Yarrowia lipolitica yeast. This manuscript describes a successful attempt at potato starch modification with a post-culture medium of Yarrowia lipolitica yeast with different concentrations of organic acids. Starch preparations produced by roasting (130 °C) and these produced by starch reaction with a synthetic acid mixture were compared in terms of the types and number of acid residues esterified with starch. The effectiveness of starch esterification was found to depend on medium composition and to be higher upon the use of a synthetic acid mixture. In addition, a higher reactivity with starch was demonstrated for citric acid than for α-ketoglutaric acid. The highly-substituted starch esters formed as a result of potato starch modification with post-culture medium were characterized by decreased values of thermal parameters of pasting characteristics, determined with a differential scanning calorimeter (DSC), and by compromised resistance to amylolysis. The intensity of these changes increased along with an increasing total percentage of starch ester substitution.This study aimed to identify the feasibility of producing highly-substituted starch esters via thermal modification of starch using a post-culture medium of  Starch—which plays the role of a storage substance in plants—is produced on the industrial scale mainly from maize, rice, tapioca, and potatoes. In 2018, global starch production was estimated to reach 133.5 mln tons .Chemical processes employed in food technology are those based on chemical reactions which require specified chemical substances and proceed without any biological factors . It mustYarrowia lipolitica yeast [Rich sources of organic acids include, among others, microbiological post-culture media deemed to be waste products after the production of acids by ca yeast . An exceca yeast ,11,12. TYarrowia lipolitica yeast containing various concentrations of organic acids, and then to determine changes in the thermal properties of the modified preparations as well as their susceptibility to amylolysis.The major objective of this study was to identify the feasibility of producing highly-substituted starch esters during thermal modification of starch using a post-culture medium of Yarrowia lipolitica yeast containing a mixture of carboxylic acids. The media were obtained from the Department of Food Biotechnology and Microbiology of the Wrocław University of Life Sciences, and their composition was as follows:ABK medium—α-ketoglutaric acid 26.5 g/L; citric acid 73.0 g/Lα20K medium—α-ketoglutaric acid 55.7 g/L; pyruvic acids 3.7 g/LY20K medium—α-ketoglutaric acid 51.5 g/L; citric acid 14.9 g/L; pyruvic acid 5.0 g/LX16M medium—citric acid 69.9 g/L; α-ketoglutaric acid 28.7 g/LPotato starch produced by PPZ Niechlów, Poland, in 2017 was modified with four post-culture media of Concentrations of acids in these media were determined using the high performance liquid chromatography (HPLC) method : citric acid, α-ketoglutaric acid, and pyruvic acid ; all acids were produced by Sigma .Starch preparations were produced according to the method used for starch citrate production described by Kapelko-Żeberska et al. . Native Starch modification with a synthetic mixture of carboxylic acids was conducted analogously to starch modification with acids of the post-culture media.+ column coupled with a UV detector (λ = 210 nm) and a refractometric detector at a temperature of 65 °C, with a liquid phase (25 nM trifluoroacetic acid) flow rate of 0.6 mL min−1. Compounds were identified based on chromatograms of standards of pure chemical substances at the Department of Food Biotechnology and Microbiology of the Wrocław University of Life Sciences [The degree of substitution was expressed in grams of acid residues per 100 g of starch preparation (substitution percentage). The content of acid residues esterified with starch was determined with HPLC  on a Carbohydrate HSciences .To prepare the samples for HPLC analysis of acid residues, 2 g of starch preparation was treated with 100 mL of 0.5 M NaOH  for 24 h under continuous stirring. Afterwards, 250 mL of concentrated ethyl alcohol  was added and the solution was left at room temperature for 24 h. Next, the solution was filtrated and the filtrate obtained was concentrated to a volume of 50 mL. Thus, prepared solutions were determined for the content of acid residues. The analysis was conducted in triplicate.The substitution percentage was used to compute an esterification yield understood as the ratio of the obtained substitution percentage to the theoretical highest substitution percentage possible at the acid dose applied ,15.The characteristics of phase transitions were determined for the starch preparations produced with the post-culture media. Native starch and starch roasted without the modifying agent were used as reference samples. Phase transition characteristics was determined using the differential scanning calorimeter DSC 822  in 100 µL aluminum crucibles with lids. Dry matter (10 mg) of the analyzed samples and water were weighed into a crucible at a ratio of 1:3. The crucible was covered with a lid, conditioned at room temperature for 30 min, and then placed in a calorimeter chamber at 25 °C and heated to 100 °C at a heating rate of 4 °C/min. The thermal curve plotted was used to determine the temperatures of the onset, maximum, and end of the phase transition, as well as the specific heat of transition. The analysis was conducted in triplicate .Analyses were carried out for the starch preparations produced with the post-culture media. Native starch and starch roasted without the modifying agent were used as reference samples. A water suspension of starch preparations (0.72 g of preparation per 100 mL of solution) was heated at 100 °C for 5 min. The resultant paste was double diluted with an acetate buffer (pH = 4.3). Flasks with mixtures were put into a water bath with a temperature of 37 °C, and a mixture of α-amylase and glucoamylase enzymes  was added. The dose of enzymes was adjusted so as to ensure complete saccharification after 20 min of native starch paste hydrolysis. The samples were incubated at 37 °C for 20 and 120 min. Afterwards, glucose content was determined in the samples using a glucose assay kit  which contained glucose oxidase and peroxidase. Following reaction with kit components, glucose contained in the samples formed a color complex, the concentration of which was measured spectrophotometrically  at a wavelength of 500 nm and compared to a standard curve. Saccharification of starch preparations exposed to a mixture of amylolytic enzymes for 20 and 120 min was calculated and used to compute the percentage of fractions of rapidly-digestible starch (RDS), slowly-digestible starch (SDS), and starch resistant to amylolysis, in the starch preparations. The analysis was conducted in triplicate ,17.Study results were subjected to statistical analysis using a Statistica 13.1 software package. Two-way analysis of variance was conducted. The significance of differences between mean values was determined with Duncan’s test at a significance level of α = 0.05.Yarrowia lipolitica yeast. This type of post-culture media management has been rarely addressed in research works.In this study, an attempt has been made to modify potato starch using a post-culture medium of Lower affinity to starch was observed for alpha-ketoglutaric acid residues compared to citric acid. No studies are available in the literature that describe the possibility of starch modification with α-ketoglutaric acid and the properties of starch esters with this acid. In all preparations produced with the post-culture media, the percentage content of alpha-ketoglutaric acid was below 0.06% . AdditioResults obtained for the substitution percentages were reflected in results of the thermal analysis . Starch Preparations produced upon starch modification with the post-culture media were also analyzed for their susceptibility to amylolysis . This suYarrowia lipolityca yeast was observed to result in starch esterification with residues of organic acids contained in the medium, the effectiveness of which depends on the composition of the medium.In this study, potato starch modification with medium remaining after a culture of Esterification effectiveness after the use of the post-culture medium was seen to be lower than that observed after the use of a synthetic mixture of acids.When the post-culture media and acid mixture were used, higher susceptibility to esterification with starch was noted for citric acid than for α-ketoglutaric acid.Yarrowia lipolytica post-culture medium were characterized by modified—respectively to the substitution degree—DSC pasting characteristics and resistance to amylolysis compared to native starch.Highly-substituted starch esters produced via potato starch modification with


The aim of this study was to evaluate the influence of multimode MDP-based primers and different application protocols on the bond strength of a representative resin cement to an yttrium stabilized tetragonal zirconia (Y-TZP) ceramic.2 particles; (2) Z-Prime Plus; (3) air abraded with SiO2 particles + Z-Prime Plus; (4) air abraded with SiO2 particles + All-Bond Universal; (5) air abraded with SiO2 particles + ScotchBond Universal Adhesive; and (6) untreated zirconia. The luting agent (Duo-Link cement) was applied on the treated dentin surface. Specimens were stored in water  and tested in shear bond strength. Data were statistically analyzed using 2-way ANOVA and Post hoc Tukey tests (α = 0.05). The occlusal dentin from 60 human molars was exposed. The teeth and zirconia cylinders (N = 60)  were divided into six groups (n = 10) according to the ceramic surface conditioning: (1) air abraded with SiO Significant effects of ceramic conditioning were found (p < 0.0001). The specimens sandblasted with silica particles followed by the application of Z-Prime Plus or All-Bond Universal presented greater bond strength values. For the untreated zirconia, several specimens failed prematurely prior to testing. Sandblasting with silica particles combined with Z-Prime Plus increased the bond strength. Yttrium stabilized tetragonal zirconia polycrystals (Y-TZP) ceramic is a material used for the restoration of teeth with primarily full coverage restorations. The zirconia crowns can be milled monolithically to full contour or alternatively the core of zirconia can be milled followed by layering of feldspathic porcelain onto the core. The layering of the porcelain onto the core of the zirconia has resulted in superior esthetics along with issues of chipping of the veneered porcelain –3.In some situations, the remaining tooth structure for a full coverage crown does not allow for adequate retention/resistance form. Therefore, the options are either increasing the clinical crown height with crown-lengthening procedures to facilitate restorative therapy , 5 or inZirconia does not contain an amorphous silica glass and as a result does not allow for traditional ceramic surface treatment , 9. VariOne of the ongoing concerns with increasing the internal surface area for bonding by mechanically altering the surface of zirconia with a diamond bur or sandblasting is the development of a phase change in the zirconia and as a result introducing flaws into zirconia microstructure –14. The The use of MDP (10-methacryloyloxydecyl dihydrogen phosphate) as a multimode functional adhesive monomer has been postulated to provide increased retention for cements used in the cementing of zirconia crowns by increasing the bond to zirconia as well as to dentin . It has The aim of this study is to evaluate the influence of various multimode MDP-based primers and different application protocols on the bond strength of a representative resin cement to a yttrium stabilized tetragonal zirconia (Y-TZP) ceramic as well as of zirconia to dentin.μm, under copious water cooling.Partially sintered zirconia disks  were used to obtain zirconia cylinders of 4 mm in height and 3 mm in diameter using a computer-aided manufacturing technique . Sixty zirconia cylinders were machined under water coolant . The cylinders were subsequently sintered in a VITA Zyrcomat 6000 MP furnace , according to the manufacturer's instructions. All specimens were polished using Diamond Lapping Films  in a decreasing sequence of abrasive size from 30, 15, 9 to 3 2. The prepared teeth were finally randomly assigned to six groups of ten teeth.Sixty noncarious human molars extracted for periodontal or orthodontic reasons were collected under ethical approval from the University of Alberta Ethics Committee In Human Research (Pro00063336). The teeth were scaled, cleaned, stored in 0.5% chloramine solution at 4°C to prevent bacterial growth, and used within 3 months following extraction. The crowns were sectioned horizontally with a low speed and intense water cooling to remove the occlusal morphology. The teeth were then ground perpendicularly to their longitudinal axes using P320 SiC paper  until all occlusal enamel was completely removed , 17. The2 particles; (2) primed with a thin layer of Z-Prime Plus ; (3) air abraded with SiO2 particles and primed with a thin layer of Z-Prime Plus; (4) air abraded with SiO2 particles and coated with a thin layer of All-Bond Universal; (5) air abraded with SiO2 particles and coated with a thin film of ScotchBond Universal Adhesive . A final group of ten specimens (6) were left as the untreated zirconia control.Prior to surface conditioning, all ceramic blocks were ultrasonically cleaned  for 5 minutes in distilled water. The ceramic cylinders were randomly divided into six groups (n = 10) with each group receiving a different conditioning treatment to the bonding surface. Group (1) was air abraded with SiOμm diameter SiO2 particles .Particle air abrasion was performed by making circular movements with the nozzle of the air abrasion unit at a distance of 10 mm, with 2.8-bar pressure for 15 s using 30 2) for 10s each from two opposite sides of the ceramic cylinder. After ten minutes following resin cementation, the specimens were transferred to storage in distilled water for 24 h at 37°C prior to shear bond strength determination.A small portion of Duo-link cement  was placed on the treated ceramic surface. The zirconia cylinder was placed on the treated dentin surface using a positioning device that allowed for the application of a consistent 750 g load on the ceramic-resin cement-dentin block. Excess cement was carefully removed using a microbrush and the resin cement light cured  and the cross-sectional area of the adhesive interface wherer, the radius of the bonding interface, was 1.5 mm.Shear bond testing was performed in a universal testing machine  with loading applied in a knife-edge configuration using a crosshead speed of 0.5 mm/min. Each specimen was rigidly positioned to ensure that the adhesive interface was parallel and as close as possible to the knife displacement axis. The shear bond strength (SBS) (MPa) was calculated from the recorded failure load  to discriminate the failure mode. Further specimens with representative fractures were selected for imaging using scanning electron microscopy (SEM). Specimens were mounted on a metallic stub, sputter coated with 7 nm of platinum in a Polaron E5100 coating unit , and observed at x20 and x500 magnifications .α = 0.05). P values less than 0.05 were considered to be statistically significant in all tests. Specimens that failed prematurely during the aging conditions were considered as 0 MPa for statistical analysis.The shear bond strength (MPa) data were statistically analyzed using a two-way analysis of variance (ANOVA) where the factors were surface finish (at two levels) and the use of a surface primer/adhesive (at four levels) followed by post hoc Tukey tests (2 particle air abrasion prior to cementation on the recorded shear bond strength (SBS) (p < 0.001). Furthermore, the SBS was significantly influenced by the use of a surface primer/adhesive but the pattern of change in SBS was associated dependent on zirconia surface condition (p < 0.001). Post hoc Tukey tests demonstrated that the use of Z-Prime Plus and All-Bond Universal to condition the SiO2 particle air abraded zirconia surface, resulted in significantly higher bond strengths (p < 0.001) than the corresponding unconditioned surface  when combined with Z-Prime Plus and also All-Bond Universal. Air abrasion with silica particles in combination with Z-Prime Plus or All-Bond Universal allowed the highest mean SBS values.A two-way ANOVA demonstrated a significant impact of modifying the zirconia surface with SiO surface . The meaA summary of failure types is presented in This study evaluated the influence on bond strength of multimode MDP-based primers and application protocols on the bond strength of resin cement to dentin and a yttrium stabilized tetragonal zirconia polycrystal (Y-TZP) ceramic.The shear bond strength test (SBS) was used to evaluate the adhesive interface between dentin, resin cement, surface treatment modalities, and the Y-TZP. Shear bond strength tests evaluate the cohesive strength of the base material better than the bond strength of the adhesive interface . The con2 was used [There is a concern with air abrasion to Y-TZP because this process may induce stresses that result in crack propagation –21. Recewas used .2 particles followed by the application of Z-Prime Plus or All-Bond Universal resulted in higher bond strength values. For the untreated zirconia, several specimens failed prematurely prior to testing which is attributed to the unstable and weak bond strength present in this group.Significant effects of ceramic conditioning were found (p < 0.0001). The specimens air abraded with SiOμm of SiO2 particle with no further surface treatment does not improve the bond strength. However, if subsequent to air abrasion a multimode MDP application is completed to Y-TZP, there is a significant difference in the bond strength. There have been various explanations provided for this fact, including (a) mechanical surface abrasion and cleaning of the surface, (b) silica deposition, (c) increased chemical reactivity energy levels, or (d) a combination of all the preceding reasons [The results of this study would indicate that air abrasion with 30  reasons .There are differences among the studies regarding sandblasting consequences likely due to the varying size of particles, pressure, distance, and angle of application. Nevertheless, surface roughness alone would not account for improved bond strength as there was no significant difference in bond strength between untreated zirconia and the specimens which were only sandblasted. Additionally, this study demonstrated that without air abrasion the application of a multimode MDP adhesive monomer did not provide an increase in bonding strength.2 deposited on the zirconia surface resulting in increased reactivity between that layer and a MDP adhesive monomer such as All-Bond Universal and ScotchBond Universal. The phosphate ester monomers from MDP have been shown to react with Y-TZP [Chen in 2011  has showth Y-TZP .Z-Prime is a zirconia primer that contains a mixture of organophosphate and carboxylate monomers. The organophoshate monomer has an organofunctional end that can polymerize with the resin cement . The synEvaluation of the mode of failure indicated that no complete failures occurred at the dentin- cement interface and this is consistent with the findings of other studies . This inFailures for SB + Z-Prime Plus took place mainly at the Y-TZP and cement interface. Given that all samples failed in the untreated zirconia at this interface this is not an unexpected finding and would indicate that given zirconia's dense tetragonal structure there is minimal mechanical retention provided by the material itself. When the zirconia was sandblasted there were less failures at the cement-zirconia interface indicating some change has occurred to the Y-TZP surface. The addition of Z-Prime Plus to the untreated surface changed the mode of failure in some samples but did not increase the bond strength for that of the untreated zirconia. When the samples were sandblasted and then conditioned with Z-Prime Plus, All-Bond Universal, or ScotchBond Universal, changes occurred to the mode of failure compared to the untreated zirconia. This factor indicates a positive effect on bond strength at the Y-TZP-cement interface.2 particles prior to conditioning the bonding surface significantly increased the bonding of Y-TZP to dentin. The SB + Z-Prime Plus protocol resulted in the highest bonding values.Within the limitations of this study, Y-TZP surface sandblasted with 30 micron SiO2 particles and primed with a thin layer of Z-Prime Plus is the recommended surface treatment before cementing a zirconia-based restoration.Zirconia surface air abraded with SiO


Several mouse models of autism spectrum disorder (ASD), including the BTBR T + tf/J (BTBR) inbred strain, display a diverse array of behavioral deficits with particular face validity. Here we propose that phenotyping these preclinical models of ASD should avoid excessive reliance on appearance validity of the behavioral observations. BTBR mice were examined in three non-diagnostic symptoms modalities, beside an anatomical investigation for construct validity. The BTBR strain displayed poor sensorimotor integration as reflected by shorter stride length and greater latency on the balance beam task (BBT) when compared with C57BL/6 (B6) controls. Also, locomotor indices in the open-field task (OFT) revealed that BTBR mice traveled longer distances with a remarkably faster exploration than the B6 group in favor of hyperactivity and impulsiveness. Furthermore, analysis of spatial performance including search strategies in the Morris water task (MWT) indicated spatial impairment in the BTBR strain due to failure to employ spatial strategies during navigation. Quantitative cytoarchitectonics and volumetric examinations also indicated abnormal cortical and subcortical morphology in the BTBR mice. The results are discussed in relation to the neuroanatomical correlates of motor and cognitive impairments in the BTBR strain. We conclude that non-diagnostic autistic-like symptoms in the BTBR mouse strain can be impacted by autism risk factors in a similar way than the traditional diagnostic signs. Patients with ASD show persistent deficits in social interaction, restricted interests and activities, and repetitive, stereotyped behaviors3. Given the highly diverse neuropathology and heterogeneous behavioral symptoms of ASD in humans, an animal model that can profile ASD-like characteristics with face validity appears useful for examining the potential relationship between neuropathology and behavioral abnormalities.Autism spectrum disorder (ASD) is typically diagnosed in the early developmental period, and its main diagnostic criteria are behavioral, including a wide range of symptoms in personal, interpersonal, and communicational domains5, a subset of preclinical models using inbred mouse strains have been proposed in recent years that superficially express traits and behaviors often overlapping with the diagnostic criteria for clinical autism. Among the existing mouse models, the BTBR T + tf/J  inbred mouse strain exhibits behavioral phenotypes analogous to core symptoms of autism, thus instituting reliable face validity for modeling ASD8. The BTBR mice exhibit social deficiencies characterized by poor social interaction and impaired communication, repetitive stereotype behaviors, and atypical vocalization10. Aside from behavioral symptoms, structural characteristics have also revealed neuroanatomical abnormalities such as complete or partial lack of inter-hemispheric connections in the corpus callosum and reduced hippocampal commissure associated with hippocampal malformation in the BTBR strain15.The use of rodents to model complex neuropsychiatric disorders with face validity proved to be challenging and comes with significant difficulties and limitations. Despite conceptual and procedural obstacles in developing animal models with core features of autism16. Although productive and informative, face validity, or excessive reliance upon the appearance in preclinical trials to determine behavioral phenotypes, may confound original findings with biased, subjective interpretations. The latter poses a potential source of discrepancy between BTBR-relevant studies5. More importantly, heterogeneity of clinical presentation in individuals with ASD is a particular impediment18. This indicates that a wide variety of other non-diagnostic behavioral symptoms  may not be considered as core symptoms in the animal models of ASD, including the BTBR strain. They are, however, still prominent in a substantial fraction of the clinical population with ASD. Accordingly, beyond face validity alternative explanations for the presence of specific behaviors should be investigated, particularly for those which are less probed or can be linked to brain morphology. In the present study, balance and coordination, locomotor activity, and spatial performance are explored in relation to neuroanatomical correlates in BTBR mice to explore further preclinical aspects of this ASD animal model.In the light of the discussion concerning face validity, BTBR mice display wide-ranging social and emotional impairments analogous to three major domains of ASD diagnostic core symptomsn = 13 and BTBR T + tf/J [BTBR]; n = 9), 3–4 months old were used in this study. No statistical methods were used to predetermine sample size, and the ample sizes are comparable to common standards reported in the field20. BTBR mice were obtained from Jackson Laboratory . Animals were housed at room temperature (21–24 °C) on a 12-h light/dark cycle (lights on at 7:30) with ad libitum access to food and water, and were handled for approximately 3–4 min daily for 5 consecutive days prior to behavioral testing. All procedures in this study were approved by the University of Lethbridge Animal Care Committee in compliance with the standards set forth by the Canadian Council for Animal Care. No randomization was applied to employ the BTBR mice. The number of animals per group used in the present study was the maximum that was approved by the University of Lethbridge Animal Care Committee, and was high enough to ensure statistical power of the analyses that were used. According to ethical regulations, one BTBR mouse was excluded from behavioral testing due to severe self-injurious behaviors.Twenty-two male and female mice  balance beam task (BBT); (2) open-field task (OFT); and (3) Morris water task (MWT). The task order was randomized and all animals were allowed to rest for nearly 24 h after being tested in each behavioral task.21; with modifications). Animals were positioned on one end of an aluminum round bar  and their home cage was located at the other end of the bar. A foam pad was placed underneath to cushion falling animals. The animals were tested for at least three trials on the bar, and their movements were video recorded from a lateral view using a digital camcorder (Panasonic HDC-SDT750) at 60 frames/s with an exposure rate of 1 ms. The latency to traverse the bar, the number of times the hind feet slipped off the bar and stride length were recorded. Each stride was defined as the distance between the takeoff and landing positions of the left hindlimb. A high-contrast point with proper vertical and horizontal edge definition was chosen on the back of the hindlimb . Stride length (cm) on the beam was measured by the number of pixels in the tracked frames traced between the takeoff and landing positions. Also, a suitable target region for tracking was determined based on a pattern that was clearly visible in every frame. If the target point did not contain a high-contrast point to track, the preprocess parameters (e.g. increasing the contrast) were adjusted to make the source image easier to track.The BBT was employed to test sensorimotor integration  was used under dim illumination. Each animal was individually placed in the central zone of the OFT and video recorded for 20 min with a camera mounted above the arena. The experimenter left the room immediately after placing the mouse in the task. Video recordings were analyzed for overall path length and path speed, path length taken by animals in the central zone (~52 cm diameter), and the number of stops by the computer tracking system . Specifically, path length and path speed were measured as an indicator of motivation level. After each animal, the apparatus was cleaned with 70% alcohol. A 5–7 min interval between cleaning and the start of the next testing session was set for alcohol odor dissipation.The OFT was used to assess psychomotor outcomes and exploratory behavior23. The task consisted of a circular pool (154 cm diameter) filled to within 20 cm of the top of the wall with water (22 ± 1 °C). The pool was located in a room enriched with distinct distal cues, which remained unobstructed throughout the duration of the experiment. A circular escape platform (12 cm radius) was visible above the water level or submerged below the water surface and located at any one of four quadrants. The platform was positioned half-way between the center and the pool wall. The animals were introduced into the water facing the pool wall at defined starting positions. All animals were required to locate the platform using either distal and/or proximal cues during navigation in the task. Each testing trial began with the mouse being placed in the pool at one of the four cardinal compass positions  around the perimeter of the pool according to a pseudo-random sequence. The maximum duration of each swim trial was 60 s, and if a mouse found the platform within the allotted time, it was allowed to remain on the platform for 5 additional seconds. If it did not find the platform during the selected time, then it was placed onto the platform for 10 s by the experimenter before being placed back into its holding cage.The MWT was used to assess place and spatial learningBecause performance in the MWT may be affected by deficits in non-cognitive domains, animals were tested by a 1-day testing protocol  using a visible (cued) platform. The platform was elevated above the water surface (0.5–1 cm) and was marked with black electrical tape. The location of the visible platform (quadrant 1) remained constant from trial to trial.Animals were required to find the hidden (invisible) platform across four 60-s trials in the task. The platform was submerged 0.5–1 cm below the water level, and was camouflaged by adding non-toxic white paint  to the water. The platform also moved to a new location (quadrant 3), and testing was not finished until four different locations were completed by each animal. The protocol allowed animals to encode the goal spatial location within a multiple-trials frameworkThe testing procedures used in this phase were identical to those described in the previous phase with the exception that mice were required to locate the hidden platform in a new quadrant (quadrant 4) for 5 consecutive days. Also, the location of the hidden platform remained constant across the testing days. The platform-reversal protocol assumes that animals establish new spatial relationships between spatial contexts and the new platform position after they have previously learned to navigate to a given goal position. Search skills obtained in the former spatial trials may also contribute in developing new search strategies with more cognitive flexibility to explore the platform in the new position. Thus, during the platform-reversal testing, the previous spatial configuration will be updated to establish more efficient navigation within the new spatial context and relationships24.Movements of the animals during the place and spatial learning including latency (time spent to find the platform), swim length, swim speed, error index (swim error or corridor percent path), and path efficiency ratio  were recorded and analyzed by an image-computerized tracking system . The error index in the present study shown in path percentage in the corridor refers to the accuracy of a mouse swim trajectory within a 20-cm-wide corridor from the start point to the platform. Any deviation from this corridor during swimming was scored as an error25. Here spatial reference memory was determined by significantly greater search time in the target quadrant compared to other quadrants of the task.Experimental groups were also subjected to probe trial testing on the eighth day of the MWT testing. This served as a transfer test to determine the extent to which the mice had learned about the location of the platform. The platform was removed from the pool and the animals were allowed to swim freely for 30 s. The percentage of time that they spent in each quadrant of the task including the target quadrant (quadrant 4) was recorded26, the overall search strategies or patterns of each mouse during navigation in the task were analyzed. Two separate investigators blind to the animals genotypes and grouping did manually categorize (modified from refs. 32) the predominant search strategies used by the mice during each trial. A certain strategy was chosen based upon swim path plotted by the tracking system and calculation of pool coverage as well as time spent in a specific area of the pool. The inter-rater reliability (level of agreement between raters) on strategy categorization was 91%. Search strategies in the present experiment were classified as follows: (1) non-spatial strategies including thigmotaxis , random swim , scanning , and chaining ; and (2) spatial strategies including directed search , focal search , direct swim , and perseverance .Also, because the conventional parameters of the MWT alone are not sufficient to draw a reliable conclusion on the hippocampal-dependent aspects of spatial performance33, and no animal was excluded from analysis for abnormal brain weight. The stained sections were imaged using a Nanozoomer 2.0RS slide scanner  for histological analysis and presentation. From the coronal sections, measurements included cortical and dorsal hippocampal (dHPC) volumetrics, neuronal density, and cortical thickness in both hemispheres. Furthermore, in order to enhance the accuracy of the histology, six animals from B6 group and one animal from the BTBR group were excluded from histological assessments and also from all other corresponding volumetric analyses because of damages to one or both hemispheres, and technical issues such as missing and/or damaged sections.Animals in both groups were euthanized and intracardially perfused when behavioral testing completed. Brains including cerebellum were removed, weighed, and fixed for coronal sectioning and cresyl violet staining. Criteria for brain abnormality was chosen based on the brain weight after extractionn = 7; BTBR, n = 8), a set of eight cross sections stained with cresyl violet was considered for cortical volumetric analysis. The most rostral section measured for cortical volume was located at ~1.78 mm anterior to bregma and the most caudal section at −2.06 mm posterior to bregma. The volume averages were calculated by dividing the sum of measures obtained from each brain by the total number of sections (shrunk brain area in mm2). The approximate volume of the cortex and hippocampus  was determined by multiplying the total area in mm2 by both the thickness of each slice (40 μm) and the sampling interval (5)34. The hippocampal volume  in each mouse was estimated using a set of four cross sections of the dHPC area, from ~−1.06 to ~−2.06 mm relative to bregma. In the case of missing or damaged sections (less than three sections for each mouse) data were calculated as average area values from preceding and following sections. For each tissue section, the contours of the bilateral hemispheres were traced and their areas were measured using ImageJ 1.47b .For each animal  was used to record up to six measurements of cortical thickness from each coronal section, three from each hemisphere. Cortical thickness in the present experiment was a mean measure of both hemispheres in seven consecutive slices.Three points  from each brain and hemisphere were selected for cortical thicknesshttp://imagej.nih.gov/ij; NIH, USA). Two regions of interest  were determined in each hemisphere. Both left and right ROIs included the same cortical regions and mainly all six cortical layers. Four approximate planes (between anterior-posterior [AP] ∼1.98 and ∼0.74 mm) of stained sections in each brain were selected. For cell counting, the red, green and blue (RGB) images were converted to 16-bit greyscale images by ImageJ, and a threshold of 90 and 180 was set for black/white background, respectively. When all particles highlighted, the particles that have merged together, were separated by Binary (watershed) option. This can often accurately cut the attached particles apart by adding a 1-pixel thick line where it seems the division should be. The binary image of the particles in black/white ROIs were counted for both hemispheres by the Analyze Particle program. It should be pointed out that because the size and circularity of the particles may affect the final counts, the size and circularity of particles in the present study were set on pixels (pixle^2) 0-infinity and 0.00–1.00, respectively. An analysis of particles within the ROIs  was separately performed for each section, hemisphere and region. All histological assessments were performed by the same person, who was blind to the groups’ assignment.Cellular density analysis (quantitative cytoarchitectonics) was performed using ImageJ 1.47b . The Mann-Whitney U test was also used to compare means of the two groups for a single dependent variable with Bonferroni correction for multiple comparisons when necessary. Correlation between variables  was analyzed by Spearman’s rank correlation. In all statistical analyses , a p-value of <0.05 (two-tailed) was chosen as the significance level, and results are presented as mean ± standard error.Because the data in the present study were not normally distributed, Kruskal-Wallis n = 9) made significantly greater average stride length compared with BTBR animals . More importantly, the average stride length in B6 mice increased across three trials (7.41 < 8.12 < 8.69 cm) suggesting that they gradually were able to reach better balance and perform longer steps across repeated trials. This profile of movement, however, was the reverse in the BTBR mice in the last trial . Accordingly, a larger stride length in the B6 group was associated with lower latency during beam walking. In contrast, because BTBR animals displayed shorter stride length, they had higher latency than B6 animals when crossing the beam. Moreover, despite a slight increase of hindlimb average slips across all trials in the B6 group, no significant difference was found between groups in the number of foot slips .All animals were able to stay and move on the balance beam Fig. . Howeverial Fig. . Also, ln = 13 and 9, respectively). Path length (distance traveled) shown by both groups indicated that the BTBR group traveled significantly more distance than B6 mice  even within the central zone of the open field  and path speed (m/s) for motivational status, and path taken by animals in the central zone of the open field along with the number of stops to assess emotionality. Representative activity traces and parameters in the open field are shown in Fig. 37, and latency can also be potentially affected by differences in swim speed26, in the present study only latency and speed were considered for preliminary spatial analysis.Spatial performance was tested by an 8-day assessment protocol Fig. . Becausen = 13 and 9). No significant difference was found between groups in trials 2–6 , except the first trial (χ2 (1) = 5.900, p ≤ 0.015, Kruskal-Wallis with a mean rank of 8.77 for B6 and 15.44 for BTBR groups; Fig. U = 19.500, p ≤ 0.009, Mann-Whitney U; Fig. p ≤ 0.05, Kruskal-Wallis with a mean rank speed of 7.38 for B6 and 17.44 for BTBR mice).The latency in both groups decreased over six trials of non-spatial testing suggesting that all mice, regardless of their experimental conditions were able to locate the cued platform at a similar rate  acquired and retrieved the location of the hidden platform in a similar manner  displayed relatively the same profile of spatial ability . The BTBR mice, however, moved consistently and significantly faster than B6 mice  will necessarily perform more accurately because their latency to find the hidden platform particularly on days 2–5 reflect a poor spatial function compared to B6 mice.p = 0.412, Kruskal-Wallis). However, although all rats showed a gradual decrease in the latency in the last trial of day 5, B6 mice located the platform more quickly than BTBR mice . Representative swim paths of two B6 and BTBR mice across the two trials  are shown in Fig. Changes in latency over 5 days of acquisition  are depicted in Fig. n = 11) spent a considerable proportion of their time (49.30%) searching in the training (target) quadrant (quadrant 4) in which the hidden platform had previously been located. In contrast, the BTBR mice (n = 8) exhibited a more diffuse pattern of searching, with much less spatial bias toward the former training quadrant  and BTBR (n = 9) groups over 7 testing days.Figure p ≥ 0.05, Kruskal-Wallis; Fig. Analysis of spatial error via corridor percent time in the MWT indicated that both groups displayed a similar swim pattern. No significant difference was found between groups . Trial-by-trial analysis of swimming error also showed that B6 mice remained more in the corridor than BTBR mice during swimming to the hidden platform in the first trial  and last trials  where B6 animals swam significantly more in the corridor than BTBR group in trials 3 and 4 on the last day of spatial testing  path to the actual path taken by animals. The BTBR mice were unable to reduce their path length to the level of B6 mice, except in the first four trials of spatial training  on visible- and hidden-platform days were grouped into either non-spatial or spatial categories . In contrast, spatial navigation on the first invisible-platform day in both groups substantially relied on non-spatial search strategies .On the visible-platform day when the platform was cued, both groups used either non-spatial or spatial strategies more or less in a similar manner. Navigation in both groups was mainly based on DSe and DSw, particularly in trials 5 and 6. Also, non-spatial strategies including T and C in BTBR animals were more common than B6 mice in the first trials Fig. . The higies Fig. . Althougp ≥ 0.05, Mann-Whitney U; Fig. U = 61, p ≤ 0.021, Mann-Whitney U). It appears that these changes in search strategy selection were the primary cause of the improved performance in the B6 group on day 5.In both groups, the use of search strategies changed over 5 days of the platform-reversal protocol. On the first day when animals were required to locate the hidden platform in the new position, the prominent non-spatial strategy use in both groups remained relatively constant across four trials . Also, ROIs for anatomical assessment were chosen based on the brain areas  involved in the neuropathology of autism38.Topographical (gross) anatomy along with routine histology was used to compare the brains of animals . Volume analysis conducted for the left and right hemispheres showed no differences in the experimental groups . However, the B6 mice overall had significantly greater cortical volumes . Correlations between cortical volume and behavioral measures revealed a significant relationship between overall cortical volume and path speed in BTBR mice within the OFT  suggesting that lower cortical volume in the BTBR animals was associated with greater path speed during exploration in OFT.There was no differences in brain weight, although B6 mice had slightly larger brains than BTBR animals , the corpus callosum was evidently absent. Because of the clear callosal agenesis, lateral displacement in the HPC, lateral septum, and striatum was detectible in the BTBR mice (data not shown). In addition to the HPC lateral displacement, the hippocampal commissure was entirely absent in all of the BTBR mice.A comparison between groups indicated that B6 mice had larger dorsal HPC volumes by ~25% compared to the BTBR group , but point because ventral cortex had reduced thickness compared to other points in both groups . Further analysis indicated that B6 mice had larger cortical thickness in dorsal and lateral cortices compared to BTBR animals  indicating that dorsal cortical thickness significantly predicts stride length on the BBT.Cortical thickness was measured in the dorsal, lateral, and ventral points of both hemispheres Fig. . CorticaU = 5, p ≤ 0.001, Mann-Whitney U). No significant difference was found between groups in terms of the number of cells in the dorsal cortex (data not shown).The number of cells in the ROIs of the left and right hemispheres were not statistically different, and no evidence of degeneration, abnormal columnar organization, and/or gliosis was observed in any experimental groups Fig. . HoweverMotor deficits and cognitive impairments represent a heterogeneous array of non-diagnostic symptoms in ASD. The present experiment was directed at a detailed analysis of three lesser-known signs of ASD, sensorimotor integration , locomotion, and spatial performance. These were investigated in relation to neuroanatomy in the BTBR mouse model of autism. BTBR mice displayed shorter stride length and longer latency when crossing the balance beam as compared to B6 control mice. In contrast, hyperactivity during free exploration in the OFT was apparent in BTBR mice as indicated by increased path speed and path length. Furthermore, measures of spatial memory revealed that failure to use spatial search strategies underlies the impaired performance of BTBR mice in the MWT. Neuroanatomic aberrations offered further insights into the functional deficits observed in BTBR mice; in addition to callosal agenesis and deficient dorsal hippocampal commissure (commissure of fornix), BTBR mice showed a strikingly reduced thickness and volume of cortex particularly in the PFC. Also, the HPC, measured in its dorsal part was about 25% smaller in BTBR mice when compared with their B6 counterparts.41. Indeed, all three core symptoms of ASD  require coordinated and synchronized neurological systems, and are established upon regulated sensory information and movement. Therefore, disturbances in motor behavior as part of autism-associated symptoms require more attention in current research.Impaired fine motor skills in patients with ASD are strongly associated with social symptomatology44, motor impairments in the BTBR strain model of autism are minimally characterized. The lack of examination of motor deficiencies in preclinical autism models is mainly due to inconsistencies in assessment and interpretation of motor impairments in ASD compared to the general focus on communication challenges and social interaction deficits. In the present experiment, we identified a substantial motor coordination deficit and postural abnormalities  that indicate that most BTBR mice moved differently than their B6 counterparts during beam walking. The reduced stride length in BTBR mice also caused a significant increase in latency on the balance beam. Apparently, reduced stride length on the beam allowed animals to maintain their center of gravity firmly within the fixed base of support. Also, longer crossing time on the beam arguably reflects a tendency to reinforce gait stability, issues that may emerge from difficulties in balance, low muscle tone (hypotonia), and even problems in proprioception. Comparable aspects of balance and coordination issues along with fragmented skilled walking patterns have been previously reported in numerous studies with clinical populations49. For instance, children with ASD exhibited abnormal limb movements, difficulties with balance, shortened steps, and increased stance times50. Interestingly, impairments in motor skills in ASD (e.g. deficient skilled motor gestures) result in difficulties of visual feedback integration to adjust and guide spontaneous skilled movements45.Although ASD patients often suffer from difficulties with posture, balance, coordination, and motor planning53. At cortical levels, however, clinical studies have shown signs of cortical thinning and neuronal loss, with no changes in overall brain volume in ASD patients56. In agreement with these findings, we report here that both dorsal and lateral cortices were thinner in BTBR mice as compared to B6 mice, while the ventral cortex remained unaffected. Also, volumetric measurements revealed no differences between BTBR and B6 groups in whole brain volume. The present correlation analysis indicated that cortical thickness in the dorsal cortex may significantly predict stride length on the balance beam. It is noteworthy that dorsal and lateral cortical areas defined for the thickness measurement in the present study mostly covered motor cortex, both somatosensory (S1) and motor cortices including primary (M1) and secondary (M2) motor subregions.Neural substrates of motor impairments in ASD include abnormalities in cerebellum and basal ganglia58. Close dialog between the M1 and S1 is essential not only for skilled motor function61, this intracortical cooperation is also supported by a wide range of afferent inputs emerging from extracortical and subcortical processing systems such as cerebellum62, basal ganglia63, and brainstem64. Although the cause of the reduced cortical thickness in the BTBR strain is unclear, such extensive morphological abnormality may be expected to severely disrupt sensorimotor integration, motor planning, and fine motor execution. Therefore, as a morphological hallmark of an undeveloped brain, cortical thinning in the BTBR strain65 reveals some key aspects of the neuropathology that may need further investigation in preclinical models of autism, particularly in relation to the non-diagnostic symptoms.The motor cortex, in general, plays an essential role in skilled movement, postural control, and balance67. Also, many behavioral symptoms such as inattention, hyperactivity, and impulsiveness seen in children with attention deficit hyperactivity disorder are also shared with ASD68. In mouse models of autism, emotion-relevant activities (e.g. anxiety-like and/or avoidance behaviors) are typically assessed based on the time spent in open vs. closed arms in the elevated plus maze. Accordingly, BTBR mice make more entries into the open arms, which has been repeatedly interpreted as lower level of anxiety compared to other strains70. However, such interpretations can be confounded by potential artifacts due to the enhanced spontaneous behaviors such as nonspecific hyperactivity and impulsiveness. The OFT in the present study was employed to rule out these confounding effects.Individuals with ASD are often diagnosed with poor emotionality71 possibly due to differences in test procedures (e.g. size of open field) and the age of animals. Also, the BTBR strain showed no reduction in exploration speed even in the last 5-min time bin interval, whereas the B6 mice exhibited significantly lower speed scores in the same period as compared with the first time bin interval. This indicates that BTBR mice failed to habituate to the arena. Hyperactivity, even in the central zone of the OFT, also can show impaired response inhibition (RI)72 in the BTBR strain, as previously seen in clinical autism73 . Therefore, the failure to habituate to the physical characteristics of the OFT along with inadequate inhibition of movement and arousal portrays a cardinal feature of ASD by which high levels of arousal lead to labile attention in the BTBR animals.During free exploration in the OFT, BTBR mice moved substantially faster than B6 controls. This finding that stands in contrast with previous reports73, the impaired RI in the OFT and intact RI on the BBT for BTBR mice support the notion that inhibitory processes seen in clinical ASD cases may be task-dependent. A second implication of these findings is that non-diagnostic symptoms , which belong to domains outside the major diagnostic triad of ASD may be impacted in similar patterns than traditional diagnostic signs.A puzzling feature of the present findings concerns the differences in overground locomotion by which BTBR mice moved faster than their B6 counterparts in the OFT while they were slow and relatively accurate on the BBT. As proposed for individuals with ASD75 that may produce excessive excitability, or to an impaired coherence across the cortico-striatal circuits in several interconnected brain regions76. This connectivity repertoire, therefore, appears to play an elemental role in both bottom-up and top-down modulation of executive control, including that involved in modulation of visuo-motor control and RI in a particular task.The reduced cortical volume may contribute to the behavioral abnormalities observed in this study. Although measures of cortical volume alone may not be sufficient to decisively explain the existing impaired RI and hyperactivity in the BTBR strain, cortical volume reduction in the present study could robustly predict higher speed of the BTBR mice in the OFT compared to B6 animals. The reduced cortical volume and thickness found in BTBR mice also may be indicative of decreased functional capacity of cortico-cortico  or cortico-subcortical  connections. Though it was not the focus of this experiment, hyperactivity along with repetitive behaviors in ASD can likely be attributed to disordered cortical connectivity77. Previously, the MWT was used to examine some aspects of perseverative behaviors or insistence on sameness in the BTBR strain2. However, the BTBR strain’s cognitive phenotype, especially spatial learning and memory is not well characterized. In the present study, we used the MWT to measure spatial performance in BTBR mice with further focus on the analysis of search strategies. We observed pronounced differences between BTBR and B6 strains in either traditional measures of spatial performance or search strategies used by animals to locate the hidden platform in the MWT. Our observations showed that goal-directed spatial behavior, which is critically HPC-dependent in humans and rodents80 was extremely impaired in the BTBR mice. All traditional indicators of spatial behaviors in the task support this conclusion. Furthermore, the BTBR strain did swim faster in both visible and hidden-platform versions of the task as compared to B6 animals. The fast searching behavior, however, could assist in decreasing the latency only in the visible-platform protocol as well as initial stages of spatial testing. More importantly, a combination of these conventional readouts and search strategies for the first time in the BTBR strain provided further explanations on many other aspects of impaired spatial function of these mice that would otherwise have not been easy to interpret. This approach was chosen to differentiate between HPC-dependent allocentric and HPC-independent egocentric search strategies.It is difficult to describe a uniform picture of spatial prowess and navigational processing in ASD on the basis of the extant literature. However, there is a growing number of claims that spatial cognition , in parallel with social behaviors and locomotion, is impacted by ASD31 because it was shown to be dependent upon the HPC structural and functional integrity30. The present study determined that BTBR mice used less effective strategies compared to B6 mice, a cognitive inflexibility that can account for the correlation between dHPC volume and the frequency of spatial search pattern. BTBR animals also displayed minimal evidence of an actual spatial reference memory in the probe trial. Furthermore, when animals were required to locate the hidden platform in a new quadrant , search strategy use in the BTBR mice did not significantly improve toward spatial patterns with 5 days of intensive training. Interestingly, there was less evidence than expected for repetitive or stereotyped behaviors in BTBR mice that might have been shown by unusual T, and/or circling and C strategies during navigation in the MWT. Overall, while B6 mice reduced the use of non-spatial strategies across spatial testing, and their navigation to the hidden platform remarkably relied upon spatial strategies (~87%) on the last day of the 5-day reversal learning period, the BTBR strain failed to exhibit the same profile of improvement (~36%).The analysis of search strategies has previously been successful in elucidating the dynamics of spatial performance in transgenic mice81. In the domain of structural defects in clinical autism, smaller HPC volume has been reported in several studies83. Nevertheless, the HPC abnormalities, either structural or functional are somewhat heterogeneous. Some studies suggest that there are differences in HPC volume and shape84, as well as HPC connectivity85 between patients with ASD and controls, whereas other studies have not found the same differences86.The neuroanatomical basis for differences in search strategy is not well known. Basically, spatial processing and behaviors are determined by the integration of information and coordination of multiple neural systems such as PFC and HPC. Moreover, spatial performance is proportional to the volume of HPC spared87 required for noncortical synchrony and integrated HPC-dependent behaviors in a given task. Regarding the callosal and commissural abnormalities, reduced dHPC volume, and the poor use of spatial search strategies in BTBR mice, it would not be unreasonable to assume that a global disorganization in HPC anatomy, arguably together with decreased cortical cell density, volume, and thickness may be responsible for disrupted search strategy use.Anatomical abnormalities related to spatial challenges may not be only limited to HPC dysfunction. A nearly complete absence of corpus callosum and hippocampal commissure in the present BTBR cohort may deprive the central processing system from an inter-hemispheric synchronized dialogIt is widely acknowledged that ASD uniquely appears in humans. Nevertheless, numerous research attempts have been made to mimic the behavioral, typically diagnostic symptoms of ASD in animal models such as the BTBR inbred mouse strain. Here we sought for non-diagnostic symptoms of autism within three motor and cognitive domains in the BTBR mice, besides the traditional behavioral phenotyping. We did incorporate face validity and construct validity in our detailed analysis, and concluded that non-diagnostic autistic-like symptoms in the BTBR mouse strain can be similarly impacted by autism risk factors as the traditional diagnostic signs. We also conclude that sensorimotor disintegration, locomotor abnormalities, and impaired spatial behavior along with the corresponding neuropathology represent effective measures of severity of impairments in one or more non-diagnostic domains. These in fact may confound final conclusions about core diagnostic symptoms linked to ASD. The underlying neuropathological mechanisms for these specific, lesser-known behaviors, and how they influence diagnostic reliability in ASD still await additional investigation beyond face validity.The sponsor had no role in the planning or conducts of the study or in the interpretation of the results.Sup LegendsSup Mov 1Sup Mov 2Sup Mov 3Sup Mov 4


Flavobacteriia occurred dominantly during both the bloom and post-bloom periods. However, bacterial community changes at the species level during the bloom and post-bloom periods occurred rapidly in a time scale of a few days. Especially, NS5, NS4 and Formosa bacteria belonging to Flavobacteriia and bacteria belonging to Halieaceae and Arenicellaceae families of Gammaproteobacteria showed a successive pattern with large short-term variation during the period. The changes in prokaryotic composition were found to be related to phytoplankton biomass and composition, as well as seawater temperature and variations in nutrients.To understand prokaryotic responses during a spring bloom in offshore shelf waters, prokaryotic parameters were measured daily at a station located in the middle of the East China Sea over a six-week period from March 25 to May 19. The site experienced a phytoplankton bloom in late April, triggering changes in prokaryotic abundance and production after a lag of approximately one week. Before the bloom, changes in prokaryotic composition were small. Both during the bloom and in the post-bloom period, successive changes among bacterial groups were apparent. A SAR11 group became more dominant during the bloom period, and diverse groups belonging to the  Daily observations at the ocean platform established in offshore waters of the East China Sea elucidated fine-scale changes in prokaryotic diversity and composition during the spring bloom. Algal blooms typically develop in the spring in temperate mid-latitude seas and are mainly triggered by a combination of higher light intensity/duration, nutrient levels, sea surface temperature and water column stratification  were conducted for nearly two months to understand prokaryotic population responses to offshore algal blooms with high resolution on a one-day time scale.This study was conducted on an oceanic and meteorological observation platform, near the middle of the East China Sea  incorporation rates (TTI) were measured using methods described in a previous study  . The filters soaked with 1 mL of STE buffer  were stored at −20°C in the field and then at −70°C in the laboratory. DNA extraction and purification were conducted as previously described  . After alignment, sequences that were likely due to PCR errors were removed using the ‘pre.cluster’ command, which permits up to four differences between sequences; then, chimeric sequences were also removed using the ‘chimera.uchime’ command. Sequence subsampling was performed to normalise the number of sequences per sample. The remaining sequences were assigned to operational taxonomic units (OTUs) using a 97% sequence identity threshold. Each OTU was classified using the naïve Bayesian classifier with a bootstrap cut-off of 80% .Reads from the MiSeq sequencing were analysed using the program mothur  acetone, was measured using a Turner fluorometer  . Seawater temperature and salinity were measured using the Castaway CTD profiler .Chl Seawater temperature tended to increase over the study period Fig. . Thermala ranged from 0.5 to 5.8 μg L−1, showing a 10 × variation over the study period  on 17 April. Similarly, chl a spikes were observed on May 2 and 4. An intense algal bloom began in late April and a relatively high chl a concentration of more than 2 μg L−1 was maintained for approximately 10 days. Thereafter, chl a gradually decreased. During the study period, chl a in the nano-sized fraction (3−20 μm) was most dominant, but tended to decrease slightly  was relatively low in late-March but gradually increased to become the most dominant in the post-bloom period. Thus, both pico- and nano-sized phytoplankton were the most dominant algal groups in the spring bloom in the shelf waters of the East China Sea.Chl iod Fig. . In mid-tly Fig. . ConversProkaryotic abundance and TTI tended to be affected directly by the spring algal bloom Fig. . The abuMost of the prokaryotic groups showed a successional change in relative abundance among groups for approximately two months in the spring and the shift occurred rapidly over a time scale of a few days . Especially, at the middle stage of the bloom, bacteria affiliated with the SAR11 clade showed a maximum dominance of up to 80%  occupied from 13% to 52% of prokaryotic sequences during the study period . Further, temporal distribution of the genotypes was variable during the bloom period. Amylibacter species belonging to the NAC11-7 clade increased after the algal bloom, while a genotype closely affiliated with Ascidiaceihabitans species (AF235129) increased in relative proportion during the bloom . By contrast, genotypes closely related to Sulfitobacter showed their maxima during the early bloom period. OTUs related to Puniceispirillum and OM38 did not show significant changes during the study .Gammaproteobacteria was the second dominant prokaryotes and their composition varied greatly during the study period . At the beginning of the bloom, as chl a increased to over 3 μg L−1, two OTUs (2116-9934-15508 and 1105-13523-5566) affiliated with Luminiphilus and Arenicellaceae species in the class Gammaproteobacteria became the dominant species for three days and then decreased  appeared again at up to 8% . A variety of Flavobacteriia groups appeared around the spring bloom period  and then tended to decrease during the bloom and post-bloom periods. However, in the initial post-bloom phase with an apparent drop in chl a to less than 2 μg L−1, other bacteria in the NS4 clade and Formosa species became dominant up to over 10%.Relative dominance of bacteria belonging to the class Roseibacillus species belonging to Verrucomicrobiae appeared to occupy up to 8% of the total reads in the post-bloom period  and two archaeal groups, Thermoplasmata and Thaumarchaeota, gradually decreased over time and nearly disappeared during the bloom and post-bloom period , respectively, tended to decrease from the early spring until the bloom period Fig. . The ChaFinely resolved prokaryotic responses to a spring algal bloom in offshore shelf waters were elucidated by daily observations for approximately two months on the IORS. The general responses seemed similar to those observed in coastal areas in previous studies. However, daily shifts in diverse bacterial genotypes (species and subspecies level) during and after the bloom were revealed by analysing daily samples using a high-throughput sequencing approach.a concentration with a scale of tens μg L−1 during the bloom  compared with those in coastal areas. Furthermore, the dominant phytoplankton during the bloom period were not large, but nano- and pico-phytoplankton. This bloom situation in offshore waters elicited a different prokaryotic response than that observed in coastal spring algal blooms. Prokaryotic abundances and production changed dynamically, and prokaryotic composition also showed a successional shift in the offshore shelf waters in the spring. The changes in prokaryotic parameters were particularly dramatic before and after the bloom. The temporal shift of dominant taxa at a species level occurred within a few days.A typical spring bloom in coastal waters, which is developed by the growth of relatively large phytoplankton such as diatoms and dinoflagellates, shows very high chl et al. et al. The algal bloom simplified prokaryotic composition in terms of both species richness and diversity in offshore shelf waters Fig. . ProkaryAlphaproteobacteria, including those of Roseobacter clade-affiliated (RCA) lineage and several groups belonging to Gammaproteobacteria and Flavobacteriia, were revealed as bloom-associated prokaryotic taxa, which can rapidly utilise various organic matter produced by phytoplankton blooms . Thus, the increase in the Ia subgroup in this study is consistent with the previous study, suggesting that phytoplankton composition is of primary importance in determining the bacterial composition during spring blooms. Therefore, a close interaction between phytoplankton and prokaryotic groups may explain the diverse responses observed during algal blooms (Tan et al. et al. et al. a were found on April 17, May 2 and May 4, probably due to advection of bloom water from another location rather than in situ growth. On April 17, the increase in chl a was due to an increase in Skeletonema diatom. However, the prokaryotic compositions on these days were similar to those on adjacent days (Fig. However, the day-to-day change in prokaryotic composition at the species level differentiated our study from the previous results in some respects. The Ia subgroup of SAR11 was found to be the most dominant bacteria during the bloom phase in this study. Given that the increase in this bacterial group occurred concurrently with the initial increase in prokaryotic abundance and production Fig. , this suays Fig. . Thus, set al. Ulvibacter, Formosa-related and Polaribacter species in the Flavobacteriia, and SAR92 and Reinekea species in the Gammaproteobacteria, increased successively to become dominant taxa at the late bloom and post-bloom stages. In another time-series study conducted in an offshore station, Formosa-related lineage, Polaribacter, and Verrucomicrobium and SAR92 taxa became most abundant in particle-attached or free-living fractions (Needham and Fuhrman Formosa-related species in the post-bloom period. However, we did not observe significant levels of Ulvibacter, Polaribacter, Reinekea or SAR92 species during that time period.In a previous time-series monitoring of the prokaryotic community during an algal bloom conducted in coastal waters (Teeling Thermoplasmata of the Euryarchaeota and ammonium-oxidising Thaumarchaeota were found at significant levels in early spring, composing up to 3% and 1% of total prokaryote reads, respectively (Fig. a increased, and became nearly insignificant during and after the spring bloom, likely due to competition by bacteria and phytoplankton for organic matter and inorganic nutrients, such as ammonia, under the bloom conditions. Similar results were also obtained in an offshore area of Southern California, where MGII euryarchaeal taxa composed up to 30% of the prokaryotic community before a bloom, but sharply dropped as the bloom began (Needham and Fuhrman The response of archaea to spring algal blooms has not yet been well-studied. Interestingly, in this study, taxa affiliated with et al. et al. et al. et al. et al. et al. et al. These shifts in prokaryotic composition during the bloom were likely affected by diverse factors, such as the quality and quantity of organic matter from primary production and subsequent prokaryotic decomposition, viral lysis, grazing and the supply of particulate organic matter from the senescence and death of phytoplankton (Smith In conclusion, daily observations at the ocean platform established in offshore waters of the East China Sea elucidated fine-scale changes in prokaryotic diversity and composition during the spring bloom. General trends in compositional shift, particularly at the class level, were similar to those observed in coastal areas. However, the responses of bloom-associated taxa at the species level rapidly shifted over a time scale of only a few days, likely due to dynamic changes in substrate conditions.Supplementary DataClick here for additional data file.


Trypanosoma cruzi infection causes Chagas disease, which affects 7 million people worldwide. Two drugs are available to treat it: benznidazole and nifurtimox. Although both are efficacious against the acute stage of the disease, this is usually asymptomatic and goes undiagnosed and untreated. Diagnosis is achieved at the chronic stage, when life-threatening heart and/or gut tissue disruptions occur in ~30% of those chronically infected. By then, the drugs' efficacy is reduced, but not their associated high toxicity. Given current deficiencies in diagnosis and treatment, a vaccine to prevent infection and/or the development of symptoms would be a breakthrough in the management of the disease. Current vaccine candidates are mostly based on the delivery of single antigens or a few different antigens. Nevertheless, due to the high biological complexity of the parasite, targeting as many antigens as possible would be desirable. In this regard, an epitope-based vaccine design could be a well-suited approach. With this aim, we have gone through publicly available databases to identify T. cruzi epitopes from several antigens. By means of a computer-aided strategy, we have prioritized a set of epitopes based on sequence conservation criteria, projected population coverage of Latin American population, and biological features of their antigens of origin. Fruit of this analysis, we provide a selection of CD8+ T cell, CD4+ T cell, and B cell epitopes that have <70% identity to human or human microbiome protein sequences and represent the basis toward the development of an epitope-based vaccine against T. cruzi. Trypanosoma cruzi . The parasite is transmitted by triatomine insect vectors  endemic to the Americas, and through vector-independent routes such as blood transfusion or mother-to-child  and nifurtimox (NFX). Although highly efficacious against the short (8–12 weeks long) acute stage of the disease, this stage is mostly asymptomatic and therefore it generally goes undiagnosed and untreated. It is at the chronic stage, which can last from years to decades, when the onset of symptoms occurs. It is usually then when the infection is diagnosed and treatment is provided, but unfortunately, the drugs' efficacy at this stage is reduced , 4. WithT. cruzi-infected humans: amastigotes replicate intracellularly in multiple host cell types and transform into circulating motile trypomastigotes that spread the infection upon bursting the host cells. The existence of these two forms in the intracellular and extracellular domains requires a broad, complete, active, and robust parasite-specific immune response to control the infection. Despite the immune system's intervention, some parasites manage to escape and, at a simple conceptual level, this is how the parasite persists  in populations of Latin America, and lack of identity to human genome and human microbiome proteins. Similarly to other studies  (T. cruzi (“antigen ID 5693”) epitopes from positive assays, which had been elicited in the course of Chagas disease (“infectious disease”) in humans. Major histocompatibility complex (MHC) Class I (CD8+ T cell epitopes), MHC Class II (CD4+ T cell epitopes), and B cell epitopes were collected separately. Information on their antigens of origin was obtained too. We only considered CD8+ T cell epitopes with nine residues as the majority of known epitopes processed by MHC I are 9-mers  . We retre 9-mers . Any nonT. cruzi protein sequences in their Open Reading Frame format (ORF_AA) from the eight parasite strains in TriTrypDB resource release 35 (7 ORF_AA FASTA entries) was generated. We excluded the bat isolated Marinkellei strain due to its phylogenetic distance to the others    to obtaiH) parameter . As a result, we obtained two masked FASTA files (one for each entropy threshold) that represented the conserved proteome of T. cruzi at those two levels of entropy.where no acids . H = 0 iposition . Gaps wede novo prediction of CD8+ T cell epitopes (peptides), we used IEDB MHC I binding prediction algorithms (http://tools.iedb.org/mhci) on the H > 0.5 T. cruzi-masked proteome. Namely, these were ANN, NetMHCpan, NetMHCstabpan  (http://tools.iedb.org/immunogenicity/) (For etworks) –39, Combetworks) , PickPocetworks) , SMM (stetworks) , and SMMetworks) . All metnicity/) .+ T cell epitopes (peptides), we used the IEDB recommended method at MHC II binding predictions tool (http://tools.iedb.org/mhcii/) (+ T cell epitopes were predicted on the T. cruzi-masked proteome file (H > 0.5). We considered for further analysis only the 0.01% top scoring epitopes that were predicted to bind more than two human leukocyte antigen (HLA) alleles.For the prediction of CD4/mhcii/) , 46. CD4We followed two procedures for the prediction of B cell linear epitopes:  a structTrypanosoma brucei brucei (PDB accession 5Y70)  . Taking  MODELER . Then, w MODELER . ResidueT. cruzi Esmeraldo-like proteins predicted to be surface exposed, either because they had a secretion signal peptide, or/and at least one trans-membrane domain, or/and a glycosylphosphatidylinositol (GPI) anchor signal. We manually curated the list taking into account annotation details to retain those proteins that were highly likely exposed, and we tagged them as “-exposed.” We fused them to the file containing all T. cruzi ORF_AA sequences to generate a file that included the “-exposed” tagged sequences as well. Again, we clustered all these sequences using CD-HIT with a shared identity threshold >90%, but this time, we kept for further analysis only those clusters with at least eight different strains that contained a “-exposed” tagged sequence. We then followed the steps outlined in sections Generation of Clusters and Multiple Sequence Alignments of T. cruzi Protein Sequences and Generation of T. cruzi Invariable Proteome and Identification of Conserved IEDB Epitopes to generate a masked (H > 0.5) T. cruzi-invariant “exposed” file. We identified the invariant regions of at least 15 residues long in it and created a FASTA file with the sequences of their corresponding antigens of origin. BepiPred2.0 was run on this file with a threshold set at >0.6 . PPCs of CD8+ T cell and CD4+ T cell epitopes for populations in Latin America was queried by “area—country—ethnicity,” selecting Mexico and South America (http://tools.iedb.org/population/). Throughout the paper, PPC is expressed as an average of the PPC computed for those two regions. We did not include Central America region in the calculation because it provided scarce information from very specific populations.We calculated the epitopes PPC with IEDB Population Coverage tool  to proteins in humans, we blasted them against human microbiome protein sequences obtained from the NIH Human Microbiome Project  and agaiWe used SignalP, TargetP, and TMHMM  to prediProteasome processing of the proposed ensemble was predicted with NetChop using C-term 3.0 method and a threshold set at 0.7 . We used+ T cell and 11 CD4+ T cell epitopes) and 2000 unique B cell epitopes. They are respectively listed in T. cruzi invariant proteome file with a threshold H > 1. Only the epitopes that perfectly matched invariant regions were considered hits. This way, we found three conserved CD8+ T cell epitopes  proteome file to the IEDB MHC II Binding Predictor  that further regulates its stability and translational efficiency. This polyA tail can be either included in the template plasmid before transcription or enzymatically added using recombinant poly(A)-polymerase after transcription.Such epitopes lists could be delivered as independent or concatenated peptides , or as gterature . Particu+ T cell response against the parasite, we would place these epitopes first in the string as those sequences located closer to the 5′-end of the construct would be expressed at higher levels, thus inducing better immunogenic responses , and predominantly a buried (B) Acc was calculated for CD8+ T cell nonamers (63.0%)  .T. cruzi strains; (iii) such protection will need to be population broad and it should be achieved with a minimum of immunizations; (iv) it will have to be developed and produced very cost-effectively; and ideally (v) it should be effective prophylactically and therapeutically.Similarly to what has been suggested for leishmaniasis vaccines , a vacciin silico methodologies to the identification of epitopes of T. cruzi toward the design of an epitope-based vaccine for Chagas disease  motif recognition  residues. Then, at least one of those key positions will always be different in any of the CD8+ T cell epitopes progressed by us to any peptide sequence of that length present in humans. Nonetheless, as it is mentioned above, cross-recognition may still occur with low similarity, so any potential cross-reactivity will have to be carefully addressed. We expect that this could be better anticipated by computational tools in the near future as structural comprehension of the epitope recognition process advances. The rationale of applying this criterion of selecting non-identical sequences to proteins in the host susceptible to vaccination has another advantage, as it lines up with the hypothesis that any peptide sequence that is non-conserved in the host will likely be more immunogenic, which is certainly a desirable attribute of any vaccine. Indeed, a lower ability to induce an immune response has been described for peptides from commensal bacteria of the human microbiome  protection. Thus, thinking about the preferred delivery system, an RNA-based strategy might be the most suitable. It would ensure direct cytoplasm processing of the epitopes supporting higher expression efficiencies and stronger immune responses in comparison to protein subunit immunizations or pDNA usage . In factT. cruzi epitopes (from IEDB) with the invariant parasite proteome (“IEDB × INVARIANT PROTEOME”) barely yielded a single B cell epitope  in the mammalian host. The T cell component consists of 18 CD8+ T cell epitopes from 17 distinct antigens and 2 CD4+ T cell epitopes from another 2 antigens that could elicit a cellular response in virtually the whole target population of such a vaccine. On the other hand, the B cell component of the vaccine encompasses a total of 10 epitopes from six different surface-exposed antigens that could elicit antibodies against the free swimming trypomastigote stage. Notably, all the epitopes in the ensemble were prioritized in agreement with their lack of identity to any protein sequences present in humans in order to preclude cross-reactivity reactions thus favoring the safety of the vaccine candidate.Chagas disease, a neglected infectious disease caused by the parasite T. cruzi-specific unique epitopes at IEDB (B cell epitope GTDEGLLLPVDNDGDESS) could be rescued out of the conserved T. cruzi proteome generated. Therefore, we had to perform de novo predictions of epitopes departing from that conserved proteome. Aware of the limitations of this procedure, we were very stringent and only selected top qualifying predicted epitopes. Nonetheless, whether they are processed and presented in the course of an infection by T. cruzi remains to be demonstrated. Even if immunogenic, we would still not know to what extent the here presented vaccine ensemble could provide protection from T. cruzi infection. This is something that we will have to evaluate in forthcoming experiments upon validation of the epitopes.We initially attempted to identify conserved epitopes that had been experimentally validated and deposited in the IEDB database, but it happened that only one of the + T cell epitope component in the suggested construct. Nonetheless, this will require further proof as well as it will be necessary to evaluate which is the optimum order and spacing to achieve maximum immunogenicity with the CD4+ T cell and B cell epitopes. In relation to the latter, we do not provide any glycan-based epitopes as they cannot be extracted with the protein-based reverse vaccinology procedure followed. We neither include any conformational epitopes since these cannot be isolated from their context and we only focused on linear epitopes. It needs to be tested whether these epitopes elicit antibodies that are capable of recognizing the native protein conformations. Omics- data repositories such as EuPathDB and IEDB . The scripts developed are available from the corresponding authors upon request.LM-T, PR, and JA-P conceived the study. LM-T and PR wrote the scripts. LM-T and JA-P analyzed the data and wrote the article. PB provided The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We applied this method to a set of four different shrimp allergens, and successfully identified several non-cross-reactive as well as cross-reactive epitopes, which have been experimentally established to cross-react. Based on these findings we suggest that this method can be used for advanced component-resolved-diagnosis to identify patients sensitized to a specific shellfish group and distinguish from patients with extensive cross-reactivity to ingested and inhaled allergens from invertebrate sources.Understanding and predicting an individual's clinical cross-reactivity to related allergens is a key to better management, treatment and progression of novel therapeutics for food allergy. In food allergy, clinical cross-reactivity is observed in patients reacting to unexpected allergen sources containing the same allergenic protein or antibody binding patches (epitopes), often resulting in severe allergic reactions. Shellfish allergy affects up to 2% of the world population and persists for life in most patients. The diagnosis of shellfish allergy is however often challenging due to reported clinical cross-reactivity to other invertebrates including mites and cockroaches. Prediction of cross-reactivity can be achieved utilizing an in-depth analysis of a few selected IgE-antibody binding epitopes. We combined available experimentally proven IgE-binding epitopes with informatics-based cross-reactivity prediction modeling to assist in the identification of clinical cross-reactive biomarkers on shellfish allergens. This knowledge can be translated into prevention and treatment of allergic diseases. To overcome the problem of predicting IgE cross-reactivity of shellfish allergens we developed an epitope conservation model using IgE binding epitopes available in the Immune Epitope Database and Analysis Resource ( The prevalence of food allergy is steadily increasing over the past decade, with ~4% of adults and up to 10% of children having some type of food allergy. The shellfish group is included among the “Big Eight” food groups that are responsible for more than 90% of all food allergy cases . ShellfiIn the sensitized individual, subsequent exposure to the shellfish allergen, via ingestion or inhalation, triggers the generation of specific IgE antibodies by activated B-cells of the immune system. These antibodies subsequently bind to immune effector cells, including mast cells and basophils, resulting in degranulation and clinical manifestation of allergic symptoms . Currentwww.allergen.org). Meanwhile, AK has been considered as the second invertebrate pan-allergen, implicating allergy cross-reactivity to shellfish, mite and insect. IgE-sensitization toward AK has been found in 10–51% of shrimp allergic patients  and the enzyme arginine kinase (AK). Tropomyosin is the major allergen of shellfish allergy with specific IgE antibody responses in 60–80% of shellfish allergic patients recognizing this protein, and it is suggested to be a good biomarker of severe clinical-reactivity to shellfish . TM has patients .The primary structure of TM's alpha-helical coiled-coil dimeric protein is highly conserved across various invertebrates and has been identified in over 150 species, including insects and mites . This stEdible crustacean and mollusc are commonly discussed as “shellfish.” However, the group of “shellfish” comprises the two invertebrate phyla arthropods and molluscs. Although all shellfish are invertebrate animals, these two groups are very distinct in evolutionary terms and subsequently contain different molecular repertoires of food allergens. In fact, crustacean are placed closer to insects and arachnids (spiders), and this seems to be the major factor for molecular sensitization and clinical reactivity between crustacean, dust-mite, insects and parasites . Severe Cross-reactivity occurs when the IgE antibodies recognize identical or very similar protein patches (epitopes) from different proteins as compared to the primary sensitizing protein . IgE croCross-reactivity of related proteins could be predicted computationally by comparing the identity of the amino acid sequence to the known allergen. Aalberse  reviewedBased on these observations, we consider that a large-scale analysis of sequential IgE epitope conservation is of great importance for predicting clinical cross-reactivity between crustacean and mollusc, as well as mite and cockroach, in allergic patients. While there are eight allergenic proteins known among different shellfish , conclusThe dataset for the subsequent analysis was built from available IgE binding epitopes from four shrimp allergens: tropomyosin (TM), arginine kinase (AK), myosin light chain (MLC), and sarcoplasmic calcium-binding protein (SCBP). The data were assembled from the Immune Epitope Database and Analysis Resource (IEDB) . The colProtein sequences were obtained from the UniProt database and aligned using MUSCLE v3.8.31 . DiffereThe conservation of amino acid residues for each allergen among the different invertebrate species was estimated using the Rate4Site algorithm in Consurf  server bThe degree of conservation of the epitopes within the sequences of the respective allergens was calculated using conservancy analysis tool  on the IData analysis was performed using GraphPad Prism version 7. One-way ANOVA was applied to determine the statistically significant difference of the conserved epitopes between groups of invertebrate species.Phylogenetic analysis was performed to determine the relationships between shrimp allergen sequences in different invertebrate groups and to infer the evolutionary trends among the wide representation of the allergens. In this study, four shrimp allergens, TM, AK, SCP, and MLC were selected based on the availability of the IgE-binding epitopes. A dataset of TM, AK, SCP, and MLC protein sequences from crustaceans and molluscs species in UniProt database was assembled to construct a tree using the Maximum Likelihood and Bayesian approach. A consensus tree generated for all protein groups showed similar topologies with good branch support (>70%) for major branches for TM and AK . These tThe degree of evolutionary conservation at individual amino acid sites of TM and AK were determined using Consurf server by applying the Rate4Site algorithm. In ConSurf, the evolutionary rate is estimated based on the evolutionary relatedness between the protein and its homologs and considers the similarity between amino acids as reflected in the substitutions matrix. The conservation grades identified using ConSurf are mapped to the query sequence and/or structure using the ConSurf color-code, with cyan-through-purple corresponding to a variable (grade 1)-through-conserved (grade 9) positions . As the To determine the likelihood of cross-reactivity between shrimp and other invertebrates, the conservation analysis of epitopes in those groups was conducted. A database of linear 176 epitopes was generated, containing 96 epitopes from TM, 39 epitopes from AK, 27 epitopes from MLC, and 12 epitopes from SCBP. These epitopes were identified to be recognized by IgE antibodies from over 100 patients with shellfish allergy as determined in previous studies , 42–45. Approximately, 91% of the IgE epitopes were found to be conserved in crustacean, 56% in cockroach and 48% in mite species. The number of conserved epitopes in crustacean, cockroach, and mites epitopes were significantly higher than in the three mollusc groups where only <20% of TM epitopes were conserved  and evenSpecies-specific conservation analysis of IgE binding epitopes was carried out to identify the allergen epitope sequences that could be termed “pan-epitope.” Of the 97 shrimp TM IgE-binding epitopes, 22 invertebrate pan-epitope were identified . These eIn contrast, only 5 of the 39 shrimp AK IgE-binding epitopes were conserved across crustacean, cockroach, mite, and mollusc  and unliAvoidance of other shellfish species including molluscs is one of the management strategies for shrimp-allergic patients. However, Of the 28 conserved TM epitopes, only 22 epitopes were present in over 50% of each mollusc classes, with three epitopes were conserved in all of the mollusc species . DetaileCombining the conserved epitopes divided the shrimp TM into three possible cross-reactivity scenarios, located on distinct areas on the TM allergen. Three regions with a total of 35 amino acids residues, or 12%, of the total 284 amino acids were conserved across all classes of the mollusc phyla , while recognition of the other allergens were not different in both patient cohorts , and therefore contained more homologous peptides of shrimp TM and AK IgE-binding epitopes. From the study of Vidal et al. , of the Gastropod and bivalve TMs share only 60% sequence identities with crustacean TMs, however, unlike cephalopod where the identity of TM amino acid sequence is very high among the group, the identity of TM amino acid sequence in the species among those two classes is very variable, particularly among bivalve species. The variability of bivalve TM is very apparent where out of 23 conserved IgE-binding epitopes, only five epitopes were shared across all species of bivalve providing a molecular basis of selective cross-reactivity . It is tBased on the abovementioned findings on the different pattern of IgE-binding epitope conservations in the three mollusc classes, we developed a decision tree to predict immunological cross-reactivity between shrimp and mollusc classes based on TM and AK. This decision tree could contribute significantly toward patient management, particularly on the aspect of food avoidance and diet. It has been well-known that shrimp allergic patients are advised to avoid all shellfish species including mollusc due to the risk of cross-reactivity. Our decision tree suggests that this advice should not be generalized as only patients sensitized to TM which account for 70–80% of total shrimp-allergic patients have a risk of cross-reacting with mollusc allergens. Moreover, the cohort of shrimp-allergic patients could be further divided into five groups based on their cross-reactivity patterns to specific IgE binding epitopes.Prediction of immunological cross-reactivity between an allergen and close related proteins based on similarity of the IgE-binding epitopes has been confirmed to be more accurate than the prediction based on similarity of the complete amino acid sequence of the allergenic proteins. Food allergens including shellfish allergens tend to have sequential IgE-binding epitopes due to digestion in the gastrointestinal tract. Thus, epitope sequence comparison is more relevant and conceivable for assessing the potential cross-reactivity of allergenic proteins, then comparing the whole protein sequence. The shrimp allergen IgE-binding epitope conservation results outlined in this study illustrate that a clear hierarchy of cross-reactivity is discovered, with TM being the most cross-reactive allergen among allergenic invertebrate species. This is most likely the main reasons that TM is one, if not the major pan-allergen in inhalant and ingestion animal allergy. The IgE binding epitopes located at the N- and C-terminal regions of TM are highly conserved and could be used as biomarkers to predict allergic cross-reactivity of shrimp-allergic patients. Unexpectedly, more than half of the TM as well as the AK IgE epitopes were found to be conserved in cockroach and mite TM and AK, respectively. In contrast, only few shrimp IgE-binding epitopes were conserved across the molluscs. This suggests a low risk of cross-reactivity of shrimp allergic patients to molluscs, while a high risk of cross-reactivity to cockroach or mite is predicted. We developed for the first time a decision tree to predict cross-reactivity between shrimp and molluscs based on the major allergens TM and AK. These fundamental findings could simplify the diagnosis of cross-reactivity among shellfish-allergic patients, thereby avoiding potential life-threatening food challenges.https://www.iedb.org/ - list of epitopes can be found in the Publicly available datasets were analyzed in this study. This data can be found here: RN, SDK, and AL conceived the study and wrote the manuscript. RN, EJ, TR, and SK performed and analyzed the experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.