The clinical spectrum of leptospirosis ranges from subclinical infection and anicteric fever to Weil’s disease, a severely fatal infection. Acute inflammation of the pancreas is a rare manifestation of Weil’s disease, and renal involvement is a hallmark in severe forms, leading to acute kidney injury (AKI) and representing a major risk factor for death. The aim of the case report was to present the clinical manifestations of Weil’s disease with acute pancreatitis and AKI and to highlight the management of its complications.A 22-year-old male patient presented to the hospital with a chief complaint of a persisting fever, abdominal pain, nausea and vomiting, decreased appetite, malaise, and urine and feces discoloration. The patient’s residence had flooded 2 weeks ago. Laboratory tests were performed and the patient was diagnosed with Weil’s disease with the complication of acute pancreatitis, AKI, hyperkalemia, hyponatremia, hypotonic hypovolemic, metabolic acidosis, and hypoalbuminemia.The patient was treated with intravenous (i.v.) ceftriaxone at a dose of 2×1 g, i.v. metoclopramide at a dose of 3×10 mg, i.v. calcium gluconate at a dose of 1 g followed by dextrose (D) 40% with insulin 2 IU for six times, avoided nephrotoxic drugs, and fluid balance was maintained at I=O+500 ml. The patient received hemodialysis due to refractory hyperkalemia. Posttreatment follow-up presented improvements in complaints and laboratory parameters.Management of severe leptospirosis or Weil’s disease with the complication of acute pancreatitis and AKI requires antibiotics and supportive therapy including adequate fluid resuscitation, proper and adequate nutrition, as well as the initiation of hemodialysis. HighlightsA rare case of Weil’s disease with acute pancreatitis and acute kidney injury is reported.The patient also had hyperkalemia, hyponatremia, hypovolemic, metabolic acidosis, and hypoalbuminemia.Appropriate antibiotics and supportive therapies avoided disease progression and improved the patient’s condition.Leptospira and most common in tropical and subtropical areas and is endemic to several places in IndonesiaLeptospira serovars are naturally carried in wild and domestic animals, especially in the renal tubules of rodents, and the transmission occurs either directly through contact with infected animal urine or indirectly through contaminated water and soilLeptospirosis is a zoonotic disease caused by bacterial pathogens of the genus World Journal of GastroenterologyLeptospira antibiotics and supportive therapy to treat acute pancreatitis. Effective treatment is strongly advised since it lowers mortalityThe spectrum of leptospirosis is wide, ranging from subclinical infection, anicteric fever, to Weil’s disease, a severely fatal infection. Leptospirosis affects multiple organs, such as the central nervous system, eyes, heart, lungs, liver, and kidneys, including the pancreasHerein, we report a case report of an adult man with Weil’s disease causing multiorgan complications and highlight the management of acute pancreatitis and AKI in Weil’s disease. This case report is reported in line with the CAse REport (CARE) guidelines criteriaA 22-year-old male patient presented to the hospital with a chief complaint of persistent fever, nausea and vomiting (three times a day with an approximate volume of 100 ml), and jaundice for a week. The fever was continuous, causing headaches, and not accompanied by chills, sweating, and rash. Upper right abdominal pain that radiates to the back persisted for a week and worsened during the supine position and when eating. The pain reduced when sitting hunched over and with knees bent. Pain in the calf was also experienced by the patient. The patient felt a decrease in appetite and malaise. The patient experienced discoloration in urine and feces. There was no history of cough or cold in the last month. A history of consciousness disorders was absent. The patient and family had never experienced this complaint before. The patient denied any history of hypertension, diabetes, or drug and food allergies. Smoking and alcohol consumption were both denied. Two weeks ago, the patient’s residence had flooded. Diet from food stalls in the last month was absent. No history of traveling out of town.The general health condition of the patient was weak, Glasgow Coma Scale score of E4V5M6, blood pressure of 130/80 mmHg, pulse rate of 100 beats per minute (bpm), respiratory rate of 20 bpm, the axillary temperature was 37.5°C, peripheral oxygen saturation was 98% room air. The patient’s body mass was 60 kg and height was 165 cm. The patient had conjunctival suffusion and icteric sclera. There were no signs of cyanosis, dyspnea, or enlargement of cervical lymph nodes. Lung and heart physical examinations were within normal limits. Abdominal examination showed a flexible abdomen (no distention), normal bowel sounds, liver and spleen were not palpable, and the pain was found in the left hypochondriac region and epigastrium. Cullen’s or Turner’s sign was absent. Extremities examination found tenderness in the right and left gastrocnemius muscle.3, neutrophils of 91.7%, lymphocytes of 3.5%, platelets of 252 000/mm3, sodium (Na) level of 131 mmol/l, potassium (K) level of 6.7 mmol/l, chloride (Cl) level of 79 mmol/l, random blood glucose of 144 mg/dl, blood urea nitrogen (BUN) of 243 mg/dl, creatinine (Cr) of 12.66 mg/dl, albumin of 2.89 g/dl, alanine transaminase (ALT) of 385 U/l, aspartate transferase (AST) of 254 U/l, direct bilirubin of 28.03 mg/dl, total bilirubin of 38.03 mg/dl, partial thromboplastin time (PPT) of 16.5 s, and activated partial thromboplastin clotting time (APTT) of 27.4 s (Table 2) of 27, partial pressure of oxygen (pO2) of 105, bicarbonate (HCO3) of 14.2, base excess (BE) of −11.7, arterial oxygen saturation (SaO2) of 98%, and PaO2/FiO2 (P/F) ratio of 500. A complete urine examination showed cloudy yellow color, pH of 5.0, specific gravity of 1.015, protein of 1+, bilirubin of 3+, normal urobilinogen, erythrocytes of 3+, leukocytes of 1+, negative for ketone and nitrite, urine albumin-to-creatinine ratio (ACR) of 150, and protein–creatinine ratio (PCR) of 0.3. The coronavirus disease 2019 antigen and polymerase chain reaction swab results were all negative. The echocardiography examination showed normal limits. Chest radiograph examination suggested a thickening of the left hilum, and the heart abnormalities were absent  of 12.2 g/dl, hematocrit (HCT) of 33.9%, mean corpuscular volume (MCV) of 80.3 fl, mean corpuscular hemoglobin (MCH) of 28.9 fl, leukocytes of 31.010/mm3 in 6 h, intravenous (i.v.) ceftriaxone at a dose of 1 g twice a day, i.v. metoclopramide at a dose of 10 mg three times a day, i.v. calcium gluconate at a dose of 1 g followed by dextrose 40% with insulin 2 IU for six times, avoided nephrotoxic drugs, and fluid balance I=O+500 ml. The consultation with the Nephrology Division suggested possible pseudohepatorenal syndrome, six times hyperkalemia correction, then a laboratory test was taken for post-correction random blood glucose and serum electrolytes, fluid maintenance with NaCl 0.9% : D10 = 2 : 2, insertion of a triple-lumen hemodialysis (HD) catheter/central venous catheter in the subclavian/jugular for parenteral nutrition, and central venous pressure was evaluated because of possible renal shutdown requiring hemodialysis. The patient then underwent hemodialysis due to refractory hyperkalemia with the following prescription: 2.5 h HD, 150 blood flow rate (QB), 500 dialysis fluid flow rate (QD), without heparin and ultrafiltration.The patient was conditioned to fast and treated with an infusion of sodium chloride (NaCl) 0.9% at a volume of 2000 cm3 per 24 h. Blood culture, urine culture, procalcitonin, laboratory tests post hemodialysis, immunoglobulin (Ig) M and IgG Leptospira serology tests were performed. Laboratory examination results are presented in Table 3 six times a day over a nasogastric tube , D10% infusion : NaCl 0.9%=2 :2, i.v. ceftriaxone at a dose of 1 gram twice a day, i.v. metoclopramide at a dose of 10 mg three times a day, i.v. calcium gluconate 1 ampoule given twice, paracetamol 500 mg three times a day over a nasogastric tube, calcium carbonate (CaCO3) dosed at 1 tablet three times, and lactulac syrup 15 ml three times a day.On the third day of hospitalization, abdominal pain was reduced, fever decreased, and the patient had no nausea and vomiting. Glasgow Coma Scale score was E4V5M6, blood pressure was 130/70 mmHg, pulse was 82 bpm, strong and regular pulse rhythm, respiratory rate of 20 bpm, axillary body temperature of 36.9°C, peripheral oxygen saturation of 98% at room air with urine production 3400 cm3 urine production for 24 h. The blood culture test and urine culture test showed no bacteria growth. Laboratory parameters are presented in Table 3 for an hour, followed by 1500 cm3 per 24 h over CVC. Sodium bicarbonate 75 mEq in NaCl 0.9% 500 cm3 (drip) over the first line of i.v., the second line was D10%+insulin of 10 IU, and other therapies remained the same.On the sixth day, complaints of weakness and fever were gone, and the patient had stable vital signs with 2600 cm3 per 24 h. Laboratory examinations are presented in Table Leptospira IgM serology was positive.Eight days after hospitalization, the patient’s condition was still weak, but there were improvements in appetite, and the patient was able to defecate. Vital signs were normal and urine production of 3000 cmLeptospira IgM serology was positive, while and Leptospira IgG yielded negative. Infusion therapy was NaCl 0.9% 1000 cm3 : D5% 1000 cm3 per 24 h, while other therapies remained the same. On day 13, the patient had no complaints and had normal vital signs. Laboratory examination results suggested a good progression of the disease Renal involvement is common in leptospirosis and its manifestations vary from mild proteinuria and changes in urine sediment to severe renal failureThis case report has strengths and limitations that need to be discussed. This is unique because Weil’s disease with acute pancreatitis and AKI is rare, and the patient was managed successfully with aggressive treatment. The diagnosis of Weil’s disease was made based on the 2012 WHO Faine modified score. Serologic tests, such as the microscopic agglutination test and enzyme-linked immunosorbent assay for leptospirosis, were not carried out on the patient due to the unavailability of the tests.In Weil’s disease, with complications of acute pancreatitis and AKI, aggressive and proper treatments with appropriate antibiotics are needed. Early hemodialysis would also benefit the patient if the indicators of AKI are present.The study is exempt from ethical approval in our institution.Written informed consent was obtained from the patient for the publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.This study has received no external funding.R.B.H.: clinical management, patient care, and manuscript preparation and editing. B.B., M.R., M.V.A., and U.H.: patient care and manuscript editing.The authors declare no conflicts of interest.None.Bramantono Bramantono.Not commissioned, externally peer-reviewed.


J. R. Soc. Interface20, 20220543. (Published online 4 January 2023) (https://doi.org/10.1098/rsif.2022.0543)The authors regret that they have found two errors in the article.P2 at generation t + 1 abruptly changes from 2 to 1 without a red star representing the mutation event, which does not accurately illustrate the model assumptions. Here, we present the correct version of the figure.1. In figure 2 of the original article, the cultural state of G can estimate…’.2. In §3.2, paragraph 5, the first sentence ‘Overall, although our heuristic algorithm can estimate…’ should read ‘Overall, although our algorithm with matrix The errors above do not affect the validity of the original article.This has been corrected on the publisher's website.


Ice templating is the mechanism by which cellular materials with hierarchical architectures are formed when aqueous solutions or slurries are directionally solidified or freeze cast. Characteristic for freeze-cast solids are lamellar cell walls with unilateral surface structures such as regularly spaced “ridges” and other complex subfeatures that do not reflect the underlying hexagonal symmetry of ice. How these unilateral structures form has remained a mystery. Combining experiments and computational modeling, we reveal that, by localizing diffusion-limited morphological instabilities to the atomically rough side of ice lamellae, anisotropic ice-crystal growth templates hierarchical architectures with complex subfeatures. Those results lay the theoretical foundation for understanding the formation of hierarchical architectures and structural subfeatures produced by directionally solidifying aqueous mixtures. Directional solidification of aqueous solutions and slurries in a temperature gradient is widely used to produce cellular materials through a phase separation of solutes or suspended particles between growing ice lamellae. While this process has analogies to the directional solidification of metallurgical alloys, it forms very different hierarchical structures. The resulting honeycomb-like porosity of freeze-cast materials consists of regularly spaced, lamellar cell walls which frequently exhibit unilateral surface features of morphological complexity reminiscent of living forms, all of which are unknown in metallurgical structures. While the strong anisotropy of ice-crystal growth has been hypothesized to play a role in shaping those structures, the mechanism by which they form has remained elusive. By directionally freezing binary water mixtures containing small solutes obeying Fickian diffusion, and phase-field modeling of those experiments, we reveal how those structures form. We show that the flat side of lamellae forms because of slow faceted ice-crystal growth along the c-axis, while weakly anisotropic fast growth in other directions, including the basal plane, is responsible for the unilateral features. Diffusion-controlled morphological primary instabilities on the solid-liquid interface form a cellular structure on the atomically rough side of the lamellae, which template regularly spaced “ridges” while secondary instabilities of this structure are responsible for the more complex features. Collating the results, we obtain a scaling law for the lamellar spacing,   Freeze casting is a directional solidification technique that exploits the phase separation of water-based solutions or slurries into pure ice-crystals and a solid solute-rich amorphous phase A. During14711Exactly how the complex ice-templated structures form is not fundamentally understood beyond the very early stages of morphological development –22. As tercooled ]. This percooled  in the mWhile the Mullins-Sekerka linear stability analysis has been extended to freeze casting –23, it h2630In contrast, the ice-liquid interface is faceted  perpendicular to the c-axis and atomically rough in the other directions contained in the basal plane , 35. As 21While anisotropic ice-crystal growth, which tends to select the fastest er scale B, with t context . Additio context –41.Herein, we present experimental and computational modeling results that focus on the fundamental question of the formation of unilateral substructures on the cell walls. We demonstrate experimentally that freeze casting of solutions with simple chemistries, in particular small solute molecules, which obey simple Fickian diffusion in the liquid, produces hierarchical architectures with elaborate substructures D similarThe presence or absence of the various cell wall features in freeze cast materials depends generally on solution and slurry composition and processing conditions –5. In thS=(1-f)λ D, where fraction . Furtherfraction  and 43, fraction ; the latSI Appendix, Fig. S1) and solute transport properties . Importantly, for both chemistries, SI Appendix, Fig. S2). As a result, in the chitosan-acetic-acid-water system, diffusive transport of the large submicron polymeric particles is too slow at dilute solute concentration  to keep up with the rate of ice-crystal growth. However, after an initial pile-up of the particles, transport becomes dramatically enhanced as the volume fraction approaches the close-packing limit due to particle-particle interactions. This enhancement is reflected in a strongly nonlinear relationship between effective diffusion constant and volume fraction  the diffusion equation in the liquid phase, where x axis in the moving frame takes the form x = 0 and t = 0.The functional dependencies of onal ice C, which symmetry , 48, 49.ocations . These pions  for the sucrose system, and diffusion coefficients, D, for sucrose, trehalose, and chitosan . In addition, we use the Clausius-Clapeyron relation for dilute alloys to estimate the analytical values of the liquidus slope . The PF-model domain is a region from 50 μm above to 300 μm below the solidification front.As input for the PF-simulations, we experimentally determined us slope , which ae growth , 54, 55 e growth  (SI Appez axis), we can reasonably assume that the preliminary ice-templated structure is represented by the unfrozen portion of the simulation domain. In addition, since crystals and grains with a large misorientation tend to be eliminated by early growth competition, the surviving ice-crystals typically grow with a small misorientation angle γ0 ≤ 10° with respect to the applied thermal gradient . By construction, this ice lamella is initially structureless on both sides  also explains, why highly aligned, continuous porosity can extend through the height of the entire sample (~75 to 100 mm), despite the local cooling rate gradually changing with distance of the solidification front from the bottom of the mold .e Top of B. Those e formed A. Indepe-axis contained within the basal plane of ice is misoriented by an angle hen α0=0 A and B, symmetry , 60. Howlamellae C, as opplamellae , 60.The cap forms between two ice-templated polymer “ridges” and becomes eliminated by their neighbors accompanied by an adjustment of the ridge spacing. Qualitatively similar instabilities are observed in directional solidification of binary alloys with non-faceted cellular interfaces where the cell elimination leads to an adjustment of the cellular spacing , 61. MeaS = w, where w is the cell wall thickness, is approximately equal to On a larger scale than the unilateral features that decorate the cell walls, which we have discussed so far, the cell walls themselves form a honeycomb-like pore structure illustrated in The first mechanism illustrated in SI Appendix, Fig. S6. Initially, at t = 120 s, the step-like defect is found near the solidification front where the rough interface of a cellular structure connects with the faceted interface through sharp cellular tips. The defect is initially formed just below a groove in between cell tips. As ice lamellae drift transversely, the defect propagates down the faceted side and eventually splits the initially smooth facet. This splitting can in turn cause secondary cells to detach from an ice lamella , resulting in a morphological transition (of the scaffold) from regular lamellae to a porous structure similar to the experimental observation in The simulation of We expect both of the above “intra-grain mechanisms” to contribute to the formation of the anisotropic irregular honeycomb-like pore structures with the first and second being more dominant during the earlier and later stages of growth, respectively. On an even larger scale of the whole sample, the simultaneous growth of grains of different crystallographic orientations is reflected in the presence of larger domains of different average lamellar orientation A. The grSI Appendix, Fig. S5 with the accompanying movie (Movie S2). Even though the simulations are 2D, we performed a few 3D simulations to check that the 2D and 3D dynamically selected spacings are nearly indistinguishable. This is not surprising because the lamellar spacing is established by a lamellar elimination process 1/2 , 26.Dendrite array structures are also known in a metallurgical context to exist for a wide range of spacing comprised in a range itions 27, 66–70. V and SI Appendix, Fig. S7 and Table S1). The variation of SI Appendix, Fig. S7 for the two global cooling rates In order to compare our simulation predictions with experiments in 3% w/v water-sucrose solutions, we need to take into account the fact that the crystal growth front velocity TC) mold , we deteTo compare experiments and PF-simulations, we superimpose in mentally , 72–75 antally 72, 38, 57 The simulations start from a monocrystalline planar interface at the liquidus temperature and are carried out at constant isotherm d, and height, h, of the “ridges”, which both increase with a decrease in applied cooling rate. In PF-simulations for 3% w/v sucrose, d varies from 42.1 ± 7.0 μm to 32.6 ± 2.5 μm with V increasing from 10 to 20 μm/s at a constant G = 12 K/cm. This result is in good agreement with the experimentally determined values for ridge spacing in 3% w/v sucrose scaffolds of d = 51.8 ± 9.2 μm at a sample position where V = 7.62 ± 0.12 μm/s and G = 9.4 ± 5.9 K/cm . In the case of chitosan (1.7% w/v in 1.0625% v/v acetic acid in water), the values increase from d = 25 ± 4 μm and h = 7 ± 3 μm at 10 °C/min to d = 43 ± 7 μm and h = 21 ± 2 μm at 1 °C/min, respectively  with a ridge spacing of d = 97 ± 4 μm, and a wavelength of 31 ± 11 μm for the “wrinkle”-like undulations  with a ridge spacing of d = 52 ± 5 μm. For comparison, experimental measurements in the ternary chitosan-acetic-acid-water system yield a wavelength of 13 ± 8 μm at a ridge spacing of d = 13 ± 2 μm for “shark-teeth”-like features, and a wavelength of 26 ± 8 μm at a ridge spacing of d = 34 ± 7 μm for “wrinkle”-like undulations  shows that cellular growth on the rough side becomes unstable, generating a disordered templated structure instead of the regular “ridges” shown in Here, our simulations reveal how different anisotropic properties of the ice-liquid interface shape 3D unilateral features that do not reflect on a macroscopic scale the underlying symmetry of ice. They show that the two-orders of magnitude slower growth along the c-axis, compared to basal directions (strong kinetic anisotropy), is responsible for the formation of a large featureless facet on one side of the inclined lamellae. Second, the weak (of the order of one percent) interface free-energy anisotropy in the basal plane enables a regular cellular array to form on the rough side. This array templates the rectilinear “ridges” . The results exhibit similar patterns as those observed in the ternary system, which supports our hypothesis. However, the question remains, whether it is the fast-diffusing solute that dominates structure formation so that it is always necessary, for example as an additive to large polymer solutions or particle slurries. Answering this important question will be possible by extending the present PF-model to treat the highly nonlinear diffusion process associated with large solutes complemented by controlled experiments. These hypotheses are important extensions yet to be explored.The ideal binary sugar-water system with the solute of small molecular size investigated here is illustrative of important phenomena. We discover that the binary sugar-water model systems with trehalose and sucrose, which contain only fast diffusing small molecules exhibit hierarchical features that are remarkably similar to those of the ternary chitosan-acetic-acid-water system, which contains two solutes, a slow diffusing large (chitosan) and a fast-diffusing small (acetic acid) polymer. To test the hypothesis that, in the ternary system, the fast-diffusing solute controls the pattern formation process, we carried out a PF-simulation treating the ternary chitosan-acetic-acid-water system as a pseudo binary system with only acetic-acid as the mobile solute  of the 1.7, 3, 5% w/v were prepared for diffusivity measurements by Diffusion Ordered Spectroscopy (DOSY). Chitosan flakes (8 g) were soaked in deionized water for 24 h, before adding 5% v/v acetic acid (0.265 M) and raising the volume to 100 mL to prepare an 8% w/v chitosan solution. For preparation of the diluted chitosan solutions of 1.2, 1.7, 2.4, 3.6 and 4.8% w/v, the 8% w/v chitosan solution was diluted down with DI water. Similarly, 1.2, 1.7, 2.4, 3.6% w/v chitosan were dissolved in deuterium oxide with acetic acid concentrations of 0.75, 1.0625, 1.5, and 2.25% v/v, respectively, for diffusivity measurements. All solutions were homogenized in a Speed Mixer  at 2,000 rpm for 2 min before use.Chitosan , acetic acid , sucrose, and trehalose  were used as received. Sucrose and trehalose solutions of 3,5 and 10% w/v in distilled water were prepared for characterizations by scanning electron microscopy (SEM) and x-ray microtomography. Sucrose and trehalose solution in deuterium oxide  and transverse (perpendicular to the freezing direction) cross sections of the chitosan, sucrose and trehalose scaffolds were prepared with a razor blade (Astra Superior Platinum Double Edge) at a height of 28 mm measured from the scaffold bottom. SEM  was performed on non-neutralized chitosan scaffolds without prior application of a conductive coating, and sucrose and trehalose scaffold sections with 10 nm gold coating. For high resolution SEM micrographs, cell walls were carefully peeled from non-neutralized, uncoated chitosan scaffolds under a stereomicroscope .For sectioning using a focused ion beam (FIB), the peeled-off cell walls were mounted on a gold-plated (10 nm) cover slip that was placed on an SEM stub before sputter-coating also this assembly with a 10 nm thick gold layer. Taking advantage of a dual-beam system , first a 100 nm thick Pt layer was deposited by e-beam over an 42 µm by 12 µm area, then a 200 nm thick Pt layer was i-beam deposited over a 42 µm by 4 µm region of interest parallel to the “jellyfish” feature to include the entire “jellyfish-cap” as well as a small distance beyond its length. To expose the feature in cross-section, a trench was FIB-milled on an area of 40 µm by 5 µm, cutting towards the center of the feature using the milling parameters of 16 kV at 0.5 nA; milling progress was monitored by SEM. Finally, the FIB-milled cross-section was cleaned at 2 kV and 70 pA, again while closely monitoring the process by SEM, until the cutting plane met the centerline of the “jellyfish-cap.” SEM micrographs, using secondary electrons, were obtained directly after FIB-milling.The 3D structure of scaffolds of two compositions, 3% w/v sucrose and 3% w/v trehalose, each freeze-cast at two applied cooling rates, 10 °C/min and 1 °C/min, was imaged by X-ray microtomography using a desktop system  operating at a source voltage of 50 kV and a source current of 200 µA. The 360° scans were performed with a step size of 0.1°, the camera size was 2,942 × 3,280 pixels. Two voxel sizes were used, 1.5 µm and 5 µm, resulting in fields of view of 4.413 mm × 4.920 mm and 14.71 mm × 16.40 mm, respectively. Reconstructions and visualizations were obtained with the Nrecon and CTVox software tools , respectively. Pore size and geometry were determined at a height of 30.5 mm and 10.0 mm measured from the scaffold bottom with ImageJ (version 1.53c) .The phase transition temperatures for chitosan solutions were determined by differential scanning calorimetry . To perform the measurement, an aluminum crucible was filled with 10 ± 1 mL of 1.2, 2.4, 3.6, 4.8, and 8% w/v chitosan solution in 0.75, 1.5, 2.25, 3, and 5% v/v acetic acid in distilled water, respectively, and the combined mass of the sample and the crucible was determined with a precision balance . To obtain the state diagram for chitosan, the samples were heated from −150 °C to 4 °C at a rate of 10 °C/min and the melting points were determined from the offsets of the peaks on the DSC curve .2O with DOSY. Bipolar gradient DOSY measurements were performed for concentrations of 1.2, 1.7, 2.4, and 3.6% w/v of chitosan in acetic acid  in D2O, 5% w/v sucrose in D2O, and 1.7%, 3%, 5% w/v trehalose in D2O, using a 500 Mhz Advance III system  retrofitted with a magnet with a 5 mm inner diameter probe  controlled by Topspin 3.2 pl6 software . The temperature was decreased in 5 °C steps from 25 °C to 5 °C for the chitosan solutions, and in 5 °C steps from 20 °C to 5 °C for the sucrose and trehalose solutions.The diffusion coefficients of chitosan, sucrose, and trehalose were determined in DV [mm/s] from the first derivative of the linear (1 °C/min) or quadratic (−10 °C) fit to the height-time plot for TCs below the fill level. The TC 1 is neglected because of its proximity to the copper bottom. The local cooling rate was determined for each TC from the 0 °C to −2 °C temperature-time gradient. A linear fit of the local cooling rates over the probe height were performed, and the corrected local cooling rates G [K/mm] were then calculated by V.A custom-made TC mold was used to determine the freezing front velocity, local cooling rates, and temperature gradients at the liquid solid interface . The timTo model the unidirectional freezing of water-based solutions, we extend to ice templating the well-developed quantitative PF formulation of binary alloy solidification , 52. Thi current , 52, whi casting , 25 in tAppendix 01 (PDF)Click here for additional data file.Movie S1.G = 12 K/cm and V = 15 μm/s, with the simulation domain size 3337 × 216 × 216 μm, the misorientation angle γ0 = 10°, and the simulation time 180 s.Three-dimensional PF simulations of ice-crystal growth during directional solidification of a 3% w/v aqueous trehalose solution with growth conditions Movie S2.G = 12 K/cm and V = 15 μm/s, with the simulation domain size 2362 × 1208 μm, the misorientation angle γ0 = 10°, and the simulation time 200 s.Two-dimensional PF simulations of ice-crystal growth during directional solidification of a 3% w/v aqueous trehalose solution with growth conditions


Weaning is a critical phase in the pigs’ life and gut health might be compromised. Gluconic acid was shown to be poorly absorbed but readily fermented to butyrate in the gut which in turn can improve gut function. Hence, a total of 144 weaning pigs were fed the experimental diets for 42 days. Three treatments were replicated in 8 pens with 6 piglets each: control; low dietary dose of gluconic acid, 9 g/kg; and high dietary dose of gluconic acid, 18 g/kg. After 21 days, one piglet from each pen was sampled for blood haematology and biochemistry, fore- and hindgut digesta characteristics and microbiota, and distal small intestinal histo-morphological indices and gene expression.P = 0.028), though the high dose did not show benefits over the low dose. Regarding d 0–42, feed intake was elevated (P = 0.026). At d 21, piglets fed 18 g/kg gluconic acid showed a trend for lower number of total white blood cells (P = 0.060), caused by particularly lower numbers of lymphocytes as compared to control (P = 0.028). Highly reduced plasma urea was found for groups fed gluconic acid, it amounted to 2.6 and 2.6 mmol/L for the 9 and 18 g/kg level, respectively, as compared to 3.8 mmol/L in control (P = 0.003). Feeding gluconic acid promoted the relative abundance of lactic-acid-producing and acid-utilizing bacteria. In distal small intestine, Lactobacillus amylovorus increased substantially from 11.3 to 82.6% for control and gluconic acid 18 g/kg, respectively (P < 0.05). In mid-colon, the butyrate producers Faecalibacterium prausnitzii (P > 0.05) and Megasphaera elsdenii (P < 0.05) showed highest abundance in gluconic acid 18 g/kg. Consequently, in caecum and mid-colon, increased relative molar percentage of butyrate were found, e.g., 10.0, 12.9 et 14.7% in caecum for gluconic acid at 0, 9, and 18 g/kg, respectively (P = 0.046). Elevated mRNA anti-inflammatory cytokine and survival signalling levels in distal small intestinal mucosa were found by feeding gluconic acid which might be mediated by butyrate.Feeding gluconic acid enhanced performance in period d 0–14 post-weaning, in particular feed intake was increased (Gluconic acid may have potential to alleviate the postweaning growth-check in pigs by altering microbiota composition and fermentation in the gut.The online version contains supplementary material available at 10.1186/s40813-023-00305-1. Piglets weaned at 3 to 4 weeks of age are exposed to nutritional, environmental, and social stresses leading to low feed intake, reduced weight gain, nutrient malabsorption, and increased occurrence of diarrhoea . Antibio6H12O7, CAS 526-95-4) is poorly absorbed in the gastrointestinal tract of the rat  ruminantium group. Agathobacter was more abundant in pigs fed gluconic acid at 18 g/kg (1.11%) than in control pigs (0.41%) (P < 0.05). Remarkable changes within Veillonellaceae occured. Genera Dialister , Megasphaera , and Veillonella  showed highest abundance in gluconic acid 9 g/kg, gluconic acid 18 g/kg, and control, respectively. As found in distal small intestine, relative abundances of species within Lactobacillaceae appeared to change by feeding gluconic acid, e.g., L. johnsonii and L. amylovorus are decreasing and increasing, respectively (both P < 0.05), although their abundance is far lower as in distal small intestine and here with the latter species less present than the former. In addition, L. prophage and unclassified Lactobacillaceae HT002 were decreased by gluconic acid 18 g/kg as compared to control (P < 0.05). Regularized canonical correlation analysis (rCCA) suggests clear correlations between pH and bacterial metabolites and bacterial species in distal small intestine , where species that were lowered showed high correlation with propionate . High acetate producers were unclassified Romboutsia and Actinobacillus spp., those were only found in significant numbers in the control treatment. Some members of the Enterobacteriaceae were highly negatively correlated with propionate. Several families within the phylum Bacteriodota  were associated with the production of branched-chain fatty acids in mid-colon . Contrary, Lactobacillaceae HT002 and Veillonella showed highly negative correlations with these metabolites, they were notably suppressed by feeding gluconic acid.Few parameters that characterise digesta in the gastrointestinal tract of piglets sampled on d 21 were found significant Table , Fig. 1.y P = 0.00 , nor any histo-morphological measurements or the number of intra-epithelial lymphocytes (IEL) differed among treatments Table . From thE. coli in the stomach and the small intestine. Indeed, a trend was observed for a dose-dependent decrease in E. coli numbers in the distal small intestine. The reduction as compared to control was 0.9 log10 CFU/g for gluconic acid at 18 g/kg. Counts of other bacterial groups including the lactic acid producing Lactobacilli and Streptococci, nor the metabolites thereof in distal small intestine were affected by treatment, which was also observed by Biagi et al. [In the current study, two levels of gluconic acid in the diet of weaned piglets were tested. In the pre-starter period, growth was improved because of both higher feed intake and better feed utilization. Gluconic acid stimulated appetite in the immediate postweaning period, and obviously these responses for feed consumption were higher for gluconic acid at 9 g/kg than at 18 g/kg. In line with this, Biagi et al.  found a i et al. .L. amylovorus increased substantially, i.e., from 11.3 to 82.6% relative abundance for control and gluconic acid at 18 g/kg, respectively, along with higher abundance of L. kitasatonis and L. delbrueckii, and lower abundance of L. johnsonii and L. prophage. As the plating assays demonstrate that the number of viable Lactobacilli did not differ across treatments in distal small intestine, congruent to Biagi et al. [L. amylovorus and others within a competitive environment like the small intestinal lumen, even though it was clearly shown that many Lactobacilli as single strains easily consume gluconic acid albeit to a different degree [V. ratti and unclassified Veillonella, both correlated with propionate as demonstrated in the rCCA. It may point to the fact that phosphoenolpyruvate is primarily converted to lactate and accumulates rather than following the succinate pathway and thus giving propionate when feeding gluconic acid [M. elsdenii and M. multacida. Lactate can be used to produce acetyl-CoA, which can be further metabolized in the crotonyl pathway with final conversion of butyryl-CoA by butyrul-CoA:acetate CoA-transferase into butyrate. This conversion utilizes exogenously derived acetate and generates butyrate and acetyl-CoA. The rCCA for mid-colon in our study showed that the genera Megasphaera and Mitsuokella were positively correlated with butyrate and even more prominent with valerate. Interestingly, while for Megasphaera the correlation with butyrate was 0.37 it was markedly higher for valerate, i.e., 0.67. Indeed, Yoshikawa et al. [M. elsdenii isolates from pig faeces in Japan produced valerate as predominant SCFA. Tsukahara et al. [M. elsdenii to the medium stimulated the production of butyrate by 60% and of valerate by 50%. Butyrate is essentially synthesized by the condensation of two molecules of acetyl-CoA, and valerate is formed by the condensation of an acetyl-CoA and a propionyl-CoA molecule. It is fair to state that in our study butyrate and/or valerate producers were favoured by feeding gluconic acid. In fact, as both butyrate and valerate were dose-dependently increased in both caecal and mid-colonic contents, it is appealing to report the sum of their relative concentrations, i.e., 12.3, 16.9, and 18.7% in caecum and 15.4, 16.7, and 18.8% in mid-colon for control, gluconic acid at 9 g/kg, and gluconic acid at 18 g/kg, respectively. In caecum, in our study the butyrate concentration reached 16.7 μmol/L when pigs were fed with 18 g/kg gluconic acid for 3 weeks, whereas Tsukahara et al. [In a rat model, Asano et al.  could cot degree . In factnic acid . Then, inic acid  and Metznic acid . Tsukahanic acid  showed ta et al.  showed ta et al.  also foua et al.  found hiBAX was downregulated in distal small intestinal mucosa by gluconic acid at 18 g/kg. The latter is in line with findings by Lacorn et al. [IFNG and IL10, whereas the lower dose upregulated IFNG. Piglets fed gluconic acid at 18 g/kg showed lower number of total white blood cells, caused by particularly lower numbers of lymphocytes in line with in vitro studies [IL10. The latter was also demonstrated by Seamann et al. [IFNG as our results are opposed to Chevassus et al. [IL10, like our results. Transcript of MUC2 was upregulated by highest dose of gluconic acid. In vitro, butyrate increased dose-dependently the expression of the MUC2 gene, again supporting the idea that current findings might be mediated by hindgut butyrate production [Lactobacilli may express to various degrees different cell surface proteins that can adhere to the intestinal mucus layer [Lactobacilli could alter mucus ecology, and potentially barrier function, and thus also be responsible for alterations in the expression of the MUC2 gene. The fact that we applied high dosages of gluconic acid as precursor of SCFA might be the reason for the very outspoken effects on gene expression of distal small intestinal tissue which are not consistently found in literature studying a wide range of butyrate-based products in livestock.Next to the well acknowledged physiological effects of butyrate , 9, recen et al.  who shown et al. . The hig studies , while an et al. . Then, es et al. . Nonetheoduction . Since Lus layer , it may Among the blood biochemical indices, highly reduced plasma urea was found for both groups fed gluconic acid. This is likely associated with the prebiotic effect of gluconic acid, i.e., providing energy from a carbohydrate source that stimulates bacterial growth consequently consuming ammonia as nitrogen source in distal small intestine and hindgut, like outcomes of other highly fermentable prebiotics such as inulin . We haveFeeding newly weaned piglets with 9 and 18 g/kg gluconic acid in the diet enhanced performance in the immediate postweaning period, though the high dose did not show benefits over the low dose. A dose-dependent increasing prebiotic effect by gluconic acid was evidenced by alterations in relative abundance of lactic acid producing and acid-utilizing bacteria, resulting in increased hindgut butyrate production. Elevated mRNA anti-inflammatory cytokine and survival signalling levels in distal small intestinal mucosa were found by feeding gluconic acid which might be mediated by butyrate. Gluconic acid may have potential to alleviate the postweaning growth-check in pigs.2 per pen, with full slatted floor) with 6 piglets each according to gender and body weight, so that each pen had similar average body weight and equal number of both genders. The 3 treatments were replicated in 8 pens each, according to a completely randomized design and were as follows: control diet, i.e., basal diet; gluconic acid at 9 g/kg, with gluconic acid source added to the basal diet at the expense of barley to obtain the intended level, and gluconic acid at 18 g/kg, with gluconic acid source added to the basal diet at the expense of barley to obtain the intended level. Source of gluconic acid was Gluconic Acid L . From d 0 until d 14, a pre-starter diet was given, followed by a starter diet until d 42. Diets were voided of antibiotics, organic acids, apart from gluconic acid in respective treatments, and supra-nutritional levels of Cu and Zn. The starter diet contained a source of 4 mol/L HCl insoluble ash as digestibility marker. The ingredient and formulated nutrient composition of the basal diets are given in Table The study was conducted in accordance with the ethical standards and recommendations for accommodation and care of laboratory animals covered by the European Directive 2010/63/EU on the protection of animals used for scientific purposes and the Belgian royal decree KB29.05.13 on the use of animals for experimental studies. A total of 144 weaning  piglets with an average weight of 8.2 kg were used in the feeding trial for 42 days. At arrival, piglets were allocated to 24 pens , ADG (g/d), ADFI (g/d), F:G (g/g), Average Daily Water Intake , and W:F (mL/g) were analysed for all periods. In addition, feed intake was registered at d 2, d 5, d 8, and d 11 to monitor immediate postweaning feed consumption increases. The same trained animal caretaker inspected the animals daily for general health, faecal consistency score and diarrhoea incidence. The faecal consistency score was visually assessed on pen level according to the following scoring system: 1 = hard or slightly moist faeces, clearly formed, normal; 2 = moist or soft faeces, but still with a definite form, sticky; and 3 = watery or liquid faeces, unformed, diarrhoea. If faeces of different consistency in a pen were observed, the highest score present was retained as data. The assessment of diarrhoea incidence  was done simultaneously by counting the piglets in the pens receiving faecal consistency score of 3 that show clear signs of diarrhoea, i.e., filthy, wet backside and tail, dehydrated, loss of condition and irritation of the skin around the anus. Faecal consistency score and diarrhoea incidence were assessed until d 21. Occasionally piglets showing prolonged and obvious health issues were treated with antibiotics if needed. Amoxicillin as broad-spectrum antibiotic was used for indications such as respiratory problems, swollen joints, unhealed wounds, severe locomotory problems, and general state of runting. Colistin was used to treat prolonged watery and severe diarrhoea. Antibiotic treatments were carefully monitored.2SO4. Further, a 1 g subsample of distal small intestinal digesta was taken for enumeration of main bacterial groups by plating. Finally, aliquots of distal small intestinal and mid-colonic digesta were snap frozen and stored at −80 °C pending metagenomic analysis. Meanwhile, a first 5 cm section of the small intestine at 3 m anterior to ileo-caecal valve was taken, rinsed with 9 g/L saline and fixated in a 4% formaldehyde solution for histo-morphological analysis. A second segment (20 cm) was obtained at the same location to assess the macromolecular permeability in Ussing chambers. A third segment (20 cm) was used to harvest mucosa by scraping with glass slide, and frozen immediately in liquid nitrogen and stored at −80 °C pending gene expression analysis.After 21 days of feeding, one piglet from each pen with body weight close to the average body weight in the respective pen was euthanized by electrocution followed by bleeding. Whole blood was drawn for haematological indices (EDTA as anticoagulant), whereas serum was employed for biochemical measurements (centrifugation at 3000 g for 15 min). Next, the whole gastrointestinal tract was removed, and contents were carefully collected from stomach, proximal small intestine , distal small intestine , distal ileum , caecum and mid-colon. pH measurement of contents was done for all sections except distal ileum. Complete contents of distal ileum were pooled per treatment, stored at −20 °C, freeze dried and used for apparent ileal digestibility determinations. Aliquots from other sections were frozen at −20 °C pending freeze drying for dry matter determination, whilst aliquots of distal small intestinal, caecal and mid-colonic contents destined for determination of SCFA, and lactic acid were acidified with 2% v/v 6 mol/L HApparent ileal digestibility of dry matter, organic matter and protein of pooled samples was assessed using the indicator method with 4 mol/L HCl insoluble ash as marker . Dry matHaematological indices and biochemical measurements  were assessed using routine methods at the diagnostic laboratory Animal Health Care .Bacterial metabolites were determined in digesta of distal small intestine, caecum, and mid-colon. SCFA and lactic acid in small intestinal contents were analysed by a GC method described by Missotten et al.  and Van 10 colony-forming units (CFU) /g fresh digesta) in digesta of distal small intestine were obtained using the ring-plate technique [E. coli , coliform bacteria  and Streptococci . Data were log10 transformed prior to statistical testing. The lower limit of detection was 2 log10 CFU/g.Bacterial counts . The Divisive Amplicon Denoising Algorithm (DADA) is based on the identification of single nucleotide sequence variants and provides higher resolution [https://www.arb-silva.de/documentation/release-1381/) was used for taxonomy assignment. Taxonomy data and metadata were merged into a phyloseq object applying the Phyloseq package  profiling was performed. Genomic DNA was extracted using PSP Spin Stool DNA Plus Kit  according to the manufacturer’s instructions and modified. Next, the 16S polymerase chain reaction (PCR) libraries were generated and processed as described by Cong et al. . The V1‐solution . The rawsolution ; https:/ 1.36.0; ) in R. LFixated sections of the small intestine were processed under standard conditions in an automatic tissue processor . Process2. Two replicate chambers were used per pig and tissues were mounted within 15 min postmortem. Fluorescein isothiocyanate–dextran 4-kDa  was used as macromolecular probe. Chambers were covered with aluminium foil to protect from light. The marker was added to the mucosal side after 20 min of equilibration to obtain final concentrations of 0.8 mg/mL. Samples were taken from the serosal side every 20 min between 40 and 100 min after mounting the segments. The fluorescent signal of FD-4 was captured using excitation filter at λ = 494 nm and emission filter at λ = 521 nm. The apparent permeation coefficient (Papp) was calculated as: Papp (cm/s) = (dc/dt) × V/c0/A, where dc/dt is the change in the marker (FD-4) concentration at the serosal side (acceptor) between 40 and 100 min, (µg/mL/s) calculated from the slope of the concentration–time curve, V is the buffer volume in the luminal side (donor) of the compartment (mL), C0 is the initial marker concentration in the donor compartment (µg/mL) and A is the exposed tissue surface area (cm2).The intestinal permeability was assessed using the Ussing chamber technique as previously described by Wang et al.  and Van MUC2, IL6, TNF, IFNG, IL10, TLR4, OCLN, TJP1, CLDN5, BAX, BCL2, NQO1, and GPX2) was performed according to the MIQE guidelines [260/280) of RNA were measured with the NanoDrop ND-1000 . One μg RNA was analysed by 1% agarose gel electrophoresis to check RNA integrity (28S and 18S rRNA bands). In addition, a minus-RT control PCR was performed using YWHAZ as primer to verify the absence of any gDNA contamination. Following this, 1 μg of high-quality DNA-free RNA was reverse transcribed in the 20 μL reverse-transcription reaction with the ImProm-II cDNA synthesis kit , containing both oligo dT and random primers. The obtained cDNA was diluted 10 times with molecular grade water and a control PCR using 2 μL cDNA was performed to verify the reverse-transcription reaction. Primers  and RPL4 (0.24) and scaled to control treatment according to Hellemans et al [RT-qPCR , and P < 0.05 and as a tendency for significance when P < 0.1. For the microbial composition following 16S rRNA amplicon sequencing, statistical analyses were performed in R using the packages Phyloseq and vegan for community analysis  and homogeneity of variances (Levene’s test) across treatments. It appeared that data for daily diarrhoea incidence were not normally distributed. Hence, data for this endpoint were evaluated with the non-parametric Kruskal–Wallis test, whereas other endpoints were tested with GLM procedures in SAS Enterprise Guide 7 . Data were analysed with the following statistical model (one-way ANOVA):n 2.5.7; ). Signifn 2.5.7; . Regularn 2.5.7; ) in R.Additional file 1: Effect of diet on faecal consistency score and diarrhoea incidence in period d 0-21.Additional file 2: Effect of diet on cumulative feed intake in the pre-starter period.Additional file 3: Effect of diet on relative abundance of bacterial taxa in distal small intestineAdditional file 4: Effect of diet on relative abundance of bacterial taxa in mid-colon.Additional file 5: Relationship between bacterial genera and pH and fermentation products in samples of mid-colon.Additional file 6: Analysed nutrient composition of experimental diets.Additional file 7: Rarefaction curves of small intestinal and mid-colonic samples.Additional file 8: Primer sequences used for reverse-transcription quantitative real-time PCR.


InAs analysis was carried out by inductively coupled plasma mass spectrometry . No sample showed contamination by OTA. AFB1 was present in 2 (4.8%) samples (1.96 and 2.20 μg kg−1), doubling the European maximum permitted level (MPL). Concerning ZEN, 88.89% of the rice samples presented levels above the LOD up to 14.25 µg kg−1 (average of 2.75 µg kg−1). Regarding InAs, every sample presented concentration values above the LOD up to 100.0 µg kg−1 (average of 35.3 µg kg−1), although none surpassed the MPL (200 µg kg−1). No correlation was observed between mycotoxins and InAs contamination. As for human exposure, only AFB1 surpassed the provisional maximum tolerable daily intake. Children were recognized as the most susceptible group.Rice is the second most important cereal crop and is vital for the diet of billions of people. However, its consumption can increase human exposure to chemical contaminants, namely mycotoxins and metalloids. Our goal was to evaluate the occurrence and human exposure of aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEN), and inorganic arsenic (InAs) in 36 rice samples produced and commercialized in Portugal and evaluate their correlation. The analysis of mycotoxins involved ELISA, with limits of detection (LODs) of 0.8, 1 and 1.75 μg kg Oryza sativa, belongs to the Gramineae family and Oryzoides subfamily. It is the second most vital cereal crop worldwide [Rice, orldwide  and an iorldwide .According to the latest report of the European Union (EU) for the “EU agricultural outlook for markets, income and environment 2020–2030”, worldwide production of rice has been progressively increasing during the last decade. Whereas in the EU, the annual per capita consumption is 6 kg, the worldwide annual per capita consumption is 54 kg [After harvest, the rice grain commonly undergoes several processing steps, such as drying, milling, and packaging, to be convenient for consumption. Rice consumption provides around 20% of the daily calories bulk for many humans ,5 and isGiven the current concern about world hunger, climate change effects, growth of the population, and food security, the production of rice is endangered, along with rice quantity and quality availability , which mAspergillus species are categorized by the International Agency for Research on Cancer (IARC) in group 1. In particular, aflatoxin B1 (AFB1), the most mutagenic, genotoxic, and carcinogenic mycotoxin, can originate hepatic lesions, cirrhosis, primary hepatocellular carcinoma, and Kwashiorkor and Reye’s syndrome [Aspergillus and Penicillium species, is associated with Balkan endemic nephropathy (BEN) [Fusarium graminaerum and Fusarium culmorum [Among several other agricultural commodities, rice was already regarded as the greatest substrate for aflatoxin production . These ssyndrome . As for hy (BEN) . Zearaleculmorum . ZEN is culmorum . It was culmorum .Arsenic (As) abounds in the soil owing to natural and anthropogenic factors such as rock weathering, mining activities, and pesticide application . It is rTherefore, these toxic elements must be controlled in rice in order to meet a high standard of quality, given its massive worldwide consumption, particularly by children . They ar−1 for individual content of AFB1 [−1 for OTA for all products derived from unprocessed cereals, including processed cereal products and cereals intended for direct human consumption. For ZEN, the cited regulation states that “given the low contamination levels of Fusarium toxins found in rice, no maximum levels are proposed for rice or rice products”. A limit of 75 μg kg−1 is set for cereals intended for direct human consumption [Current EU legislation does not establish maximum permitted limits (MPLs), specifically in rice, for either AFB1, OTA, or ZEN. However, according to the legislation in force, Regulation (EU) No. 165/2010 sets a maximum level for cereals of 2 μg kg of AFB1 . Commisssumption .−1 was set for non-parboiled milled rice (polished or white rice) and 0.25 mg kg−1 for parboiled and husked rice [Regarding arsenic, Commission Regulation No. 2015/1006, amended Regulation No. 1881/2006 with regard to maximum levels of arsenic in inorganic form (sum of As (III) and As (V)) in foodstuffs, setting limits to the presence of inorganic arsenic in rice and rice products. A maximum of 0.2 mg kgked rice .This research aimed to evaluate, for the first time, if AFB1, OTA, ZEN, and InAs co-occur and correlate in rice acquired in Portugal. Moreover, we also aimed to confirm the compliance with the maximum permitted levels of the European legislation and evaluate the exposure of the Portuguese population, more specifically children, adolescents and adults, according to their consumption of this cereal so as to assess their potential risk.In the present study, from the 36 samples analyzed, none showed contamination by OTA. Contamination of OTA in rice is generally lower than in wheat or corn .−1, with a mean of 0.12 μg kg−1. Both positive samples were branded commercially acquired samples. One sample was of long wild rice, and the other was of short white rice, with their origins in Canada and Portugal, respectively. One should note that only one sample surpassed the European maximum limit of 2 μg kg−1.AFB1 was only found in 2 samples (4.8%) in concentrations of 1.96 and 2.20 μg kgThe occurrence of aflatoxin in brown rice is often slightly higher than the MPLs established. However, after removing the husk, that content generally decreases below the maximum levels . In the −1 in Vietnam [−1, regards a Pakistan study [−1 [−1 [Comparison with other scientific studies is difficult, namely because most of the reported studies are from countries with different weather and production conditions. However, when comparing AFB1 results with previously published scientific studies, as presented in  Vietnam . The maxan study . Nonethetudy [−1  and in C [−1 [−1 . With th [−1 [−1 , our ave−1, averaging 2.75 ± 2.26 µg kg−1. Comparing private and white label samples, a significant difference was found (p = 0.0196), with white label samples showing a higher average (2.91 µg kg−1) than the branded ones (2.08 µg kg−1). Comparing brown and white rice, there was no statistical difference, but the p-value was near 0.05 (p = 0.0557), with brown samples showing a higher average. No other significant comparisons were observed.Regarding ZEN, 88.89% (32 out of 36) of the samples presented levels above the LOD, up to 14.25 µg kgFusarium spp. cause major reductions in rice quality due to environmental conditions. High moisture and high-temperature conditions favor field Fusarium growth, which may also develop when rice is stored [Fusarium proliferatum dominates [−1 in Côte d’Ivoire [−1 in Brazil [s stored . Howeverominates , which jominates . As seend’Ivoire  and 143 n Brazil . HoweverMycotoxins are subject to regulation in many countries worldwide to limit their presence in foods. Specifically, the strict European legislation obliges the application of good practices in rice production, storage, and distribution, which may justify the current results . On the −1, with an average of 35.3 ± 28.2 µg kg−1. None exceeded the MPL established by the EU.Bearing in mind the results shown in −1, respectively; a significant statistical difference was observed with a p = 0.0236. Comparing white and branded labels, a significant statistical difference was also observed (p = 0.0220), with average levels of 10.5 vs. 37.5 µg kg−1, respectively.When comparing supermarket samples versus those provided by rice producers, it was found that the average contamination was higher in the latter, with 25.9 and 41.2 µg kg−1 (p = 0.0480), up to 100.0 µg kg−1. Long grain had a lower average of 26.9 ± 26.1 µg kg−1 and a lower maximum value of 90.0 µg kg−1.Regarding long vs. short grain, it was verified that average levels were significantly higher in the latter, with a value of 41.9 ± 28.6 µg kg−1, while short grains presented a mean concentration of 122 μg kg−1. Therefore, short rice showed a concentration of around 30% higher As  than long rice [−1 (n = 8) [−1 [−1); however, sticky rice showed lower mean contamination values.Accordingly, other authors  reportedong rice . In Finl (n = 8) , while i= 8) [−1 . On the p-value, <0.0001, was obtained when brown and white rice were compared; brown rice ranged between 23.0 and 100.0 µg kg−1, with an average of 55.1 ± 27.7 µg kg−1, while white rice ranged between >LOD and 80.0 µg kg−1, with an average of 22.6 ± 20.5 µg kg−1 (The lowest  µg kg−1 .−1) [InAs concentrations in rice decrease in the following order: hull > bran polish > brown rice > raw rice > polished (white) rice. Therefore, commercially available polished white rice presents lower arsenic levels than whole grains and is safer for consumption . In the −1) . Other a−1) . Polishi−1) ,59.−1. Comparing rice from Portugal and abroad, it was found that Portuguese rice presented significantly higher average levels, 38.3 ± 26.1 µg kg−1, against 26.1 ± 33.9 µg kg−1 (p = 0.0272). According to data reported in the scientific literature, InAs levels were quite constant in temperate, subtropical, and northern hemisphere tropical regions [Finally, between the production regions of Tagus, Mondego, and Sado, there were no statistical differences, with average contamination values of 59.1 ± 28.0, 19.0 ± 11.3, and 32.9 ± 24.1 μg kg regions .−1 for milled rice, 250 μg kg−1 for brown rice, and 100 μg kg−1 for rice intended for consumption by infants and young children. However, we can consider that the sample contaminated at 100 μg kg−1 should not be used in the diet of infants and young children. This sample was of short brown rice originating in the Tagus River provided by a rice producer.Legislation sets maximum concentration levels of inorganic arsenic present in rice and rice derivatives . Among tOne should note that the present study was done on uncooked rice. Pre-cooked rice presents less than 50% of the As and InAs levels of dry rice. The fact that pre-cooked rice has already been boiled leads to partial arsenic removal. It has also been stated that washing the rice before cooking or boiling it with abundant water may decrease arsenic concentrations by up to 60% .The co-occurrence of many kinds of mycotoxins/contaminants may increase the risk to human health . Two bra−1 b.w./day, calculated for children when the UB–95th consumption scenario was considered. Viewing the LB–average consumption scenario, the EDI values were 0.26 ng kg−1 b.w./day for children, 0.18 ng kg−1 b.w./day for adolescents, and 0.12 ng kg−1 b.w./day for adults.For children, adolescent, and adult populations, different EDI assessments were carried out, bearing in mind different scenarios, including the LB approach, the UB approach , and both combined, with the average and 95th percentile consumption . For eve−1 b.w./day, for adults and children without hepatitis B, and 0.4 ng kg−1 b.w./day for those with hepatitis B virus. As seen in −1 b.w./day. The PMTDI of 1 ng kg−1 b.w./day was only surpassed when the UB approaches were considered. As for the lower PMTDI of 0.4 ng kg−1 b.w./day, even the LB approach with 95th percentile consumption of children surpassed it.Risk assessment was achieved through the comparison of the obtained EDI with the Kuiper–Goodman PMTDI values , 1.0 ng Regarding the MOE approach, and considering that a value inferior to 10,000 represents a concern for human health , one can−1 b.w./day, a value much higher than the EDI obtained in the present study.According to a Pakistani study , the meaOne should note that the exposure and risk evaluation of the present study were estimated in uncooked rice. Previous studies reported that cooking or food processing can significantly reduce mycotoxin levels, including AFB1 . Specifi−1 b.w./day would increase the incidence of cancer in the liver at 0.013 cancers/year/100,000 inhabitants, so the EDI obtained is quite of concern to the health of consumers.As mentioned, according to epidemiological data on primary liver cancer collected by JECFA, an intake of 1.0 ng kg−1 b.w./day, established by the EFSA Panel for Contaminants in the Food Chain (CONTAM Panel) in 2011, is based on its oestrogenicity in pigs [−1 b.w./day, a value obtained when considering the worst-case scenario (highest concentration) and the 95th consumption of children, with consequently higher risk values, 31.1%. Nonetheless, in every case, the EDI was far from the established TDI, ranging from 0.00275 µg kg−1 b.w./day and 0.078 µg kg−1 b.w./day, corresponding to a calculated risk of 1.10% and 31.1%, respectively.The current ZEN tolerable daily intake (TDI), 0.25 µg kg in pigs . RegardiWhen considering the average concentration (AC)/average consumption approach, lower EDI and risk values were obtained, as expected, ranging between 1.10 and 2.40%.Again, in every approach considered, children presented higher risk values considering this TDI value. Accordingly, other authors described children as a susceptible group to most contaminants .−1 b.w./day, obtained for children when the HC–95th consumption scenario was considered. When considering the AC–average consumption scenario, the EDI values were 0.0770, 0.0535, and 0.0351 µg kg−1 b.w./day for children, adolescents, and adults, respectively.For every population in the study, different EDI assessments were attempted based on different scenarios, including the AC approach, the HC approach, and both combined, with the average and 95th percentile consumption . As show−1 b.w./day, established by the EFSA’s Panel on Contaminants in the Food Chain [010.3/EDI, using the AC-average consumption scenario, was 3.90, 5.61, and 8.54 for children, adolescents and adults, respectively. The MOE decreased when the 95th percentile was considered for values lower or close to 1:0.55, 0.89, and 1.28, for children, adolescents, and adults, respectively. Certainly, when considering the BMDL01 to be 8 µg kg−1 b.w./day, higher MOE values were obtained, ranging between 14.65 for children when considering the HC–95th consumption scenario and 227.61 for adults considering the AC–average consumption scenario.Risk assessment  was accood Chain , obtaini−1 b.w./day) established by the EFSA in every exposure scenario considered  [−1b.w./day, but in no scenario, it was above the upper BMDL01 of 8 µg kg−1 b.w./day established by EFSA [Compared with other published data, other authors also found that toddlers and infants presented the highest dietary exposure to total As, 4.08 and 3.99 μg/day, respectively. However, in contrast with the other population groups, the major contributor was organic arsenic, and none of the population groups surpassed the lower limit of the BMDL01 range (0.3 μg kgand low) . Other s by EFSA .−1 b.w./day for children [On the contrary, in Finland, others observed that the InAs exposure from the consumption of long grain rice and rice-based baby food in all age groups is close to the lowest BMDL0.1 value, considering conservative worst-case scenarios estimations, reaching a maximum of 0.67 μg kgchildren .This pilot survey showed that rice is widely contaminated with InAs and ZEN, with frequencies of contamination of 100% and 88.89%, respectively. Nevertheless, none of these contaminants surpassed the established MPL in the European Union. Conversely to the field mycotoxin, the surveyed storage mycotoxins surveyed were scarce. Indeed, OTA was not detected, whereas AFB1 was found in two single rice samples. It is noteworthy that the two AFB1-contaminated rice samples featured levels close to and even slightly higher than the MPL.p = 0.0196). White-label samples presented higher values and thus presented a higher contribution to the exposure of consumers.No correlation was found between the mycotoxin and InAs contamination of rice samples. Nevertheless, statistical analysis showed a significant difference regarding mycotoxin levels when comparing private and white-label samples (p < 0.0001) was observed in the contamination by InAs. Brown rice featured higher values in both maximum and average levels. A similar tendency was observed for ZEN, although without statistical significance.Furthermore, when brown and white rice were compared, a significant difference  were commercially acquired as available for regular consumers from different Portuguese supermarkets. The latest included 8 private labels  and 6 white labels .A total of 36 rice samples intended for human consumption were collected between November 2019 and February 2020. A total of 22 samples were kindly provided by Portuguese rice producers, while the remaining samples (n = 22), brown rice (n = 13), and wild rice (n = 1). Regarding grain length, 20 were long grain, and 16 were short grain rice. Concerning origin, most samples were produced in mainland Portugal (n = 27), while 9 samples were imported (Canada (n = 1), China (n = 1), India (n = 4), Thailand (n = 2), and Uruguay (n = 1)).Convenience sampling gathered different types of rice: white rice  before their expiration date.For the quantification of AFB1, an immunoenzymatic test in a competitive format was performed according to the enclosed instructions of the commercial test kit .For OTA quantification, the competitive enzyme immunoassay kit RIDASCREEN Ochratoxin A 30/15, R1312  was used following the manufacturer’s instructions.FOR ZEN determination, the RIDASCREEN Zearalenon (Art. Nr.: R1401) kit was applied, following the instructions of the manufacturer.−1; 0, 0.03, 0.1, 0.3, 1, and 3 μg L−1; and 0, 0.05, 0.15, 0.45, 1.35, and 4.05 μg L−1 respectively. Mycotoxin quantification was achieved using the software RIDASOFT.Win.net. The calculation performed in double determinations used a cubic spline function. According to the manufacturer, the limit of detection of the enzyme immunoassays was 1 μg kg−1 for AFB1, 0.8 μg kg−1 for OTA, and 1.75 μg kg−1 for ZEN.The standard curves in the enzyme immunoassays, AFB1, OTA, and ZEN were obtained with the mean values of each of the six duplicated concentration levels: 0, 1, 5, 10, 20, and 50 μg L3 prior to analysis by inductively coupled plasma mass spectrometry (ICP-MS) XSERIES-2, ThermoUnican, with peak jumping acquisition mode, at 1370 W (m/z 75), using the following conditions: number of main runs—5; extraction lens potential—−106 V; RF forward power—1370 V; dwell time—0.02 s; sampling depth—108 mm; nebulizer gas—0.87 L min−1.For InAs analysis, 5 g of each minced sample was vortexed and digested with 50 mL of 1% nitric acid (1%) for 10 min, followed by ultrasound extraction for 15 min and centrifugation at 2880 g for 15 min. After filtration of the supernatant with a 0.45 μm filter, the extracts were diluted with 0.5% HNORegarding the AFB1 standard curve, the mean coefficient of variation (CV) was 2%, with the highest value being 7%. As for OTA, the mean CV was 2.5%, with the highest value being 6%. For ZEN, an average CV of 5.2% was obtained, with a maximum of 6.3% and a minimum of 2.6%.−1, respectively, it is perceived that the LODs of the ELISA’s applied methodologies are adequate [Considering the AFB1, OTA, and ZEN maximum permitted levels in cereals for human consumption, 2, 3 and 75 μg kgadequate ,18.−1 of arsenic prepared in 0.5% HNO3. An internal standard of scandium (Sc) (m/z 45) was used at 25 μg L−1. The correlation coefficient obtained was 0.9999.The calibration curve was performed with the following standards: 0.2, 0.4, 0.8, 1.0, 2.0, and 5.0 μg L−1 and 10 μg kg−1, respectively [The LOD and LOQ were determined by taking 3.3 and 10 times the standard deviation, respectively, plus the mean obtained for 10 blanks. The LOD and the LOQ obtained were 3.3 μg kgectively .Accuracy, calculated using 5 recovery assays, was 99%, 114%, 106%, 111% and 89%, with a precision of 10.8% .−1 and a LOQ of 38 μg kg−1, with an accuracy of 108.7% [−1, and a recovery of 91.8% [Compared with validation results reported by other ICP-MS studies in rice, the currently applied methodology satisfies. Other authors using similar methodologies obtained a LOD of 11 μg kgf 108.7% , a LOQ oof 91.8%  . To assess if the datasets were Gaussian-distributed, D’Agostino–Pearson normality, Shapiro–Wilk and KS normality tests were applied. Given that the datasets were not normally distributed, non-parametric tests were used. To compare InAs concentrations in rice of different origins, the Kruskal–Wallis test with Dunn’s post-test was applied. For other comparisons, the Mann–Whitney test was used. The statistical significance level was established at −1), C is the mean annual intake estimated per person, N is the number of analysed samples, D corresponds to the number of days in one year, and K stands for the body weight (kg).The estimated daily intake (EDI) was calculated through a deterministic method  using the equation:According to the last report of the “National Food Survey and Physical Activity, IAN-AF 2015–2016”, the adult population averaged 25.1 kg of rice ingested annually per capita. Regarding children and adolescents, the annual ingestion was 19.1 and 28.3 kg/year, respectively. Concerning the 95th percentile, the annual ingestion was 47.9, 62.8, and 59.0 kg/year for children, adolescents, and adults, respectively . For intThe mean body weight taken into account for the Portuguese adult population was 69 kg . For chi−1 b.w./day for adults and children without hepatitis B and 0.4 ng kg−1 b.w./day for those with the hepatitis B virus since this is a genotoxic carcinogenic compound and, therefore, no official TDI is set. As such, the AFB1 risk was also characterized by calculating the margin of exposure (MOE) approach. The MOE corresponds to the ratio between the lower benchmark dose (BMDL) for the critical effect and the exposure dose. An increased MOE implies a smaller risk, and a value below 10,000 indicates a concern for human health [−1 b.w./day, set by EFSA, was applied for calculating the MOE MOE [For AFB1 exposure estimation, and given that the percentage of censored data (results reported under LOD) was above 50%, two different scenarios, as reported by the EFSA, were considered: (1) the lower-bound (LB) approach, implemented by replacing the results below the LOD with zero, and (2) the upper-bound (UB) approach, implemented by replacing the results below the LOD with the LOD value . The AFBn health . The BMD MOE MOE .−1 b.w./day [Regarding ZEN, two exposure scenarios were considered. In the first one, the average concentration (AC) of ZEN in rice was considered. In the second, the worst scenario was verified using the highest concentration (HC) found. The risk was calculated, in percentage, through the ratio between the obtained EDI and the current tolerable daily intake (TDI) of 0.25 µg kgb.w./day . The obt−1 b.w./day for a higher risk of cancer of the lungs, skin and bladder, as well as skin lesions [Concerning InAs risk assessment, the EFSA Panel on Contaminants in the Food Chain established a BMDL01 ranging between 0.3 and 8 µg kg lesions . The MOE lesions . Risk as


Bovine enterovirus (BEV), bovine coronavirus (BCoV), and bovine rotavirus (BRV) are still the major worldwide concerns in the health care of cattle, causing serious economic losses in the livestock industry. It is urgent to establish specific and sensitive methods to detect viruses for the early control of diseases. Droplet digital PCR (ddPCR) has been proposed to effectively detect viral particles, and it does not involve Ct values or standard curves. In this study, we designed specific primers and probes, based on conserved regions of viral genomes, to optimize protocols for a dual ddPCR assay for detecting BCoV and BRV and a multiplex ddPCR assay for BEV, BCoV, and BRV. Sensitivity assays revealed that the lower limit of detection for qPCR was 1,000 copies/μL and for ddPCR for BEV, BCoV, and BRV, 2.7 copies/μL, 1 copy/μL and 2.4 copies/μL, respectively. Studying 82 samples collected from diarrheal calves on a farm, our dual ddPCR method detected BCoV, BRV, and co-infection at rates of 18.29%, 14.63%, and 6.1%, respectively. In contrast, conventional qPCR methods detected BCoV, BRV, and co-infection at rates of 10.98%, 12.2%, and 3.66%, respectively. On the other hand, studying 68 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV at rates of 14.49%, 1.45%, 5.80%, and 1.45%, respectively. Our multiplex ddPCR method detected BCoV, BRV, BEV, co-infection of BCoV and BEV, and co-infection of BRV and BEV. at rates of 14.49%, 2.9%, 8.7%, 2.9%, and 1.45%, respectively. Studying 93 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV was detected at rates of 5.38%, 1.08%, 18.28%, and 1.08%, respectively. Co-infection of BCoV, BRV, BEV, BCoV, and BEV, and co-infection of BRV and BEV, were detected by multiplex ddPCR methods at rates of 5.38%, 2.15%, 20.45%, 1.08%, and 1.08%, respectively. These results indicated that our optimized dual and multiplex ddPCR methods were more effective than conventional qPCR assays to detect these viral infections. BEV In 1985, Mullis and Faloona  reported2.2.1.Samples were obtained from 82 diarrheic calves from a cattle farm in Ürümqi, Xinjiang, China; 68 anal subsamples were obtained from diarrheic cattle from a cattle farm in Bole, Xinjiang, China; and 93 anal formulae were collected from cattle in cattle farms in Ürümqi County, Xinjiang, China.TRIzol was purchased from Invitrogeng; 2× Taq PCR Mix, Common Agarose Gel DNA Recovery Kit (DP209), DH5α receptor cells, Rapid Plasmid Small Extraction Kit, and SuperReal Fluorescence Quantitative Premix Reagent (TaqMan) were purchased from Tiangen Biochemical Technology Co., Ltd. ; RT EasyTM II Reverse Transcription Kit was purchased from FIC Biotechnology Co., Ltd. ; T-Vector pMD19 vector kit was purchased from TaKaRa; 2× ddPCR SuperMix for Probes was purchased from Bio-Rad.2.2.pro, and the BRV-VP6 gene, the sequences were sent to Sangon Bioengineering (Shanghai) Co., Ltd. BEV, BCoV, and BRV PCR were cloned into pMD18-T, pGEM-T Easy Vector, and pUC18 vectors (purchased from ADDGENG), sequenced for validation, and mapped , and BRV-VP6 (GenBank No. AB573082.1) genes for qPCR and ddPCR reactions  software was used to design specific primers and probes for the conserved regions of BEV E (GenBank No. MN607031.1), BCoV Neactions . The pro2.4.6 copies/μL were selected as templates, and other conditions were controlled constants. qPCR reactions were performed at the upstream and downstream primer concentrations, selected as 200, 300, 400, 500, 600, 700, 800, and 900 nM concentration gradients, to screen for the best concentration of primers to use. The qPCR reactions were performed when the final concentrations of probes were set to 150, 200, 250, 300, 350, 400, and 450 nM, respectively, to determine the optimal probe concentrations for BEV, BCoV, and BRV. The initial concentrations of 1 × 1010 copies/μL of BEV, BCoV, and BRV plasmid standards were diluted in a 10-fold gradient (1 × 109–1 × 102 copies/μL), qPCR reactions were performed, standard curves were constructed, and sensitivity, specificity, and reproducibility tests were performed. The PCR program was 94°C for 3 min, 94°C for 7 s, 60°C for 30 s, and 40 cycles.According to the SuperReal Pre Mix (TaqMan) kit instructions, plasmid standards of BEV, BCoV, and BRV at a concentration of 1 × 102.5.9–1 × 102 copies/μL), and a negative control was set up; each concentration was repeated three times. qPCR reactions were performed using the optimized reaction conditions to determine the sensitivity of the method.BEV, BCoV, and BRV were diluted in a 10-fold gradient  of Ct values in intra- and inter-batch replicate experiments was calculated using the formula CV = standard deviation/mean, and the CV was used to respond to reproducibility.The optimized method was used to perform qPCR reactions using 1 × 102.8.The ddPCR reaction was performed using the QX200 ddPCR system of the Bio-Rad Company, program was 94°C for 10 min, 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 40 cycles, 98°C for 10 min, and 4°C for storage. The reaction system was configured according to the ddPCR SuperMix for Probes kit (Bio-Rad) to ensure complete mixing and no air bubbles. The droplets were placed into the reader for low signal readings. Sensitivity, specificity, and repeatability experiments were carried out.2.9.2O was supplemented to 20 μL, and the reaction conditions were the same as above.According to the ddPCR workflow and method, primers and probes for BCoV and BRV were used. The FAM channel detected the target BCoV, and the HEX channel detected the target BRV. The duplex ddPCR reaction system was: 2× ddPCR SuperMix for Probes 10 μL, two pairs of upper, downstream primer concentrations were 450 nM each, probe concentrations were each 250 nM, the template was 2 μL, ddH2.10.The annealing temperature was set to 58°C, the primers were all 500 nM, and four groups of different probe concentrations were set. The conditions of the reactions were optimized. The specific probe sequences and concentrations are shown in 2.11.Stool samples were added to three times the volume of sterilized phosphate-buffered saline (PBS), vortexed and mixed, and centrifuged at 10,000 g for 10 min at 4°C. The supernatant was collected, penicillin-streptomycin liquid was added and left for 4 h in a refrigerator at 4°C, and the virus treatment solution was obtained by filtration using a 0.22 μm filter. The total RNA of the samples was extracted using TRIzol (Invitrogen), the concentration of total RNA was determined, and the total RNA was reverse transcribed to cDNA using a reverse transcription kit.2.12.The 82 fresh samples collected from diarrheal calves from the cattle farm in Ürümqi were added to the sterilized PBS, vortexed, and mixed. The extracted RNA was reverse transcribed into cDNA. These samples were then tested using the constructed qPCR and duplex ddPCR methods. The 68 fresh samples collected from diarrheal calves from the cattle farm in Bole were treated with sterilized PBS, vortexed, and mixed, and RNA was extracted and reverse transcribed into cDNA. These samples were then examined using established qPCR and multiplex ddPCR methods.3.3.1.y = −0.3295x + 42.281; the correlation coefficient (R2) of the standard curve was 0.9996, and the amplification efficiency was 99.68% ; Autonomous Region Major Science and Technology Project (grant number: 2020A01001-2); National Natural Science Foundation of China (grant number: 32260881); National Student Innovation Project (grant number: 202110758005), Autonomous Region Postgraduate Innovation Project (grant number: XJ2022G131).DL was employed by Tecon Biology Co., Ltd.The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.