These physical and microstructural changes of the inherent starch promoted the shift in gelatinization temperatures to a higher temperature and reduced the gelatinization enthalpy. The study demonstrated that PEF can be utilized to change the starch fraction of cassava flour, which is driven by electric field strength and specific energy input, causing changes in the starch-related properties leading to increased digestibility.The research aimed to investigate the effect of pulsed electric field (PEF) treatment on cassava flour at mild intensities  combined with elevated levels of specific energy input (250–500 kJ/kg). Influences on starch digestibility, morphological characteristics, birefringence, short-range order and thermal properties were evaluated. Application of PEF at energy input no greater than 250 kJ/kg had negligible influence on the different starch digestion fractions of cassava flour but raised the rapidly digestible starch fraction at a combined electric field strength >1 kV/cm and energy input >350 kJ/kg. Morphological evaluation revealed that at this PEF combination, cassava starch’s external structure was consistently altered with swelling and disintegration, albeit some granules remained intact. Consequently, this led to disruption in the internal crystalline structure, supported by progressive loss of birefringence and significantly lower absorbance ratio at 1047/1022 cm Pulsed electric field processing is an emerging technology that involves the application of high-intensity electric pulses for a short duration (nano to microseconds) inside a treatment chamber confined between electrodes ,2. A sigPEF also has the potential to modify the microstructural and functional properties of biomacromolecules, such as starches . PhysicaThere have been recent attempts to utilize PEF in starch-rich matrices and to understand their impact on inherent starch, e.g., potato , black bCassava, for example, is a highly valued agricultural crop in the developing countries of Asia, South America, and Africa. Compared to other crops such as rice, corn, and sorghum, cassava produces more energy per hectare . Its undTo have a better understanding of the effect of PEF on cassava flour, key processing parameters previously identified to have a dominant influence on starch properties, electric field strength (EFS) and specific energy input (SEI) ,28, wereAspergillus oryzae α-amylase (30 U/mg) and porcine pancreas pancreatin  was manufactured by Sigma, St. Louis, MO, USA. Porcine stomach pepsin  was from AppliChem, Barcelona, Spain and porcine bile extract (SC-214601) from ChemCruz, Dallas, TX, USA. The GOPOD reagent and total starch kit was purchased from Megazyme, Wicklow, Ireland.Flour from Philippine cassava cultivar NSIC Cv-44 was kindly provided by PhilRootcrops of the Visayas State University, Leyte, Philippines, and had a starch content of 85.83%. ® HVP 5 PEF system  using a batch configuration. The chamber consisted of two parallel stainless-steel electrodes with a 40 mm gap. Square wave pulses were supplied at a constant pulse width of 20 μs with a 100 Hz pulse frequency. This was monitored using an oscilloscope  during each treatment.PEF treatment was carried out using the ELCRACKThree electric field strengths  combined with four levels of specific energy inputs  were selected for this study. These combinations induced notable progressive physical changes (turbidity and viscosity) with no or minimal electrical arcing based on preliminary trials. The pulse count and energy delivered by the system was used to calculate the total specific energy input according to Alpos et al.  using Eqw/w) flour suspension. The temperature of the suspension was kept between 8–15 °C to ensure actual pulse energy was maintained especially at higher specific energy input requirements. To guarantee a similar level of water imbibition, a delay of 3 min was enforced from water addition to flour, mixing and start of PEF treatment [Flour suspension was prepared by weighing 1.05 g of cassava flour into the PEF chamber and then 33.95 g of cold distilled water was added to make a 3%  NaCl and 2.1 g KCL (28 mM) in 1 mM HCl (pH 3). To a 100 mL of gastric solution, 4 g porcine stomach pepsin was added to make the “gastric juice”. The “intestinal juice” was prepared by adding 1 g of porcine pancreas pancreatin and 0.8452 g of porcine bile extract into 100 mL of 0.1 M NaHCO3 (pH 7). All solutions with enzymes were prepared on the day of the assay and kept chilled until use. Additionally, 1 M HCl and 1 M NaOH were also prepared.The solutions were prepared according to the work of Abduh et al. . “SalivaFor stage 1 or the oral phase, the hydrated flour samples were mixed with 8 mL saliva juice and incubated at 37 °C  for 5 min on a shaker  with a rocking motion (55 strokes/min). Afterwards, 2 mL of α-amylase solution was added and incubated for another 5 min. Then, the pH of the solution was adjusted to 3 with 1 M HCl to deactivate the amylase. In stage 2 or the gastric phase, the acidic mixture was added with 8 mL gastric juice and incubated for 120 min with rocking. Aliquots of 0.5 mL were collected at 0, 60, and 120 min, with immediate heat shocking in boiling water for 10 min for enzyme deactivation. Then, pH was adjusted to 7 with 1 M NaOH to deactivate the pepsin. For stage 3 or the intestinal phase, 16 mL of intestinal juice was added into the neutralized digest and incubated with rocking. Aliquots of 0.5 mL were again collected at 0, 20, 40, 60, 90, 120, 180, and 240 min, followed by immediate heat shocking. All the collected digests were added with 2.5 mL of 100 mM sodium acetate buffer (pH 5), vortexed, and stored at 4 °C until glucose analysis which was conducted within 24 h.g  for 20 min at 20 °C. Supernatant aliquots of 50 μL, which contain all the glucose released during enzymatic hydrolysis, were transferred into microtubes. To this, 1.5 mL of GOPOD reagent was added, mixed, and microtubes were incubated in a water bath  at 50 °C for 20 min. The absorbance of the mixture was measured using a microplate reader at 510 nm  and the glucose produced was calculated against a 100 mM sodium acetate buffer (pH 5) blank and a glucose standard . Starch digestibility was assessed through the determination of the percentage of rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) produced. These were calculated according to Equations (2)–(4):20G and 120G is the amount of glucose released during intestinal starch hydrolysis at 20 min and 120 min, respectively; FG is the amount of inherent free glucose in the flour; 0.9 is an adjustment to convert glucose to anhydroglucose (as occurs in starch); and TS is the total starch content of the flour [The diluted digests were centrifuged at 2056× he flour . The iniC is the amount glucose released at digestion time t, 0C is the amount of glucose at the start of the small intestinal phase (0 min), and k is the rate constant of starch digestion through glucose release (min−1). The model fitting and estimation of kinetic parameter k was estimated using R software (v.4.0.4 2021) and R Studio . The goodness of fit of the kinetic models were evaluated by their R2 value and through residual (random distribution of error) and parity plots.To estimate the rate of starch digestion through the amount of glucose released in the whole digest per gram (dry basis) of flour during the small intestinal phase, a linear regression model was used (Equation (5)) :(5)C=k×w/v) with ultrapure water, transferred to glass slides, and viewed under the Olympus BX41-P microscope  at 400× magnification. The micrographs were taken by an attached Canon EOS 1100D camera, while polarized images were captured through a rotatable polarizer .Flour suspensions were diluted to 0.05% . Then, the spectral region of 1200–875 cm−1, associated with the short-range ordered structure of starch, was deconvoluted based on the second-order derivative peak identification [−1 was used to quantify the ordered and amorphous structure of the flour’s starch [The FTIR spectra of cassava flour samples were obtained by using a Bruker Optics FTIR Spectrometer  with an ATR platinum diamond one accessory. Cleaning of ATR crystal with isopropanol-soaked delicate wipes and background scanning were performed prior to each sample scan. The spectrum was scanned from 400 to 4000 cmfication ,32 usings starch ,34,35, ws starch ,36.w/v) and allowed to stand for at least 2 h at room temperature prior to analysis. Using the TA Instruments Q2000 differential scanning calorimeter , samples were equilibrated at 25 °C for 1 min then heated to 130 °C at a 5 °C/min rate with an empty sealed pan as a reference and a nitrogen flow of 50 mL/min. The transitional thermal properties were determined using the TA Instruments Universal Analysis 2000 Version 4.5A software ; onset, peak, conclusion temperatures, and gelatinization enthalpy (ΔH). The temperature range of gelatinization (Trange) was calculated using Equation (6) [In a hermetically sealed aluminum pan, approximately 5 mg of flour was dispersed with distilled water (1:3 tion (6) , while tResults are presented as mean value from 3 replicates ± standard deviation. One-way ANOVA was performed to compare the means of the studied parameters for the untreated and treated flours at a 5% significance level using SPSS version 28 , while Tukey’s HSD was used for post hoc analysis. If assumptions were not met, appropriate non-parametric alternative tests were used, i.e., Welch’s ANOVA with Games–Howell post hoc tests.The actual electric field strength and total specific energy input supplied by the PEF system are shown in ϕ 0.25 mm) allows almost complete dispersion of ions when prepared as a suspension in the water prior to PEF treatment.The conductivity increase after PEF treatment is mainly due to membrane electroporation and ion diffusion into the medium , but thek) of the control and PEF-treated cassava flours was estimated  at the small intestinal phase and SDS fraction. The effect of PEF at mild intensity combined with elevated levels of specific energy input on the in vitro digestibility of the starch will help explore suitable application for PEF-treated cassava flour and the probability of creating novel products with tailored digestibility. At present, this is the first report on starch digestibility of PEF-treated cassava flour.Overall, the present study showed that PEF treatment enhanced the susceptibility of inherent starch in cassava flour to digestive enzymes, producing more glucose than native starch when digested but without affecting the rate of starch digestion  and a wide range of sizes . There wA few of the highly swollen starches showed granular disintegration. These aggregated with presumably hydrated non-starch components, thereby trapping other non-gelatinized or partially gelatinized starches . The conPolarized images revealed a Maltese cross pattern on the granule, as starch is birefringent owing to the natural assembly of amylose and amylopectin in the form of semi-crystalline granules . Figure PEF 2 at 500 kJ/kg and 4 at 450–500 kJ/kg had a significantly lower 1047/1022 value than the control sample, while other treatments were not different from the control . The higWhereas no significant difference was found for 1022/995 absorbance ratio. Notably, PEF 2 and 4 at 500 kJ/kg had the highest average value. For 1022/995, a higher number means lower molecular order of starch double helices . This maIn general, there was a shift to higher gelatinization temperatures and reduction in gelatinization enthalpy with the application of PEF at 2–4 kV/cm and at an elevated SEI . PEF 4 aNonetheless, a decrease in the gelatinization enthalpy (ΔH) with increasing EFS as observed in this study agrees with previous work on PEF-treated pure starches ,12,28,41The effect on ΔH corroborates with the degree of gelatinization result, wherein PEF 4 at 500 kJ/kg > PEF 2 at 500 kJ/kg > PEF 2 at 450 kJ/kg and PEF 4 at 450 kJ/kg > other treatment combinations. In summary, the results showed that the microstructural integrity of cassava starch was reduced from SEI 450 kJ/kg and upwards, with increasing impact at higher EFS. This was supported by the polarized images showing loss of birefringence, reduction in the 1047/1022 absorbance ratio value, and decreasing ΔH. However, at a lower EFS of 1 kV/cm, increasing the SEI up to 500 kJ/kg was unable to induce changes in the flour or in its inherent starch. Further study is necessary to study the critical SEI that could promote a shift in thermal properties at this intensity.In conclusion, the findings from this study demonstrated that PEF treatment enhanced the starch digestibility of cassava flour. In particular, an increase in the RDS, decrease in RS, and no changes in the SDS fraction and rate of starch digestion in the small intestinal phase can be achieved with mild electric field strength but at high-energy input for cassava flour. Detailed investigation has shown that PEF treatment caused morphological damage and transformed the short-range structure of inherent cassava starch to a more amorphous one through gelatinization; thereby shifting the gelatinization temperatures to higher temperatures and reducing the enthalpy change. At mild intensities (2–4 kV/cm), PEF was able to induce changes in the flour’s starch properties, which were formerly achieved at higher electric field strengths for pure starches (25–50 kV/cm) but given sufficient specific energy input is applied (≥450 kJ/kg). This study further establishes that PEF can be utilized as a nonthermal and clean technology to change cassava flour’s starch digestibility and its related properties. However, potential new applications delivered by PEF require a detailed cost–benefit analysis to be conducted before recommending its adoption at commercial scale. The difference in magnitude of PEF impact on flour from previous reports further supports the claim that electric field strength and specific energy input are important processing factors to report in order to achieve reproducible results, along with inherent sample factors and other processing conditions. Further research on comparing flour and its isolated starch would be helpful to decrease this information gap.


Aortoenteric fistula is a rare condition. Atypical presentations may cause significant management delays. We present the case of a 64-year-old male who experienced a pathological femoral fracture as an initial presentation of an underlying aortoenteric fistula. The aortoenteric fistula, possibly related to a poor graft tunneling technique, induced femur osteomyelitis and the associated pathological fracture. Aortoenteric fistula is a rare condition. It presents with the high complexity of management and a high mortality rate.A 64-year-old male patient underwent an aortobifemoral bypass, using a polyethylene terephthalate (PET) graft, in another institution owing to lifestyle limiting claudication.Seven months after his primary aortic surgery, a diaphyseal femoral fracture occurred while he was resting. Given the absence of trauma or stress, the fracture was considered pathological.To treat his femoral fracture, external osteosynthesis was performed along with extensive purification and drainage because of intraoperative purulent exudation around his femur .Fusobacterium nucleatumandParvimonas micra).The intraoperative periosteal inflammatory tissue culture revealed two strains of oral gram-positive anaerobic cocci  scan was performed but was reported without any pathological findings regarding abdominal or other system infection.Three months after orthopedic surgery, while being afebrile, his external osteosynthesis was removed. After repeating a debridement, antibiotic-impregnated cement and a distal femur plating system were introduced .Escherichia coli,Enterococcus faecalis, andProteus mirabilis.Despite initial improvement, a localized mass appeared in his left popliteal fossa in the following weeks. The mass was irrigated and a culture isolatedOwing to fever along with extensive edema in his left thigh and elevated inflammatory blood markers ], the patient was readmitted to the orthopaedic service with a diagnosis of osteomyelitis. Radiographic images confirmed the diagnosis; however, blood cultures were negative. Despite an extended antibiotic regimen, the swelling in his left popliteal fossa relapsed. This finding was accompanied by an additional palpable mass in his left inguinal ligament. He was then referred to our vascular surgery department.The patient underwent a new CT angiography scan that revealed a thrombosed left limb of his aortobifemoral PET graft, with the impression that the thrombosed segment, as well as the right limb of the aortic graft, took an intraluminal course at the proximal end level of his sigmoid colon and cecum, respectively . MoreovInflammatory markers normalized . A course of intravenous antibiotics (ceftazidime/avibactam and tigecycline) was instituted, and a transperitoneal approach exposed the aortic graft under general anesthesia. In addition to excessive fluid accumulation around the aortic graft, it was found that the right and left iliac limbs of his PET graft transversed the cecum and sigmoid colon, respectively.Following total explantation of his aortobifemoral graft, accompanied by wide debridement and retroperitoneal irrigation, an appendectomy and sigmoidectomy were performed.Considering the extensive inflammation in his left inguinal area, and the absence of ischemic symptoms despite the thrombosed left limb of the PET graft, we proceeded with a bailout procedure by creating a composite graft consisting of his harvested right femoral vein and a short segment of tubular silver-impregnated PET graft. The length of the autologous graft alone was inadequate . The tuDespite initial postoperative improvement, sigmoid colon necrosis was observed on day 3 and Hartmann's colectomy procedure was thus performed. Owing to the freedom of the aortofemoral bypass from the above pathology, no reintervention to the graft was attempted. Eventually, the patient died of sepsis because of peritonitis after a prolonged stay (16 days) in the intensive care unit.Aortic graft infection is a rare condition, with a reported incidence of 1 to 4%, while aortoenteric fistula has an incidence of 0.4 to 2%.The clinical presentation of aortic graft infection varies, depending on the time of symptom onset after the primary aortic surgery.Conversely, in late-onset cases, symptoms are manifested from 4 months up to 10 years after the primary aortic surgery and are associated with less virulent but more fastidious microorganisms. In these cases, patients complain of nonspecific symptoms such as malaise, fatigue, weight loss, intermittent fever, intermittent claudication, back pain, localized inguinal mass, and gastrointestinal bleeding when aortoenteric fistula is also present.In our case, the malposition of the iliac limbs of the graft tangentially with the cecum and sigmoid colon gave rise to the aortoenteric fistula during the tunneling.Based on the literature, there have already been reports of iatrogenic perforation of the colon during tunneling of an aortobifemoral graft; however, our patient exhibited a unique presentation.In conclusion, poor graft tunneling technique caused an aortoenteric fistula that induced femoral osteomyelitis and an associated femoral fracture. Regarding the underlying pathology, aortic graft infection may exhibit an unusual presentation; therefore, vigilance regarding aortic surgery complications is crucial.


Estimating liver function reserve is essential for preoperative surgical planning and predicting post-hepatectomy complications in patients with hepatocellular carcinoma (HCC). We investigated hepatic viscoelasticity quantified by tomoelastography, a multifrequency magnetic resonance elastography technique, to predict liver function reserve.c, m/s), which represents stiffness, and loss angle , which represents fluidity. Both were correlated with the ICG-R15. A prediction model based on logistic regression for major hepatectomy tolerance (ICG-R15 ≥ 14%) was established.One hundred fifty-six patients with suspected HCC  underwent preoperative tomoelastography examination between July 2020 and August 2021. Sixty-nine were included in the final analysis, and their 15-min indocyanine green retention rates (ICG-R15s) were obtained to determine liver function reserve. Tomoelastography quantified the shear wave speed (n = 50) or ICG-R15 ≥ 14% (n = 19) group. Liver c (r = 0.617) and φ (r = 0.517) were positively correlated with the ICG-R15 (both p < 0.001). At fibrosis stages F1–2, φ was positively correlated with the ICG-R15 , but c was not (p = 0.104). At stages F3–4, c  and φ  were both positively correlated with the ICG-R15. The optimal cutoffs of c and φ for predicting ICG-R15 ≥ 14% were 2.04 m/s and 0.79 rad, respectively. The area under the receiver operating characteristic curve was higher for c (0.892) than for φ .Patients were assigned to either the ICG-R15 < 14% (Liver stiffness and fluidity, quantified by tomoelastography, were correlated with liver function and may be used clinically to noninvasively assess liver function reserve and stratify treatments.The online version contains supplementary material available at 10.1186/s13244-022-01232-5. Hepatic biomechanical properties were sensitive to liver function reserve.Liver stiffness and fluidity quantified by tomoelastography could be potential biomarkers for liver function assessments.Liver fluidity showed unique sensitivity to liver function reserve at early fibrosis (F1–2) stage.Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor and ranked as the third leading cause of cancer death worldwide . HepatecChild–Pugh classification is the most commonly used method in clinical practice to evaluate liver function and enables partial evaluation of the surgical risk . Some inWith the development of imaging technology, several recent studies have used ultrasound elastography to measure liver function reserve. Liver stiffness enables quantifying liver fibrosis of mixed etiologies to various degrees , 13. As Magnetic resonance elastography (MRE) is a noninvasive imaging technique for quantifying the biomechanical properties of tissue , 21. MREc, m/s) and loss angle of the complex shear modulus , which are surrogate indicators of stiffness and viscosity (or fluidity), respectively, through postprocessing. Tomoelastography has been applied for the biomechanical characterization of a variety of diseases in vivo, including pancreatic diseases [Tomoelastography, an advanced multifrequency MRE technique, is an emerging noninvasive imaging modality used to characterize biomechanical properties of the tissue. With multifrequency data acquisition and wavenumber-based inversion method, the parameter maps provided by tomoelastography can reveal rich anatomical details . Tomoeladiseases , 36, neudiseases , prostatdiseases , 38, recdiseases , inflammdiseases , liver tdiseases , 40, anddiseases , 42. Becdiseases .Thus, in this study, we aimed to (1) investigate correlations between the liver mechanical properties derived from tomoelastography and liver function reserve evaluated by the ICG test and (2) develop a tomoelastography prediction model of liver function reserve for patients with a low tolerance for major liver resection.n = 50; mean age, 58 ± 10 years; 45 men, 5 women) or ICG-R15 ≥ 14%  group , and all participants provided written informed consent. From July 2020 to August 2021, 156 patients with radiologically suspected HCC, who underwent preoperative tomoelastography, were enrolled in this prospective study. The exclusion criteria were (a) lack of ICG results, (b) iron deposition, and (c) suboptimal image quality on tomoelastography. Eighty-seven patients were excluded, and 69 were included in the final analysis . Based on their ICG-R15 values, the final 69 patients were categorized into either the ICG-R15 < 14%  and 3 × 3 × 5 mm3 resolution were acquired during free breathing. Additional imaging parameters included echo time (ET) = 59 ms; repetition time (TR) = 2050 ms; parallel imaging with GRAPPA factor 2; MEG frequencies of 43.48 Hz for the 30, 40- and 50 Hz vibration frequencies and 44.88 Hz for the 60 Hz vibration frequency; and a MEG amplitude of 30 mT/m. The total acquisition time was approximately 3.5 min.Tomoelastography examinations were performed with a 1.5T scanner . The setup was similar to that described in Shahryari et al. . Brieflyhttps://bioqic-apps.com. Full FoV high spatial resolution maps of shear wave speed (c) and phase angle (φ) were generated. Because c is proportional to the square root of the storage modulus and φ continuously changes from 0 (solid properties) to π/2 (viscous properties), they are considered surrogates for stiffness and fluidity, respectively. Throughout the text, we use c and φ to provide quantitative information and “stiffness” and “viscosity” (or “fluidity”) to discuss qualitative changes.Multifrequency wave field data were processed using the processing pipeline available at c- and φ-map slices. The measurements were averaged and used as the representative parameters. Two radiologists (rater #1 with 8 years of experience and rater #2 with 1 year of experience) analyzed the tomoelastography data independently for all patients to test interobserver variability.Regions of interest (ROIs) were manually drawn using tomoelastography magnitude images to encompass as much of the background liver as possible on three consecutive sections with the largest liver cross-sectional coverage on the central The ICG clearance test was performed before surgery and within 1 week of tomoelastography scanning. After fasting for 6 h, ICG was injected through a peripheral venous access at 0.5 mg/kg body weight, and the injection was completed within 10 s. ICG-R15s were registered via a digital pulse densitometer, which was connected to a DDG-3300 K device  through a near-infrared finger piece sensor. ICG-R15 values < 14% were considered safe for major resection , and we Liver tissue samples were obtained via surgical resection. Liver fibrosis grades were analyzed by a pathologist with 10 years of experience in hepatic pathology, who was blinded to all radiological and clinical results.U test were used for quantitative data. Interobserver agreement was analyzed for biomechanical parameters by using intraclass correlation coefficients (ICCs). Pearson rank correlation was performed to analyze the relationships between normally distributed variables; Spearman correlation was used for non-normally distributed variables. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic efficacy of the parameters, and area under the ROC curves (AUROCs) was calculated with 95% confidence intervals (CIs). All statistical analyses were performed with SPSS software , GraphPad Prism software , and MedCalc software . Two-tailed p values < 0.05 were considered statistically significant.The chi-square test was used to compare qualitative parameters between two groups. Student’s t test or the Mann–Whitney p = 0.007), lower PAB (p < 0.001) and PLT (p = 0.002) levels, and increased PT (p < 0.001) and INR (p < 0.001) levels. Liver c  and φ  values differed significantly between the ICG-R15 < 14% and ICG-R15 ≥ 14% groups. No other demographic or laboratory results differed significantly between the groups.We included 69 patients with HCC in our study. Table Sixty-eight patients were categorized as Child–Pugh class A, of whom 47 had a score of 5 and 21 had a score of 6. The remaining patient, who had a score of 8, was categorized as class B owing to their biochemical data and clinical symptoms. In the Child–Pugh class A patients, the ICG-R15s ranged from 1.3 to 48%; the patient in class B had an ICG-R15 of 20.7% (Table c and φ were significantly higher in participants with higher fibrosis grades of F3–4 than in those with liver fibrosis grades of F1–2 (Table p = 0.021) and PLT (p < 0.001) values and increased PT (p = 0.042) and INR (p = 0.039) values.ICG-R15, liver c and 0.949 (95% CI 0.920–0.968) for φ, suggesting good concordance and data consistency  for c and φ (Table c (r = 0.617) and φ (r = 0.517) were positively correlated with the ICG-R15  was positively correlated with the ICG-R15, but liver c (p = 0.104) was not. In liver fibrosis stages F3–4, liver c  and liver φ  were positively correlated with the ICG-R15. Figure c and φ maps for three patients with their corresponding ICG-R15 data and fibrosis stages.Blood samples were taken from all patients. TB, AST, PT, and INR were positively correlated, and PLT, ALB, and PAB were negatively correlated with both liver  φ Table . Liver c p = 0.10 was not.c and φ cutoff values for predicting the ICG-R15 ≥ 14% group were 2.04 m/s and 0.79 rad, respectively. The AUROC was higher for c than for φ . However, combining c and φ did not significantly increase the AUROC compared with that of c alone  stage which might be associated with inflammation. Liver stiffness and fluidity may be potential biomarkers for noninvasively assessing liver function to allow making informed treatment decisions. Future studies in large patient cohort with postoperative follow-ups are warranted.Additional file 1. Table S1. Characteristics of the related works. AUROC: area under receiver operating curve, TE: transient elastography, HCC: hepatocellular carcinoma, LS: liver stiffness, ICG-R15: 15-min indocyanine green retention rate, ARFI: acoustic radiation force impulse, MRE: magnetic resonance elastography, HBV: hepatitis B virus.


The sweeteners were sucrose, xylose, ribose, glucose, galactose, fructose, mannose, mannitol, L-sorbose, xylitol, tagatose, allulose, maltose, lactose, isomaltulose, isomalt, sorbitol, maltitol, and raffinose. Retrogradation rates and amounts were compared by Avrami equation rate constants (k = 0.1–0.7) and absorbance values measured on day 28 (Abs = 0.1–1.0), respectively. Both sweetener concentration and type significantly affected retrogradation. Gels made with sugar alcohols and high sweetener concentrations (≈≥40%) tended to retrograde more and faster, whereas gels made with sugars and low sweetener concentrations tended to have lower retrogradation rates and amounts. Sweeteners with more equatorial and exocyclic hydroxyl groups  and those with larger molar volumes  tended to increase the rate and amount of retrogradation, particularly at higher concentrations. The impact of sweeteners on retrogradation was a balance of factors that promoted retrogradation  and inhibiting behaviors , which are influenced by sweetener concentration and structure. Understanding which sweeteners at which concentrations can be used to promote or inhibit retrogradation is useful for product formulation strategies.Starch retrogradation is desirable for some food textures and nutritional traits but detrimental to sensory and storage qualities of other foods. The objective of this study was to determine the impact of sweetener structure and concentration on the retrogradation of wheat starch gels. The effects of 20 sweeteners selected based on common food usage and stereochemical structures of interest, and ranging in concentration from 10 to 50% Native starch granules are water insoluble and relatively inert in foods, but in the presence of water (or other low molecular weight plasticizers such as glycerol or sugar solutions) and sufficient heat to reach the gelatinization temperature, the crystalline regions of the starch granule melt and there is an irreversible loss of the native structure ,2. Durinto water ,4.Upon cooling after gelatinization, amylose and amylopectin begin to retrograde, reassociating via chain entanglements that lead to the formation of ordered crystalline regions . The ratg) of amorphous fraction) [The rate and amount of retrogradation are dependent on many factors such as the botanical source and starch fine structure ,9,10, reraction) ,12, storraction) ,14, heatraction) ,16, starraction) ,18, and raction) . Thus, braction) , Hoover raction) , Fu et araction) , and Zobraction) . A recenraction) .Pueraria lobata (kudzu) [The effects of sweeteners  on starch retrogradation have also been investigated; however, the findings are contradictory. For example, sweeteners were reported to promote the retrogradation of oat , normal  (kudzu) , and ama (kudzu) . These c® P wheat starch, an unmodified, highly purified native wheat starch  [Aytexamylose)  was donaamylose) ) from BEw/w (when possible based on sweetener solubility limits) were prepared in 10 mL volumes in 15 mL centrifuge tubes and stored at −20 °C when not in use for extended periods. The solubility limits for mannitol and lactose prevented production of the higher solution concentrations. Moisture contents of the sweeteners that were crystal hydrates were accounted for when preparing solutions. To defrost and encourage sweetener dissolution, solutions were heated at 60–80 °C in a heating block for 5–15 min, followed by rotational mixing on a Scientific Industries Roto-Shake Genie . This was repeated until the sweetener was fully dissolved. Solutions were not used if crystals were visibly present after overnight storage at ambient conditions.Sweetener solutions at 10, 20, 30, 40, and 50%w/w), and 4 μL of a 25%w/w calcium propionate solution (preservative). Slurries were made on a starch weight-to-solution volume ratio to achieve similar gelatinized starch volumes in each gel. Slurries were vortexed until fully mixed, immediately poured into the well of a folded hexagon polystyrene weigh dish (4.7 cm inner diameter), then 40 μL of slurry was pipetted into each of 6 wells of a clear polystyrene flat bottomed 96-well plate using a 6 channel pipettor. The plates were sealed with a thermal adhesive sealing film for polymerase chain reaction plates and heated for 90 min at 99 °C (to gelatinize the starch) in a digital AccuTemp-09s vacuum oven  at ambient pressure. Preheated metal blocks were also placed on top of each plate to maintain the seal and for better heat transfer. After heating, the plates were held at ambient temperature for 1 h to allow for the initial amylose retrogradation and vapor condensation inside the wells to take place. Water that condensed on the inside of the film was knocked back into the wells by gently tapping the plate to maintain the original moisture contents. The plates were then loaded into a Beckman Coulter AD 340  Microplate Reader, and the absorbance values at 620 nm were measured (day 0). Plates were resealed with a new adhesive sealing film and stored at 4 °C. Absorbances were measured on days 1, 3, 7, 14, 21, and 28, and plates were resealed with a new adhesive sealing film after each absorbance measurement. The controls were starch with water and water alone. The retrogradation measurement method using spectrophotometric absorbance values of starch gels was adapted from Jacobson et al. . An advaThe gelatinization temperatures of the starch-sweetener slurries across all sweetener concentrations were determined using differential scanning calorimetry, as described by Allan, Rajwa, and Mauer . Additiot was the predicted absorbance at time t, Abs∞ was the absorbance at day 28, k was the calculated rate constant, t was days of incubation, and n was the Avrami exponent. Rate constants (k) of retrogradation were calculated using absorbance measurements and the Curve Fitting Application in MatLab R2019a  for the 620 nm absorbances (y-axis) plotted with respect to time . The Avrami exponent (n) was set to 1 to compare retrogradation rate constants (k) despite the slightly weaker fit than with a free n variable in Equation (1). The Avrami exponent (n) for starch retrogradation has been previously calculated to be 1, which assumes that starch crystallization was “rod-like growth from an instantaneous nuclei” [k values were used to model the absorbances of the starch gels. In addition to determining rate constants (k), the amount of retrogradation was documented by using Abs∞ (absorbance at day 28) and the change of absorbance from day 0 to day 28 (Abs(∞-0)) to offset for initial absorbance differences. A modified Avrami equation adapted from Berski, Ziobro, Witczak, and Gambus  was used nuclei” . Equatiok, Abs∞, and Abs(∞-0)) were analyzed using 2-way ANOVA (α = 0.05) in the Standard Least Squares “personality” in JMP Pro 14.0.0 . Statistical differences between the onset temperatures, peak temperatures, and enthalpies measured by DSC were compared by one-way ANOVA with a Tukey post hoc test (α = 0.05) in JMP Pro 14.0.0.The effects of categorical sweetener solution properties  on retrOH,effN) and effective volume fraction of the solvent ϕw,eff of sweetener solutions were calculated as described in van der Sman and Mauer [g, molar volumes  and amounts ) for the sweetener-starch gels were grouped by sweetener concentration, then individual Pearson correlation coefficients (r) were calculated between the retrogradation properties (y-axis) against the numerical sweetener properties (x-axis). For example, r was calculated for the linear correlation between the k values of gels made with 10% sweetener solutions against the molar volumes of the sweeteners. Significances of r values were assessed using two-tailed t-statistics with Microsoft Excel 365 .The molar effective number of hydroxyl groups , as wel620nm values over time, and the initial absorbance values (day 0) were greater with increasing sucrose concentrations  varied even though all samples were heated above the Tgel. Wheat starch pasting temperatures in 43% (1.5M) and 55% (2M) sucrose solutions are less than 95 °C, but the starch in these solutions does not fully swell as indicated by lowered peak pasting viscosities with no viscosity breakdown [620nm values. Wheat starch gels made with 0 to 50% sucrose solutions exhibited increasing Abstrations . The Abser light . Starch er light  and paster light ,56. Incrolutions ; therefok) decreased with sucrose concentrations up to 20% but then increased with increasing sucrose concentrations in gels made with >30% sucrose solutions (k and 22% reduction in Abs∞) but increasing sucrose at higher concentrations will promote retrogradation (≈2 fold increase in k and Abs∞). In the absence of sugar, Huang, Chao, Yu, Copeland and Wang [gel, and resulted in increases in retrogradation, which were likely due to enough residual short-range molecular order to serve as nucleation sites and enough molecular mobility of the glucan chains to facilitate retrogradation. However, this does not explain the inhibition of retrogradation at the lower sucrose concentrations compared to the water control.Increases in absorbance over time are attributed to amylopectin retrogradation. As starch retrogrades, intra- and intermolecular starch–starch junction points grow into crystalline zones, resulting in lower starch solubility and dispersed particles large enough to scatter light . The ratolutions . This suand Wang  found thThe melting enthalpies of wheat starch–sucrose gels determined by DSC analysis of the same sucrose-containing gels analyzed in the spectrophotometer are reported in The melting peak temperatures of retrograded sucrose-starch gels ranged from 55.07 to 60.92 °C, and only gels made with 40 and 50% sucrose had higher melting temperatures than gels made with water . Retrogrw/w concentrations) on the retrogradation rate constants (k) and amounts (Abs∞ and Abs(∞-0)) in 10% wheat starch gels were compared  values, respectively. Starch–sweetener gels with the highest rates and amounts of retrogradation, exhibiting k, Abs∞, and Abs(∞-0) values in the top quartile, were made with 50% glucose, 30 and 40% isomalt, 40% isomaltulose, 40 and 50% maltitol, 30% raffinose, 40 and 50% sorbitol, 50% sucrose, and 40 and 50% xylitol solutions. Gels in which retrogradation was inhibited, for which k, Abs∞, and Abs(∞-0) were in the lowest quartile, were made with 30% maltose, 20% mannose, and 10% sucrose solutions. The effects of sweetener solutions  values of these gels increased with trehalose concentration; however, k values remained similar to the control (≈0.1 day−1). Isomalt and isomaltulose were two of the strongest retrogradation promoters and both have an α(1–6) glycosidic linkage, which allows for greater molecular extensibility and flexibility [gel of starch at the 30% and 40% concentrations  was ≈ 0.0, the calculated k values were artificially high  values; the number of carbons was significant for Abs(∞-0) but not k or Abs∞; and if the sweetener was a reducing sugar, it was not a significant factor  and k values. The gels that retrograded the most  values) and the fastest  were predominately made with sugar alcohols at high concentrations (40 and 50%), whereas the gels that retrograded the least and the slowest were made with sugars at low concentrations. Baek, Yoo and Lim [To better understand why different sweeteners affected retrogradation differently and at different concentrations, the effects of categorical sweetener solution properties  on starct factor . Signifi and Lim  also repw,eff was also moderately but significantly negatively correlated to Abs∞, but to a lesser extent than Tgel. Sweetener solutions that resulted in the highest onset Tgels also tended to result in the most retrogradation. Increasing the concentration of all of the sweeteners studied increased the Tgel, although the extent of Tgel elevation varied across the different sweetener types attributed to structural differences between the sweeteners, including measures of their intermolecular hydrogen bonding ability and ϕw,eff [gel and the preparation of the gels (99 °C) affected retrogradation, as reflected in the distribution of Abs∞ values at any given onset Tgel value  values were investigated  values at 20 to 50% sweetener concentrations. The e-OH was more influential than the OH,effN. The equatorial and exocyclic hydroxyl groups in a monosaccharide have been shown to be more reactive than axial hydroxyl groups [k) in a first-order kinetic equation of 6% sweetener and 30% rice starch gels and the number of equatorial hydroxyl groups in the sweeteners. This is in agreement with the retrogradation behaviors of wheat starch gels made with 10 and 20% sweetener solutions because there were also negative correlations with the retrogradation rate constants (k) and the number of equatorial and exocyclic hydroxyl groups in a sweetener  values of gels made with 20, 30, and 40% sweetener solutions (∞ and Abs(∞-0)) without greatly affecting retrogradation rates (k). The molar volume is the space the solute takes up in solution [Sweetener molar volumes  were alsolutions . Howeverolutions  were ≈10olutions , indicatsolution , so conck, Abs∞, and Abs(∞-0) values of retrogradation were also compared with the dry glass transition temperatures (Tg) of sweeteners . For example, the Tg of the wheat starch gel made with a 50% sucrose solution was estimated to be ≈−63 °C (210 K) using the Fox equation . Therefgel of the starch and retention of residual short-range molecular order in the gelatinized starch [gel relative to the water control, yet some sweeteners delayed retrogradation compared with the control. These sweeteners that delayed retrogradation likely functioned as starch crystallization impurities and contained structures less favorable for intermolecular interactions. The sweeteners that inhibited retrogradation  tended to have the least effect on elevating the Tgel ) of starch gels made with sucrose, glucose, mannose, galactose, fructose, maltose, and maltitol were not linear with concentration but instead were primarily “U”-shaped . Huang, Chao, Yu, Copeland, and Wang [gel. Higher sweetener concentrations increased Tgel and would have retained more residual starch structure, which likely served as nucleation sites and promoted retrogradation. At the lower sweetener concentrations, despite having higher Tgels and, therefore, more residual short-range order than the water control, the number of equatorial and exocyclic hydroxyl groups in the sweeteners was negatively correlated with retrogradation. It could be that the favorable intermolecular hydrogen bonding between these sweeteners and starch resulted in interference with crystal growth in these conditions, but not in systems containing higher amounts of these sweeteners and more residual short-range order.The concentration-dependent influence of some of the sweeteners on inhibiting retrogradation at lower concentrations then promoting retrogradation at higher concentrations underscores that more than one factor affects starch retrogradation in the presence of the same sweetener. The retrogradation behaviors  and with sweeteners with molecular configurations favorable for intermolecular interactions, such as more equatorial and exocyclic hydroxyl groups, α linkages, larger molar volumes, and sugar alcohols. The sweeteners that promoted retrogradation also tended to elevate the T


Philip Alexander, MD, is a native Texan, retired physician, and accomplished musician and artist. After 41 years as an internal medicine physician, Dr. Phil retired from his practice in College Station in 2016. A lifelong musician and former music professor, he often performs as an oboe soloist for the Brazos Valley Symphony Orchestra. He began exploring visual art in 1980, evolving from pencil sketches—including an official White House portrait of President Ronald Reagan—to the computer-generated drawings featured in this journal. His images, which first appeared in this journal in the spring of 2012, are his own original creations.Methodist DeBakey Cardiovascular Journal, submit your creation online at journal.houstonmethodist.org as a “Humanities” entry.If you would like to see your art published in the


One year ago, we had the honor of writing our first Editorial for the Brazilian Journal of Otorhinolaryngology, entitled “The Challenge of the Past”. The text attempted to convey the responsibility of managing the Publications Department of our Journal, and continue this long and successful tradition of our major means of scientific disclosure. We thought we could anticipate the challenge we would have ahead of us. However, it was only after one year of work that we could appreciate the true size of such challenge and the repercussions of our actions.During this first year, we tried to implement editorial and administrative changes in our journal. The first and foremost administrative measure was to centralize the stages of editing, formatting and printing management all in one single company, and this brought about an incredible speed to all the stages of our “production line”, with a substantial reduction (of about 45%) in lead time between paper submission and approval. Moreover, such measure is very much in tune with ABORL-CCF management goals and paramount for the financial balance of our Publications Department, and consequently, for that of our institution. In these regards, we also achieved an important increase in publicity space, of more than 30 %.Late last year, our journal was indexed by the MedLine database, a final touch to the efforts of the previous administration, and this led to an important leap in the disclosure of our papers. Just to illustrate this fact, we have recorded about 25,000 hits per week in our Internet site in Portuguese, and about 1,700 hits in our site in English. And, finally, another action of great impact has been the updating of our journal at the National Library , that requires, by Federal Law, since 2004, the submittal of all the issues of any publication printed in our country. In order to meet this challenge, we counted on the support of some colleagues, who have kindly donated some past issues that we no longer had in our library. We appreciate it and deeply thank them for this.From the editorial stand point, the first change is right at the face. The new cover layout, more sober, tried to follow on the modern trend of important international journals, highlighting one image from the ones present in the papers that make the journal body. For that, we count on the outstanding scientific quality of the papers submitted and their images, which reflect the competence of Brazilian Otorhinolaryngologists in the production of scientific knowledge. The “Letter to the Editor” section has been an important asset introduced by our chief editor, allowing better interaction between subscribers, journal staff and authors – making room for productive discussions on controversial themes. The expansion of our editorial body, coupled to new case report submission standards, have also contributed to further enhance our editorial process.And finally, the last novelty the authors enjoyed was to turn the Proceedings of our our Conventions into a scientific supplement to our journal. From now on, in selection processes, in competitive exams and, mainly, for research grant agencies and post-graduation programs, the summary of papers we present in our annual meetings will enjoy the status of being indexed in Exerpta Medica, Lilacs, SciELO and MedLine. And, there is much more to come.And in closing, we would like to especially thank our Associate Publisher, Prof. Dr. Regina Helena G. Martins, with whom we have shared the pleasure of working in our department; our Chief Editor, Prof. Dr. João Ferreira de Mello Jr., who have strenuously dedicated himself to our journal; our associate editors, greatly responsible for the final scientific quality of each issue; our secretary Fernanda Vieira, who works hard in numerous activities; GN1 Genesis Network, most especially Márcio Argachof and Daniel Marcoto, who have truly “embodied our Journal” and, most especially, all the authors and co-authors who represent the main success pillars or our dear publication.Silvio Caldas NetoPublisher


Unfortunately, we had different results than before.This led to inaccurate conclusions in this manuscript”.https://pubpeer.com/publications/51FACD700BCEB2DE71D4748E96BE72>.This denouncement consisted of potential data falsification and/or inaccuracy.In addition, the manuscript was questioned by independent journalists from the “PubPeer”website <Thus, the Editors decided to retract this article to avoid further damage to thescientific community. The Brazilian Journal of Medical and Biological Research remainsvigilant to prevent misconduct and reinforces the Journal’s commitment to goodscientific practices.


Diabetes, dermatological diseases, primary glaucoma, and sex were not associated with corneal graft failure (p > 0.05). Conclusion: Chronic loss of the endothelium was the primary cause of graft failure in individuals who underwent PK. The high-risk transplant has up to 4.65 times higher risk of complications compared to the indications with a good prognosis.Purpose: this study aimed to assess the frequency of complications related to corneal grafts, including epithelialization disorders, wound dehiscence, infectious keratitis, recurrence of herpetic keratitis, graft rejection, late graft failure, and infectious and noninfectious corneal melting, while also considering risk factors, particularly indications. Methods: this retrospective analysis of corneal graft failure included a chart review of the hospital records of patients who underwent penetrating keratoplasty (PK) between January 2016 and December 2020 at the Department of Ophthalmology of the District Railway Hospital, Katowice, Poland. Results: Between 2016 and 2020, a total of 758 PK procedures were carried out at the ophthalmology department. Bullous keratopathy (20.58%), keratoconus (18.07%), and corneal perforation (13.32%) were the primary indications for keratoplasty. Secondary glaucoma was diagnosed in 99 patients (13.06%). The success rate of PK was 72.43% (494). The most frequent treatment complication was secondary glaucoma (13.06%), followed by late endothelial failure, perforation (4.1%), and bacterial keratitis (3.23%). Patients in the high-risk group were 4.65 times more likely to develop complications than those in the low-risk group. Multivariate regression analysis showed that concomitant ophthalmic diseases (odds ratio (OR): 3.12, confidence interval (CI): 1.60–6.08,  The need for a corneal transplant can arise for a variety of reasons, such as corneal ectasia, bullous keratopathy, corneal stromal dystrophies, Fuchs’ endothelial dystrophy, scarring, perforation, and corneal graft failure. The frequency of certain indications varies over time and is influenced by the demographics of the patients as well as the socioeconomic background of the country . DespiteKnowledge of risk factors and their influence on transplant survival allows undertaking various preoperative, intraoperative, and postoperative preventive measures and also helps to improve patient prognosis for graft success. This study assessed the frequency of complications related to corneal graft, considered the risk factors, especially indications, which are the primary determinants of transplant success, and attempted to provide updated information on this topic.2 test. The influence of the studied factors on the occurrence of complications was assessed by generating a single and multifactor logistic regression model with selected risk factors included. Data for the model are presented as ORs with 95% confidence intervals (CIs). p values < 0.05 were considered significant. Statistical analysis was performed using the RStudio software in the R language.This retrospective, observational study included a chart review of the hospital records of patients who underwent PK between January 2016 and December 2020 at the Department of Ophthalmology of the District Railway Hospital in Katowice, Poland. The study adhered to the tenets of the Declaration of Helsinki. The data collected for the study included patients’ age and sex, date of surgery, reason for surgery, corneal transplant technique, preoperative visual acuity, concomitant conditions, eye surface disorders, number of previous corneal transplantations, lens status, presence of glaucoma before and after keratoplasty, postoperative visual acuity, occurrence of any complications following the surgery, and any surgical interventions after keratoplasty. The complications found in the analyzed group were classified as follows: epithelialization disorders, wound dehiscence, infectious keratitis, recurrence of herpetic keratitis, graft rejection, secondary glaucoma, late graft failure, and infectious and noninfectious corneal melting. The incidence of these complications in the “low-risk” and “high-risk” graft failure groups was compared. Patients with conditions such as ocular burn and limbal stem cell deficiency, perforation, keratitis, at least one graft decompensation, or a history of herpetic keratitis were considered to be at high risk. In addition, the number of unique complications, depending on the indications and other risk factors such as connective tissue diseases, accompanying eye diseases, and previous corneal transplants, and the odds ratio (OR) of complication occurrence in low- and high-risk transplants were examined. Data are presented as the number of cases with a percentage value. The relationships between the qualitative variables were compared using the Pearson chiA total of 758 PK procedures were carried out at the Ophthalmology Department of the District Railway Hospital in Katowice between January 2016 and December 2020. Of these patients, 76 skipped more than one follow-up. The main indications for keratoplasty were bullous keratopathy (20.58%), keratoconus (18.07%), corneal perforation (13.32%), Fuchs’ dystrophy (12.27%), graft failure (10.16%), dystrophies other than Fuchs’ (7.39%), traumatic scar of the cornea (5.01%), bacterial keratitis (3.17%), fungal keratitis (1.72%), herpetic keratitis (1.19%), chemical burn of the cornea (0.53%), and acanthamoeba keratitis (0.26%) .The primary procedures were performed in 557 (73.48%) patients, the second corneal transplant in 127 (16.75%), the third in 19 (2.5%), the fourth keratoplasty in 7 (0.92%), the fifth in 1 (0.13%), and the sixth also in 1 (0.13%). Primary glaucoma was diagnosed in 170 patients (22.42%), while secondary glaucoma was noted in 99 patients (13.06%). Before the keratoplasty, 398 (52.24%) patients were phakic, 291 (38.39%) were pseudophakic, and 22 (2.9%) were aphakic. The majority of patients (90.24%) did not have any comorbid ophthalmic conditions; the rest had had at least one of the following: a history of uveitis, prior herpetic keratitis, chemical burn, eyelid disorders, ocular synechia, multiple vitrectomies, dry eye syndrome, limbal stem cell deficiency, or bullous pemphigoid. Thirty (3.96%) patients had suffered from diabetes, 23 (3.02%) from connective tissue disease, and 5 (0.65%) from dermatological diseases .The complications related to corneal graft are listed in The failure rate of PK depended on the complications .Patients with acanthamoeba keratitis (0%), herpetic keratitis (12.5%), fungal keratitis (30.8%), or perforation (35.2%) had the lowest success rate, while those with lattice corneal dystrophy (94.1%), keratoconus (91.0%), macular corneal dystrophy (87.5%), granular corneal dystrophy (85.7%), and graft decompensation (83.8%) had the highest success rate .p = 0.00), indication for keratoplasty (p = 0.00), concomitant ophthalmic diseases (p = 0.00), and connective tissue diseases (p = 0.00) were significantly associated with an increased risk of graft failure. On the other hand, diabetes, dermatological diseases, primary glaucoma, and sex were not linked with the occurrence of complications (p > 0.05).Following PK, 85 (12.46%) patients required another ophthalmic surgery, including corneal regraft (8.2%), amniotic membrane transplant (2.05%), evisceration (1.76%), and wound suturing (0.15%). Patients in the high-risk group were 4.65 times more likely to develop complications than those in the low-risk group. Previous corneal graft failure (p = 0.03), high-risk group , previous corneal transplants , concomitant ophthalmic diseases , connective tissue diseases , and diabetes .Several factors were included in the univariable analysis . The folp = 0.00), concomitant ophthalmic diseases , and connective tissue diseases .Multivariate regression analysis  revealedDespite the increasing success of lamellar keratoplasties, PK is widely used in many countries, including Poland . The maiOur study showed that the success rate of keratoplasty was 72.43%, which is comparable to other reports ,8. The mIt is highly likely that among patients with endothelial failure were those who have had a graft rejection but did not show up quickly enough to diagnose it. Corneal graft rejection and inflammation are significant factors of endothelial graft failure . Severe most corneal transplants are performed in our region, a lot of patients with severe corneal conditions are referred to us, and the incidence of perforation is significant. Furthermore, it is well known that corneal transplantation has a low success rate [Surprisingly, perforation was found to be the second most common complication after PK in the study period. This fact should be explained in the framework of indications in the study group, with perforation being the third most common complication. Because our hospital is a tertiary referral hospital where ess rate . Based oIn our study, the incidence of infectious keratitis was 3.23%, which is within the range of this complication being reported in developed countries (1.5–11.6%) ,17,18. IThe study shows that fungal keratitis, herpetic keratitis, acanthamoeba keratitis, and perforation are associated with the worst prognosis after keratoplasty , which cAnother intriguing finding was that as many as 45% of patients with complications related to corneal button needed surgical intervention, of which the vast majority (29.79%) required regraft. In addition, the primary indication for almost half of the patients who required retransplantation was perforation (44.64%).According to some reports, transplant failure is determined by both donor and recipient factors. The indication, prior corneal transplants, neovascularization, prior graft rejection, and type of transplant are known to influence the graft outcome . AdditioKnown risk factors such as previous corneal graft, indication, other ophthalmic diseases, and connective tissue diseases were found to be significantly associated with an increased risk of graft failure .Although diabetes and sex are controversial risk factors for graft failure , glaucomNeovascularization was not examined in our study, although it is another recognized factor that is known to cause graft failure and graft rejection ,31,32. TAssessment of the correlation between many variables and the occurrence of graft complications using the multivariate analysis led to the identification of three important variables: a high-risk group, concomitant ophthalmic diseases, and connective tissue diseases. As the high-risk group represents patients with specific indications, it can be assumed that the indication for corneal transplantation is a significant risk factor, as reported in several previous studies ,34,35,36In summary, one of the main causes of graft failure is chronic loss of the endothelium after PK. The occurrence of complications is mainly dependent on the indication for corneal transplantation, and, even after years, it remains a major treatment challenge.The high-risk transplant is up to 4.65 times more likely to develop complications than the indications with a good prognosis. It is essential to take efforts to improve the chances of graft survival, including advancements in corneal preservation methods, surgical techniques, and the use of more potent antirejection drugs. However, it appears that a key strategy is to ensure appropriate patient follow-up and educate patients to identify severe corneal conditions at an earlier stage.


This work aims to improve the mechanical properties of starch-based hydrogels using a double-network (DN) strategy. The single network (SN) starch hydrogel was first prepared using glutaraldehyde as a crosslinker. The compressive properties of the SN hydrogels were influenced by both crosslinker content and crosslinking time. The SN starch hydrogel possessing the best mechanical properties was then fabricated into DN hydrogels. Poly (PVA) and borax were used as a secondary polymer and a crosslinker, respectively. The PVA–borax complexation partly enhanced the DN hydrogel’s compressive modulus by 30% and its toughness by 39%. DN hydrogels were found to have denser microstructures than SN hydrogels. To be specific, their walls thickened and grew more continuous while their pores shrank. The increased crosslinking density resulted in changes to the microstructure, which were well correlated with their porosity and water uptake capacity. An in vitro cytotoxicity test of the DN hydrogels revealed that they were non-toxic to chondrocytes. This work demonstrated that double networking is a simple but effective strategy for improving mechanical properties of starch-based hydrogels without sacrificing their biocompatibility. This approach can be used to tailor hydrogel properties to fulfill requirements for biomedical applications, such as tissue engineering and other related fields. Starch-based hydrogels have drawn substantially attention in the field of tissue engineering in the recent years . Starch In tissue engineering application, hydrogels of starch are attractive because they are functionally resemblant to natural tissue with a high degree of biocompatibility . The sofco-2-hydroxyethyl methacrylate) or P(AM-HEMA) as the primary and secondary polymers, respectively. Borax and N,N′-methylenebisacrylamide were used as crosslinkers for the primary and secondary polymers, respectively. The resulting DN hydrogels were strong and stretchable. The improvement in tensile strength to 60% higher than that of the single network PVA hydrogel was due to the complex formed by PVA–borax on the P(AM-co-HEMA) network [Numerous approaches have been used to develop a hydrogel with enhanced mechanical properties. Designing of a hydrogel by choosing different type and number of crosslinker were conventionally done. In the recent year, different strategies have been developed to create novel types of hydrogels. These include a hydrogel with a topological structure (TP), a hydrogel with a hybrid structure, a hydrogel with a nanocomposite structure (NC), and a hydrogel with double network structure (DN). Among these, making a double network hydrogel is a relatively new concept that has gained significant attention in the recent years. These DN hydrogels are formed by intertwining two different polymer networks together, resulting in a hydrogel with altered properties that are contributed by both polymer networks. Various properties of the DN hydrogels were reportedly improved over those of the SN hydrogels of their constituents . For exa network .Choosing the second network is crucial in order to improve mechanical properties of the fragile starch-based hydrogels . SynthetCrosslinking agents for PVA include ethylene glycol diamine methacrylate (EGDMA) , bis. Deionized water was used in this study.Native cassava starch was purchased from Kriangkrai Co., Ltd. . GA (25 wt% in aqueous solution) was purchased from Thermo Fisher Scientific . PVA  was purchased from Chem-Supply Pty., Ltd. . Sodium tetraborate decahydrate (borax) was purchased from QRëCPreparation of single network (SN) starch hydrogelsTo prepare a SN Starch hydrogel, a 10 w/v% starch solution was gelatinized at 90 °C and mechanically stirred for 30 min until a transparent starch solution was obtained. Then, 50 mL of 2.5 M GA solution was added to the gelatinized starch solution and stirred for 2 min. The mixture was poured into round disk molds with a diameter and a thickness of 15.0 cm and 1.5 cm, respectively. Then the filled molds were placed in an oven at 60 °C for different crosslinking times: 2, 4, 6, 8, 10, and 24 h. After a predetermined crosslinking time was reached, the starch hydrogel samples were removed from the molds and left to dry at room temperature for 3 days, until they reached a constant weight. The samples were kept in a dry place at room temperature before test and characterization.To prepare SN starch hydrogels with varying GA concentrations, GA stock solution with 2.5 M was used. GA working solutions were prepared from the stock solution at different amount, i.e., 10, 20, 30, 40, and 50 mL and DI water was added to the solutions to make a constant final volume of 50 mL. The hydrogels with various concentrations of GA were designated as SN10, SN20, SN30, SN40, and SN50, respectively.Preparation of double network (DN) starch hydrogelsSN starch hydrogel possessing the highest compressive modulus and strength was used to fabricate into DN hydrogels. Borax was used as a secondary crosslinker to form a 3D network of PVA with the primary starch network. To prepare a DN hydrogel, the dried SN starch hydrogel was immersed in 50 mL of 3 wt% PVA for 24 h at room temperature. The product of this stage was called an interpenetrating network (IPN) hydrogel. This immersion method of DN hydrogel preparation was adapted from Dixit et al. . After i−1 with a resolution of 4 cm−1 and a number of scans of 64 [Chemical functional groups of SN and DN starch hydrogels were identified by a Bruker Tensor 27 Fourier transform infrared (FTIR) spectrometer . Dried samples were ground with analytical grade KBr and pressed into transparent disks prior to the measurements. Their spectra were recorded over a wavenumber range of 4000 to 400 cmns of 64 .The morphologies and porous structures of hydrogels were investigated by a FEI Quanta 450 Scanning electron microscopy (SEM) . Before characterization, hydrogel samples were frozen at −60 °C for 24 h before being lyophilized for 48 h in a Gamma 1-16 LSCplus freeze dryer . The dried hydrogel cross-sections were then sputtered with a thin layer of gold. This SEM characterization method was adapted from the work of Podhorská et al. . The por1) were immersed in pure ethanol for 24 h. After that, the hydrogels were removed from the ethanol, blotted with tissue paper before being weighed again (W2). The mass of hydrogels was weighed by a ML204/01 analytical balance . The percentage of porosity was calculated using the following Equation (1): 1 and W2 are the weights of a hydrogel before and after immersing in absolute ethanol, respectively. ρ is the density of ethanol and V the volume of a hydrogel [The porosity of a hydrogel sample can be quantified by measuring the amount of ethanol that penetrates the sample. Since ethanol is non-solvent for starch hydrogel, it simply filled the pores and the free space in the network structure of the hydrogel. The porosity of hydrogels was measured using the solvent replacement method. The hydrogel discs with known weight (Whydrogel .s) (about 24 h). After that, the hydrogels were dried at 60 °C in a hot air oven and reweighed (Wd) [Water uptake of a hydrogel is the total amount of the water absorbed by the material. The water resides in the pore and between polymer chains. Due to the microporous structure and hydrophilic nature of the hydrogels, water can enter both the pores and the free space in the network structure of the pore wall. Therefore, water uptake by a hydrogel is typically used as an indicator for the degree of crosslinking. Water uptake capacity of hydrogels was measured by soaking the dried hydrogel samples in distilled water until they reached an equilibrium swelling weight (Whed (Wd) . The mas® software .Compressive properties of hydrogels were determined using a TA.XT Plus Texture analyzer . A fully swollen hydrogel sample was cut into a cylindrical disk with a diameter of 11 mm and a thickness of 2 mm. Compressive tests were performed using a 1 kgF load cell at a strain rate of 0.05 mm/s. The testing method was adapted from Llorens-Gámez et al. . Five re2 cell culture flasks  under standard culture conditions of 37 °C and 5% CO2.A preliminary study on the cytotoxicity of the prepared hydrogels was conducted in accordance with ISO standard 10993-5: Biological evaluation of medical devices. As cultivated cells, primary human chondrocytes (HC) from articular cartilage  were employed. Dulbecco’s Modified Eagle Medium  with 1% penicillin-streptomycin, 1% L-glutamine, and supplemented with 10% fetal bovine serum  was used as a culture medium. Prior to starting the assays, the HC cells were grown to 80% culture confluence in 75 cm® syringe filters .The tests were conducted on the extracts of culture media in serum-free medium (SFM) using a similar approach as Pankongadisak et al. . The hyd®, St. Louis, MO, USA). The cells were plated at a seeding density of 5 × 104 viable cells/cm2 in SPL 96-well culture plates (flat bottom) using a multichannel pipette. After an incubation period of 24 h at 37 °C with 5% CO2, the culture medium was removed and replaced with the extract media. Cells were then incubated for 24 h at 37 °C and 5% CO2 before being tested for viability using the 3--2,5-diphenyltetrazolium bromide (MTT) assay. The viability of cells cultured by the fresh SFM was used as the control. After the treatment, the medium was discarded, and the samples were washed with PBS. 100 µL of MTT solution (0.5 mg/mL) was added and incubated for 3 h. After decanting the MTT solution, 100 µL of dimethyl sulfoxide was added to dissolve the formazan crystals. After 2 min of agitation, the solutions’ absorbance at 570 nm was determined using a MultiskanTM GO Microplate spectrophotometer . Cell viability was calculated according to the following Equation (3):570A is a value of the measured optical density of the treated cells, and OD570B is a value of the measured optical density of the non-treated cells.Cells were counted using the Trypan blue (Sigma-Aldrichn = 5), whereas the other three tests  were conducted on three replicate samples (n = 3). All quantitative data were analyzed using one-way ANOVA and Turkey’s post hoc comparison test. To identify statistical differences between the comparison groups, a value of p < 0.05 was employed as the level of statistical significance.Statistical analysis was performed using the IBM SPSS Statistic, version 24.0 . The means and standard deviation of each result were displayed. The compressive test was performed on five replicate samples  and 947 cm−1 (symmetric stretching band), 661 cm−1 (bending of the B-O-B bond in the borate network), 833 cm−1 4−) [−1 (asymmetric stretching of B-O-C) was correlated to the hydroxyl groups that form complexes (crosslinks) with borate ions. These results suggested that the complexation of PVA and borate ions took place, which led to the formation of a physical crosslinking of the secondary network in the DN hydrogel.FTIR spectra of PVA, GA, Borax, SN, IPN and DN starch hydrogels are shown in ectively . The C=Of starch . For thef starch ,34. NoneB(OH)4−) ,35. The SEM micrographs of cross-sections of freeze-dried SN30 hydrogels, prepared at various crosslinking times and those of SN hydrogels, prepared at various GA contents are shown in In SEM images of SN starch hydrogels at various GA contents after 24 h of crosslinking time are shown in As can be seen in the SEM micrographs in The porosity of SN starch hydrogels prepared at various crosslinking times and GA contents was determined and illustrated in The equilibrium water uptake of SN starch hydrogels at the different crosslinking times and GA contents are shown in Stress–strain curves of SN starch hydrogels at various crosslinking times and GA contents are shown in 3 to 69.93 ± 2.17 kJ/m3. This indicated that increasing the crosslinking time allowed for a greater interaction between starch and GA molecules to occur.As crosslinking time increased from 2 to 24 h, the compressive strength of the prepared starch hydrogels increased from 111.52 ± 5.26 kPa to 443.46 ± 5.76 kPa while their compressive modulus increased from 8.64 ± 1.84 kPa to 26.49 ± 0.69 kPa. Additionally, the increased crosslinking time increased the toughness of these hydrogels from 18.80 ± 5.23 kJ/mAt constant crosslinking time of 24 h, an increase in the GA content up to 30 mL resulted in increases of the compressive strength, compressive modulus, and toughness of the SN hydrogels, as shown in A similar observation was also reported by Bi Long et al. . In theiIn our case, the mechanical strength of the hydrogels seemed to be largely dependent on their microstructure. Specifically, mechanical properties of a porous materials are largely influenced by the size of its pores, and smaller pores contribute to the enhancement of the mechanical strength of engineered structures ,41. NoneIn this study, 30 mL of GA was optimal content for preparing SN starch hydrogel with highest compressive properties . The hydIn summary, the important findings from the investigation of effects of crosslinking time and GA content on mechanical properties of SN starch-based hydrogels were that the optimal crosslinking time was 24 h and GA content was 30 mL. The final mechanical properties were mainly dependent on the microstructure of the porous hydrogels.To further enhance the mechanical properties of the starch-based hydrogel, the SN hydrogel with the highest compressive properties  was chosen to be used as a primary network for fabricating the DN starch hydrogels. In this study, we developed DN starch hydrogels by introducing poly (PVA) as a secondary polymer and borax as a crosslinker. The PVA chains interpenetrated into the SN starch hydrogel network that served as the primary structure. The secondary network was then formed by the interaction of borate ions holding PVA strands together.SEM micrographs and EDS spectra of SN30 and DN starch hydrogels prepared with various borax concentrations are shown in In In The porosity of SN hydrogel (SN30@24h) and its DN starch hydrogels prepared at various borax concentrations is shown in p < 0.05, compared with SN30). Moreover, the DN0.15 hydrogel had the lowest water uptake, measuring at 497.58%, as shown in After 24 h of immersion in deionized water, water uptake percentages of the DN hydrogels were determined. It was found that the water uptake of the DN was linearly dependent on the amount of borax concentration used. The water uptake capacity of DN hydrogels significantly decreased with increasing borax concentration , compressive modulus (33.99 ± 2.71 kPa), and toughness (90.53 ± 3.38 kJ/m3). The results revealed that the presence of the secondary network in the DN hydrogel increased polymer chain entanglement and crosslink density, reducing chain freedom in comparison to a single network hydrogel [Stress–strain curves of DN hydrogels are shown in hydrogel ,47,48. Ihydrogel . The resThe MTT cell cytotoxicity assay is one of the in vitro screening tests used to evaluate the effects of materials on cellular growth at various extraction ratios. In this study, chondrocyte cell viability in the hydrogel extractions was investigated. The hydrogel extractions at concentrations of 0.5, 5, 10, and 50 mg/mL were compared against a fresh culture media. Starch hydrogels have been proven to be promising candidates for usage in medical applications. Their limitations, however, are generally due to poor mechanical properties, which need to be addressed before their efficacy can be realized. We have demonstrated in this study that the mechanical properties of starch hydrogels could be improved by using a double network strategy. We synthesized crosslinked double-network hydrogels where starch was a primary network and poly was a secondary network. We reported the parameters affecting the hydrogels’ properties. For SN starch hydrogel, the compressive properties increased with increasing crosslinking time. We found the optimal content of the primary crosslinker (GA) content in SN starch hydrogel to be 30 mL, the SN30, which gave the highest compressive properties. The SN starch hydrogel prepared with the optimal condition was further improved by synthesizing into DN hydrogels. The compressive properties of DN hydrogels increased with increasing borax (the secondary crosslinker) concentration. The DN hydrogel prepared with 0.15 M of the borax solution exhibited the highest compressive properties. The values were higher than those of the SN starch hydrogels. The mechanical properties, porosity, and water uptake capability of the hydrogels were all well related to their microstructures. In addition, an in vitro cytotoxicity test revealed that the stable DN hydrogels were safe to chondrocytes. The optimal crosslinker concentration and crosslinking time are essential parameters that influence the final properties of the hydrogel. It is crucially important to systematically determine these parameters in order to achieve a hydrogel with the desired properties. This study successfully proved that a double networking strategy could be used as a tool to improve the mechanical properties of starch-based hydrogels. Further improvements are required to obtain hydrogels with properties suitable for tissue engineering applications. Our ongoing research focuses on the fabrication of tougher hydrogels using combined strategies, i.e., double network together with nanocomposite. The preliminary results show the promising development of hydrogels with improved mechanical properties and enhanced osteoblast cell growth.


Phaeocystis globosa is a marine-bloom-forming haptophyte with a polymorphic life cycle alternating between free-living cells and a colonial morphotype, that produces high biomass and impacts ecological structure and function. The mechanisms of P. globosa bloom formation have been extensively studied, and various environmental factors are believed to trigger these events. However, little is known about the intrinsic biological processes that drive the bloom process, and the mechanisms underlying P. globosa bloom formation remain enigmatic. Here, we investigated a P. globosa bloom occurring along the Chinese coast and compared the proteomes of in situ P. globosa colonies from bloom and dissipation phases using a tandem mass tag (TMT)-based quantitative proteomic approach. Among the 5540 proteins identified, 191 and 109 proteins displayed higher abundances in the bloom and dissipation phases, respectively. The levels of proteins involved in photosynthesis, pigment metabolism, nitrogen metabolism, and matrix substrate biosynthesis were distinctly different between these two phases. Ambient nitrate is a key trigger of P. globosa bloom formation, while the enhanced light harvest and multiple inorganic carbon-concentrating mechanisms support the prosperousness of colonies in the bloom phase. Additionally, colonies in the bloom phase have greater carbon fixation potential, with more carbon and energy being fixed and flowing toward the colonial matrix biosynthesis. Our study revealed the key biological processes underlying P. globosa blooms and provides new insights into the mechanisms behind bloom formation. Phaeocystis globosa is a marine phytoplankton that causes blooms in coastal seas of temperate and tropical regions [P. globosa blooms, the colonies produce massive amounts of biomass and dominate the phytoplankton communities, thus impacting food web structures and aquaculture, threatening the safety of coastal nuclear power plants, and influencing global biogeochemical cycles and even climate regulation [P. globosa has caused harmful algal blooms (HABs) almost every year and has become the primary problematic species in Chinese coastal waters [ regions . It has  regions ,3. When gulation ,5,6. In l waters ,7.Phaeocystis species differ from other marine phytoplankton in their ability to form large gelatinous colonies with a unique colony matrix, which plays a significant role in their success and uniqueness in the environment [Phaeocystis a competitive advantage against other species when resources are scarce and variable [Phaeocystis blooms.Previous studies have demonstrated that ironment . This coironment ,10,11. Mvariable ,13. TherP. globosa grows on a temperature spectrum spanning from 2 to 30 °C, and optimally at 15 to 20 °C [Phaeocystis colony matrix, thus inhibiting GAG accumulation and colony formation [P. globosa competes well for nitrogen but poorly for inorganic phosphate in light-saturated conditions [P. globosa blooms, according to both field investigations and mesocosm experiments [P. globosa bloom outbreaks are a combined result of the environmental conditions that induce colony formation and the strong competitiveness derived from the colony matrix structure. However, little is known about the intracellular biological processes that drive the bloom process, and the mechanisms behind P. globosa blooms remain unclear.Temperature is one of the most important environmental factors that triggers outbreaks and shapes the field distributions of marine phytoplankton. to 20 °C ,15. Moleormation . Irradiaormation . Interesormation ,18. Reganditions . Nitrogeeriments ,22. OverP. globosa bloom occurred and lasted for approximately half a month in Dapeng Bay off the southern coast of China. Field investigation showed that the morphology and distribution of P. globosa colonies were obviously different as the bloom developed. These variations are likely associated with some intrinsic biological processes, and the proteins involved in these processes should contain crucial information related to bloom formation, since proteins are the actual executants of life functions. Therefore, in this study, we selected in situ P. globosa colonies from the bloom and dissipation phases, and compared their proteomes using a tandem mass tag (TMT)-based quantitative proteomic approach. Based on these findings, the functions of differentially abundant proteins between these two phases were characterized, and key biological processes that might be associated with bloom development were analyzed. The purpose of this study was to identify the key biological processes driving P. globosa blooms and to reveal the molecular mechanisms underlying bloom formation.Previously, a According to the field investigation, colony density increased sharply in the early blooming stage and reached the highest level of approximately 18 colonies/L on the third day . In thisP. globosa bloom formation, according to previous studies [According to the physical oceanographic parameter measurements, the pH and salinity of seawater remained stable throughout the whole investigation A. Seawat studies ,7.p ≤ 0.05 for both nitrate and ammonium) on 26 January and a significant increase (p ≤ 0.05 for nitrate and p ≤ 0.01 for ammonium) on the last two days of the survey (Inorganic phosphate remained stable at a low level (0.074 ± 0.03 μmol/L) B, which e survey B.http://proteomecentral.proteomexchange.org, accessed on 5 September 2022) via the iProX partner repository [The mass spectrometry experiment output 585,523 spectra, 45,941 of which were matched to 18,773 peptides. Using the MASCOT search engine, 5540 proteins were identified from 16,739 unique peptides derived from the matched spectra. Functional annotation of these identified proteins based on KEGG and NCBInr databases is presented in pository  with theOf the 5540 proteins identified, 300 proteins were differentially abundant, 191 were more abundant in the bloom phase, and 109 displayed higher abundances in the dissipation phase. The functional classification of these differentially abundant proteins is presented in a oxygenase (PAO), which regulates chlorophyll catabolism, displayed higher abundance in the dissipation phase , carbonic anhydrase (CA), and phosphoenolpyruvate carboxykinase (pckA) . Consiston phase . For niton phase . Additioon phase . In addion phase . These pP. globosa bloom. Hundreds of P. globosa proteins were differentially abundant between the bloom and dissipation phases. These proteins participate in a variety of biological processes, such as photosynthesis, nitrogen metabolism and GAG biosynthesis, and are closely associated with bloom development.In this study, a TMT-based quantitative proteomic approach was utilized to analyze the key intracellular biological processes underlying a Functional analysis showed that proteins with higher abundances in the bloom phase were dominated by photosynthesis-related proteins, and these proteins covered both light-dependent and light-independent reactions of photosynthesis. Among them, light- harvesting proteins were components of the light-harvesting complex; additionally, photosystem II proteins, ferredoxin and cytochrome were proteins involved in light harvest and electron transportation. Higher abundances of these proteins suggest a greater energy production potential in blooming colonies. Moreover, it is noteworthy that ribulose bisphosphate carboxylase (RubisCO), carbonic anhydrase (CA), and phosphoenolpyruvate carboxykinase (pckA) displayed higher abundances in the bloom phase. These proteins mainly participate in light-independent reactions of photosynthesis and are the key enzymes of inorganic carbon-concentrating mechanisms (CCMs).Phaeocystis can reach 40 g carbon/(m2·month) during the bloom period, and the high biomass is actually one of the prominent features of P. globosa blooms [2 actually limits inorganic carbon accumulation from seawater, and the affinity of RubisCO for CO2 cannot match the high rates of photosynthesis [2 environments, have evolved high-CO2-affinity RubisCO and, most importantly, have evolved CCMs, such as CA and C4 CCM [P. globosa [3− to CO2, which then diffuses across the membrane to support photosynthesis and growth. This CA CCM benefits P. globosa in interspecific competition, especially in situations of pH increase and CO2 decrease [4 CCM of phytoplankton [P. globosa possesses multiple CCMs, which may explain why the inorganic carbon acquisition of Phaeocystis is equal to or more efficient than that in diatoms [P. globosa blooms.It is estimated that the rate of carbon fixation of a blooms . Howeverynthesis . To overd C4 CCM ,27. Prev globosa . It catadecrease ,30. The  diatoms . OverallPhaeocystis demonstrated that colonies could regulate their buoyancy according to the ambient light level, which conferred an advantage when accessing nutrients [Phaeocystis blooms [In addition, the variation in photosynthetic efficiency might be associated with the different vertical distributions of colonies between the bloom and dissipation phases . A previutrients ,18. Thisutrients ,32. In ts blooms .a oxygenase (PAO), which regulates chlorophyll catabolism, displayed significantly higher abundance during the dissipation phase. PAO is one of the two proteins catalyzing the degradation of chlorophyll to colorless linear tetrapyrroles, and its expression is positively correlated with cell senescence [P. globosa blooms since it supports the high energy transduction and carbon fixation of blooming colonies.Consistent with the higher photosynthetic efficiency, proteins involved in photosynthetic pigment biosynthesis also displayed higher abundances in the bloom phase. As the major photosynthetic pigments, chlorophyll and carotenoids play essential roles in photosynthetic light harvesting and energy transduction in marine phytoplankton . Chloropnescence . The higPhaeocystis colonies, and thus mediates field bloom processes [Nitrogen is an essential nutrient for phytoplankton because of its contribution to building chlorophyll, amino acids, and nucleic acids. Laboratory experiments have demonstrated that nitrogen regulates the cell growth and colony formation of rocesses ,21,22. Irocesses .Phaeocystis usually blooms in environments where nitrate is the major nitrogen source, while ammonium supports the growth of solitary cells rather than colonial cells [P. globosa blooms and that the enhanced nitrate utilization of colonial cells is essential for bloom maintenance.In this study, NRT and NR were more abundant in the bloom phase, while an ammonium transporter (AMT) and GS displayed higher abundances during the dissipation phase, which indicated that nitrate utilization was more active during the bloom phase and that ammonium assimilation was enhanced during the dissipation phase of the bloom. This result was consistent with the nutrient investigation that showed a sharp decrease in the nitrate concentration during the bloom period B. Differal cells ,41. Our Emiliania huxleyi, which is a phytoplankton species related to P. globosa, ambient amides were confirmed to be able to be transported into the cell and subsequently degraded to ammonia by aliphatic amidase [In addition, it is noteworthy that a protein aliphatic amidase (amiE), which is involved in organic nitrogen metabolism, was differentially more abundant in the dissipation phase. This enzyme participates in short-chain aliphatic amide hydrolysis and produces the corresponding organic acids and ammonia, which provides the cell acetamide as both a carbon and a nitrogen source . It help amidase ,45. The Phaeocystis, the colony morphotype is arguably the most salient feature because of its exceptional physiology and ecological advantages in interspecific competition [Phaeocystis colonies, thousands of cells are distributed evenly within the gelatinous matrix by which the colonies can dominate the phytoplankton community or even the entire ecosystem with massive biomass production during the bloom period [For petition . In Phaem period ,47.Phaeocystis was released in the form of GAGs [Phaeocystis cells via exocytosis, in which a condensed polymer matrix first packs the output products, then encapsulates them in secretory granules, and finally releases them from the cell [2+ signal [P. globosa colony formation.Biochemical analysis confirmed that the gelatinous matrix of colonies is synthesized by the combination of a large number of cell-secreting GAGs . For mar of GAGs . After bthe cell . Further+ signal . TherefoP. globosa bloom was traced in Dapeng Bay along the Shenzhen coast  from 23 January to 3 February  in the laboratory. Physical oceanographic parameters of seawater  were measured in situ with a YSI meter . Additionally, to investigate the colonial movement during the bloom period, a GoPro camera  was utilized to capture the vertical distribution of the colonies from the surface to the bottom at the sampling site. During the field investigation, a cruise was conducted at a fixed time (10:00–11:00 a.m.) every day from 23 to 28 January and every two days for the last period. For each survey, 3 × 1 L surface water (0.5 m) was collected for colonial density monitoring. Large For nutrient concentration, seawater was first filtered through a 0.45-μm membrane (Millipore). Then, the inorganic nitrogen and phosphate levels were determined using the diazo coupling method and phosphomolybdenum blue spectrophotometric method, respectively . For proteomic analysis, colonies of surface water were collected in situ using a sampling mesh, transferred to petri dishes with a dropper pipette, rinsed with sterilized seawater, and frozen immediately with liquid nitrogen before transfer to −80 °C storage in the laboratory.P. globosa bloom [P. globosa reached the highest level on 25 January and started to dissipate from 27 January. Therefore, in this study, samples from 25 January were selected as the bloom phase (BP), and those from 3 February were selected as the dissipation phase (DP) of the P. globosa bloom. Three biological replicates were used to compare the proteomes of these two phases.According to the field investigation results and a previous study of the same sa bloom , the colProteins were extracted using TRIzol reagent following a previously reported protocol and quantified with the Bradford assay, in which bovine serum albumin (BSA) was used as a standard . After em/z were dynamically chosen for subsequent higher-energy collisional dissociation (HCD) fragmentation. During this process, the parameters were set as follows: automatic gain control (AGC) target at 3 × 106, maximum injection time of 10 ms, dynamic exclusion duration for 40.0 s, survey scan resolution of 70,000 at 200 m/z, HCD spectrum resolution of 17,500 at 200 m/z, isolation width of 2 m/z, and normalized collision energy of 30 eV. In addition, the underfill ratio that specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%.LC-MS/MS analysis was performed for 90 min with a Q-Exactive mass spectrometer  coupled to an Easy nLC instrument . Positive ion mode and peptide recognition mode were set for the mass spectrometer operation. MS data acquisition was conducted using the data-dependent top 10 method, in which the most abundant precursor ions of the survey scan at 300–1800 P. globosa [The output MS/MS spectra were searched using the MASCOT search engine  embedded in Proteome Discoverer 1.4. Protein identification was performed using a self-built database consisting of 61,148 peptide sequences that translated the sequences of unigenes from a previous transcriptomic study of  globosa . All pep−5 with allowance for up to two missed cleavages. Fixed modifications were set as TMT labeling and carbamidomethylation, and a potential variable modification was set as oxidation (M). In addition, an automatic decoy database search was performed in MASCOT to estimate the false discovery rate, in which random sequences were generated and then tested for spectra in the decoy database and the real database. A protein was considered identified if it contained at least one unique peptide.For protein identification, the search parameters were set as follows: fragmented ion mass tolerance of 0.1 Da, peptide mass tolerance of 2 × 10For protein functional annotation, the amino acid sequence of each protein was searched against the Kyoto Encyclopedia of Genes and Genomes (KEGG) and National Center Biotechnology Information nonredundant protein sequences (NCBInr) databases using BLAST client software.p value ≤ 0.05) of DP/BP ≥ 1.5 (higher abundance in the DP group) or DP/BP ≤ 0.67 .For protein quantitation, the protein ratios were calculated as the median values of their corresponding unique peptides. Protein abundances were compared in DP/BP, and the average ratio of three replicates was calculated as the differential protein abundance. A protein was considered to be differentially abundant if it had a significance ratio (t test P. globosa bloom that occurred along the Chinese coast and, for the first time, studied the key intrinsic biological processes associated with the in situ bloom process using a TMT-based quantitative proteomic approach. Proteins involved in photosynthetic processes, pigment biosynthesis, nitrate utilization, and matrix substrate biosynthesis displayed higher abundances during the bloom phase. The colonies in the bloom phase have greater carbon fixation potential, and more carbon and energy are fixed and flow to the matrix biosynthesis. Ambient nitrate is a key trigger of P. globosa bloom formation, while enhanced light harvest and multiple CCMs support prosperous colonies in the bloom phase. Our study revealed the key biological processes underlying the blooming of P. globosa and shed new light on the mechanisms driving the formation of P. globosa blooms.Our study investigated a