPEP-UWM database . The preComputer-aided quantification parameters of chickpea proteins were calculated using the tool “Calculation” available in the BIOPEP-UWM database . The foli—the number of repetitions of ACE-I-inhibitory fragments in the protein sequence.N—the number of amino acid residues of the protein.k—the number of different ACE-I-inhibitory fragments.N—the number of amino acid residues in the protein sequence.50i–the half-maximal effective concentration.ECLegumin and provicilin sequences were submitted to in silico enzymatic hydrolysis using the “enzymatic action” tool available in the BIOPEP-UWM database . Single E) and (2) the ACE-I inhibitory potential of peptides released by a given enzyme (BE). Higher values of AE correspond to more peptides released with ACE-I inhibitory activity. Contrary, lower values of BE equal a higher ACE-I inhibitory potential of peptides released by a given enzyme. Parameters AE and BE were calculated as follows:The following quantification parameters were calculated from the peptides released from the enzymatic hydrolysis: (1) the frequency of peptides released with ACE-inhibitory activity by a given enzyme  (accessed on 15 December 2021). The ACE-I structure was prepared for molecular docking by removing the ACE-I inhibitor lisinopril and the water molecules. The ACE-I model included polar hydrogens and charges [The three-dimensional (3D) structures of the predicted ACE-I inhibitory peptides were obtained from the PubChem or SATPdb databases and prepared for molecular docking (adding the polar hydrogens and assigning charges). Peptides without 3D structures available on PubChem or SATPdb databases were excluded from the molecular docking analysis. The crystallographic structure of human ACE-I in complex with the ACE-I inhibitor lisinopril (PDB ID: 1O86)  was obta charges . Incompl charges . Docking charges ,15. The  charges . The selADMET is an important set of pharmacokinetics properties for identifying bioactive peptides with the potential to exert in vivo activity. In brief, ADMET properties of ACE-I inhibitory peptides with EC50 values <200 µM were predicted using the ADMETlab 2.0 server . The tooIn this study, the ACE-I inhibitory potential of legumin and provicilin peptides from chickpea were predicted as well as the ACE-I inhibitory peptides released by simulated enzymatic hydrolysis. Additionally, the potential interactions between the ACE-I inhibitory peptides and the active site of ACE-I were evaluated through molecular docking, and the potential pharmacokinetics of the peptides were assessed. The peptides with ACE-I inhibitory activity predicted by the “profiles of potential biological activity” tool within the sequences of legumin and provicilin are shown in E values of 0.0605 and 0.0442, respectively. Contrarily, trypsin hydrolysates had the lowest AE values (0.0101 for legumin and 0.0362 for provicilin), but the peptides released from legumin after trypsin hydrolysis had the most potent ACE-I inhibitory potential (BE = 0.00018). Pepsin hydrolysis can release potent ACE-I inhibitory peptides from provicilin (BE = 0.00011). Papain and alcalase hydrolysates of legumin and provicilin generated ACE-I inhibitory peptides  at higher frequency than human proteases (E = 0.00018–0.00302) than the ones generated with papain (BE = 0.00302–0.00139) and alcalase (BE = 0.00273–0.00067). Previous studies have reported that lower ACE-I IC50 values are obtained after in vitro gastrointestinal digestion (140–229 µg/mL) than after in vitro papain and alcalase hydrolysis  of two chickpea variates [Papain and alcalase hydrolysates from different food sources could have antitumor, antioxidant, antidiabetic, and antihypertensive activities ,18. Thusroteases . Howevervariates .https://doi.org/10.6084/m9.figshare.19710343) (accessed on 4 May 2022). The binding energy between legumin or provicilin peptides with the ACE-I active sites ranges from −5.7 to −9.2 kcal/mol. The main molecular interactions between the ACE-I inhibitory peptides and the amino acid residues from the ACE-I active sites were conventional hydrogen bonds and attractive charges (distances ranges from 1.92 to 3.08 Å) . Docking results are shown in  3.08 Å) . These fThe ADMET properties of ACE-I inhibitory peptides with an EC50 < 200 uM were screened using the ADMETlab 2.0 platform . CompliaPapain, alcalase, pepsin, trypsin, and chymotrypsin hydrolysates of legumin and provicilin from chickpea are potential sources of ACE-I inhibitory peptides. The molecular docking interactions of the ACE-I inhibitory peptides from chickpea proteins suggest strong interactions with residues from the active sites of ACE-I through hydrogen bonds and salt bridges. Furthermore, ADMET prediction analysis indicates that CP are potentially absorbed at the intestinal level, are bioavailable, are adequately distributed in systemic circulation, and are nontoxic. Further studies evaluating the antihypertensive effect of chickpea hydrolysates in vivo are warranted. The use of databases and in silico assessments as the first steps in the search for bioactive peptides derived from food sources could reduce time and optimize resources in research laboratories.


Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus. Upon assessing their antibiofilm activities, the ten tested gliptins significantly diminished biofilm formation. In particular, sitagliptin exhibited the most efficient antibiofilm activity, so it was chosen as a representative of all gliptins to further investigate its antivirulence activity. Sitagliptin significantly protected mice from P. aeruginosa and S. aureus pathogenesis. Furthermore, sitagliptin downregulated QS-encoding genes in P. aeruginosa and S. aureus. To test the anti-QS activities of gliptins, a detailed molecular docking study was conducted to evaluate the gliptins’ binding affinities to P. aeruginosa and S. aureus QS receptors, which helped explain the anti-QS activities of gliptins, particularly sitagliptin and omarigliptin. In conclusion, this study evaluates the possible antivirulence and anti-QS activities of gliptins that could be promising novel candidates for the treatment of aggressive Gram-negative or -positive bacterial infections either alone or as adjuvants to other antibiotics.The development of bacterial resistance to traditional antibiotics constitutes an emerging public health issue. Promising approaches have been innovated to conquer bacterial resistance, and targeting bacterial virulence is one of these approaches. Bacterial virulence mitigation offers several merits, as antivirulence agents do not affect the growth of bacteria and hence do not induce bacteria to develop resistance. In this direction, numerous drugs have been repurposed as antivirulence agents prior to their clinical use alone or in combination with traditional antibiotics. Quorum sensing (QS) plays a key role in controlling bacterial virulence. In the current study, dipeptidase inhibitor-4 (DPI-4) antidiabetic gliptins were screened for their antivirulence and anti-quorum sensing (anti-QS) activities against Gram-negative  Since their discovery, antibiotics have revolutionized the treatment of infectious diseases; however, this great achievement has been fading due to the cumulative ubiquity of microbial resistance, constituting a major global health challenge ,2. With Bacteria recruit an extensive panel of virulence factors and employ diverse systems to regulate the expression of these factors. The bacterial arsenal of virulence factors extends from acquiring bacterial structures, such as capsules, pili, flagella, and others, to the production of a diverse array of destructive extracellular enzymes, such as protease, elastase, urease, hemolysins, and others ,8,21. BaSeveral tactics have been suggested to target bacterial virulence by utilizing a wide array of natural and synthesized chemical moieties ,32,33,34Serratia marcescens virulence factors, such as biofilm formation, bacterial motility, and the production of protease, catalase, and hemolysins, and diminished its pathogenesis in vivo . T. T78]. Ting site ,96,97,98ing site ,96,97,98ing site ,92,97,98ing site ,94,95.Despite the fact that AgrC can be crystallized in its holo state , none of the crystallized AgrC PDB files deposited within the protein data bank revealed ATP electron density ,87. The The latter compounds can generally adopt more extended conformations, which would cause significant steric clashes with the pocket G2 box/α4 helix residues. Conformation analysis of the crystallized AgrC protein illustrated that both α3 and α4 positions are more likely to hinder large substrate binding due to their relative conformations and their presence at the active site entrance . In thesInvestigating the differential gliptin binding modes showed that the top docked ligands with almost comparable high docking scores  formed extended polar contacts with key pocket residues . These lOMR showed a very low docking score , which is likely due to significantly fewer polar contacts with the ATP-binding site. Furnishing hydrogen bonds with conserved G1-box Asp373 and Asp374 mainchain NH groups and a single polar contact with the ATP-lid Glu391 residue permitted relative ligand stability inside the active site F. DespitOn the contrary, ADP-β-N showed highly extended polar networking with the pocket residues, translating into a significantly high docking score . Polar contacts with the Asp373 mainchain and Gln423 sidechain are important in anchoring ADP-β-N at the nucleotide-binding site to involve several polar contacts between the ligand’s phosphate groups and mainchains of Ser387, Gly392, Arg393, and Leu397 residues G. BesideS. aureus cytoplasmic response regulator AgrA  ,99S. aurAlanine-mutated AgrA showed lower DNA binding affinities with increased Kd values of almost 40-, 10-, and 90-fold for His169, Asn201, and Arg233, respectively, as compared to the wild-type protein . NeverthS. aureus AgrA inhibitors for disrupting agr-driven quorum sensing [For a validated molecular docking study, a reference control inhibitor was adopted since none of the PDB-deposited AgrA high atomic resolution structures show bound ligands. Savarin (SAV) is one of the most cited  sensing ,104,105. sensing . AdditioThe docking results showed comparable orientations as well as conformations for the docked gliptins and the reference control at both sites C. The toIn our docking study, the control reference SAV showed similar reported Site I–specific binding patterns, which have been depicted in several reported studies ,102,105.Similar Site I/Site II docking preferentiality was also illustrated for both the top active investigated gliptins (SIT and TRG) and the modest control, OMR. Higher and more extended polar interactions were observed for SIT at Site I, where the ligand’s triazolopiperazine ring managed to form contacts with the sidechains of Arg218 and Arg233, as well as Asn201 and Val232 mainchains E. AdditiA comparison of the highest and lowest docked gliptins, TRG and OMR, showed that the docking scores appear to correlate with the extent of polar interaction networking as compared to hydrophobic contacts. Due to TRG’s non-linear topology, the ligand adopted radial proximity to the interface between Site I and Site II F. The TRBased on the modeling data, the top active gliptins are capable of anchoring at the AgrA-LytTR DNA-binding site within SAV, suggestive of a promising biological profile. Moreover, Site I- over Site II-docking preferentiality holds the advantage of interacting with a wider range of residues that are reported to be responsible for AgrA transcriptional and DNA-binding machinery. These include Arg233 and Asn201, which mediate specific direct DNA-binding contacts, as well as others, such as His200 and Tyr229, which are reported as being important for protein transcriptional activation and DNA affinity.P. aeruginosa and S. aureus. However, all gliptins showed significant antibiofilm activities; sitagliptin and omarigliptin showed the most significant biofilm inhibition. Sitagliptin was chosen for further antivirulence evaluation. Sitagliptin at sub-MIC significantly protected mice against P. aeruginosa and S. aureus. The anti-QS activity of sitagliptin was assessed, and it significantly downregulated P. aeruginosa QS-encoding genes and S. aureus QS- and biofilm-formation-encoding genes. A detailed in silico monocular docking study was conducted to test the ability of different gliptins to hinder the QS receptors in P. aeruginosa and S. aureus. The molecular docking demonstrated the ability of sitagliptin and omarigliptin to bind to different QS receptors. The current study evaluated the preliminary possibility of gliptins and other related chemical moieties to serve as antivirulence and anti-QS agents; however, further pharmacological and toxicological studies are necessary prior to the use of gliptins as adjuvants to traditional antimicrobial therapy.Counteracting bacterial resistance requires developing new innovative approaches, and targeting bacterial virulence is one of them. Mitigating bacterial resistance confers several merits, as it does not influence the bacterial growth or induce the bacteria to develop resistance and, at the same time, facilitates their eradication by immune cells. Gliptins are dipeptidyl peptidase-4 inhibitors (DPI-4) that are used to control blood glucose levels in diabetes mellitus. In the current work, the antivirulence activities of gliptins were evaluated against two bacterial models,


Fruit is a kind of plant food which is rich in nutrients and immune-regulating ingredients. A meta-analysis has demonstrated that fruits have a protective effects against asthma. On the other hand, clinical syndromes of allergic reactions to fruits manifest as an oral allergy syndrome. We aimed to investigate the patterns and associated factors of fruit allergen-specific IgE (sIgE) sensitization among patients with suspected clinical symptoms. Data were extracted from the Chang Gung Research Database. Fruit sensitization in Taiwan was evaluated using the presence of IgE antibodies against specific fruits. The overall prevalence of positive sIgE responses to fruit allergens in Taiwan, in order of decreasing importance, was pineapple, kiwi, banana, and papaya. Children aged 0–18 had a higher positive rate of allergic responses to pineapple, kiwi, banana, and papaya than adults over the age of 18. Positive specific IgE for kiwi, banana, or papaya was more frequent in younger than in older children and children with a higher total IgE of both logarithmic (log) and arithmetic values. The analysis of log IgE for pineapple positive vs. negative children determined an optimal cutoff value, log IgE 2.2, with both sensitivity (0.9) and specificity (0.5). Dermatitis was significantly more prevalent in children with positive IgE for pineapple, kiwi, banana, and papaya than negative specific IgE. The highest positive rate of sIgE against fruits was pineapple among children. Even in older children, the positive rate of pineapple allergens was high. IgE discriminates with and without sIgE for pineapple, with an optimal cutoff of 158.5 U/mL. Allergies constitute a global public health burden, and food allergies have increased worldwide ,2,3. TheIt has been argued, by Hua et al., that the diversity of allergenic foods in infancy, including fruits, may protect infants from IgE sensitization at the age of one year . Fresh fChallenge tests found that the prevalence of allergy to fruits greater than 1% included apples, pineapples, and kiwi, and the skin prick tests found only apples to be greater than 1% . The preData about fruit sensitization in children, especially tropical fruits, is scarce. In vitro testing of serum allergens in patients is an important method for identifying allergens. Although the number of hospital visits for patients with suspected allergy symptoms has decreased and dietary habits have also changed during the COVID-19 pandemic, the positive rate of food sensitization has not changed ,18. WhilWe adopted a multi-center hospital-based  approach. Specific IgE was retrospectively collected from an administrative database. All fruits in the Chang Gung Research Database (CGRD) were analyzed. In CGRD, testing for allergen fruit was entirely performed at the discretion of the individual clinician. Data consisted of all age groups of patients with allergic diseases who had asthma, dermatitis, or rhinitis and higher IgE, eosinophil counts, or positive phadiatop allowed by insurance claims during the period 2015–2019 in Taiwan. The guidelines for sIgE at the Chang Gung Memorial Hospital (CGMH) followed the rules established by Taiwan Health Insurance, which do not support asymptomatic sIgE testing. Specific IgE data have only been integrated with CGMH since 2015. Information on the patients’ age, sex, total IgE, and diagnosis was also obtained. We further analyzed the positive rate of various fruit allergens and the associated factors of specific allergens. The numbers of patients investigated and the positive percentage were listed in the Specific IgE levels of patients’ sera samples were measured against allergens according to the standardized procedure guidelines . In CGRD, the test (immunoCAP) identified allergen-sIgE against fruits in serum . The sIgThe demographic information on patients and allergic comorbidities  were compared between the negative and positive sIgE groups for fruits. All allergic illnesses were followed at the outpatient clinics, inpatient ward and/or emergency rooms. We defined the seasons as the time of sIgE determination: spring as March–May; summer as June–August; autumn as September–November; and winter as December–February .2 tests. p values <0.05 were defined as statistically significant. Continuous variables were presented as the mean ± standard deviation.To obtain the best cutoff value of pediatric logarithmic (log) IgE, which was the coordinate point corresponding to the point closest to the upper left corner of the receiver operating characteristic curve, we selected the best critical point according to the largest Youden’s index . Statistp = 0.004; kiwi, p < 0.001; banana, p < 0.001; papaya, p < 0.001) . Detailed data and the number of patients tested for each top allergen on sensitizing fruits are presented in p < 0.001, for banana 4.9 ± 4.5 and 6.9 ± 5.5 years old, p < 0.001, and for papaya 4.7 ± 4.3 and 7.1 ± 5.5 years old, p < 0.001) (p < 0.001) (The most common fruit sensitizations in children were pineapple (specific antibodies detected in 10.6% of subjects), kiwi (10.2%), banana (10.1%), and papaya (7.2%) . For the< 0.001) . In the < 0.001) . In cont< 0.001) .The analysis of the log IgE values of pineapple with significant differences for positive vs. negative sIgE allowed us to determine the optimal cutoff value of 2.2 (IgE 158.5 U/mL) with excellent sensitivity (0.9) and modest specificity (0.5) . The anan = 302, 48.1%, p < 0.001), asthma combined with dermatitis , dermatitis combined with rhinitis , and asthma combined with dermatitis and rhinitis . Regarding the above clinical appearance and sIgE, children with positive sIgE for kiwi, banana, or pineapple have higher rates of dermatitis, asthma/dermatitis, dermatitis/rhinitis, and asthma/dermatitis/rhinitis than those with negative sIgE (p < 0.05). Children with positive sIgE for bananas have higher rates of asthma combined with rhinitis .Common risk factors of fruit sensitization in children include dermatitis, concurrent asthma/dermatitis, and dermatitis/rhinitis . Clinicap < 0.001 between pineapple and orange; 2.3% of mango and 2.1% of melon)  . We analTo the best of our knowledge, this large-scale database study is the first to investigate fruit sensitization-based sIgE tests in Taiwanese children. As a result, we found that fruit sensitization was significantly associated with various factors including age, IgE, and clinical presentations. Applying judicious testing may be helpful in younger patients with symptoms indicating clinical allergy and higher total IgE who were likely to have fruit sensitization. The most important approach is to not indiscriminately remove tolerable fruit from one’s diet simply because they are related to a mild reaction . A comprThe top four fruits with positive sIgE were all locally grown tropical fruits from Taiwan that were highly ingested by Taiwanese. Since the positive rates of challenge tests for pineapple and kiwi were the highest among fruits, the sensitization of these particular two needs special attention . Of thosAge and total IgE but not sex were significant risk factors for positive sIgE against fruits. The risk of sIgE was higher in allergic diseases among teenagers, and the sIgE of food was the second-highest among allergic diseases in Taiwanese children, second only to dust mites . The reaPollen exposure plays a role in the prevalence of cross-reactive food sensitization mediated by IgE antibodies . Both biAn important limitation of the present study is its selection bias in pediatric patients with higher IgE in the database. The reason may be that clinicians tended to check the specific IgE of the fruits for those patients with high IgE. Another limitation was the correctness of the diagnostic code by physicians in the database. Due to the lack of standard operating procedures and considering the low willingness to participate in challenge tests, no further skin prick or challenge tests could be performed ,30. In tThis study highlighted a higher sensitivity rate of fruit allergens to pineapple, kiwi, banana, and papaya in children inhabiting the Taiwanese region rich in tropical fruits. Leading complications of hypersensitivity in children were dermatitis and dermatitis combined with asthma or rhinitis. An analysis of sIgE tests revealed that the sensitivity rates for fruits were higher in children than in adults. Further prospective or national health and nutrition examination survey studies are warranted to confirm our findings and identify any clinical relationships.