Implications of Polystyrene Microplastics on the Gastric Digestion of Bovine Milk

The prevalence of microplastics (MP) pollution in different zones of the environment has been established by several studies [1]. Due to its widespread presence, MP have found its way into food items. Fish, shellfish, water, milk, salt, and sugar are just some examples of the food we commonly consume that are contaminated with MP [2]. Human ingestion of MP is already well-established but there is limited data regarding how MP affect human gastric digestion of food components, especially proteins. In this study, we investigated the effects of polystyrene (PS) MP on pepsin, the major protease in human gastric digestion. Pepsin activity was tested during exposure to two different sizes -10 μm (PS10) and 100 μm (PS100), and three different quantities- low count (142 particles), moderate count (1420 particles), and high count (14200 particles), of PS using haemoglobin as substrate. Results showed that exposure to PS100 has no effect on enzyme activity. However, exposure to high count PS10 considerably reduced pepsin activity from 2957 ± 310 U/mg to 1674 ± 270 U/mg. To test the effect on food digestion, high count PS10 was added to a sample of commercially available liquid bovine milk (defatted). In this case, the static in vitro simulation of gastric digestion was followed to mimic human digestion of food [3]. Milk digesta at different time points (5, 10, 15, 20, 30, 60, 90, 120 minutes) were obtained to monitor the progress of protein degradation. SDS-PAGE showed no difference in the peptide bands from 30-120 minutes. However, bands corresponding to caseins were not observed at 5 minutes when PS10 was present. Additionally, 14 kDa fragments were not observed at 10-20 minutes. Washing of the PS particles followed by SDSPAGE revealed a faint pepsin band from all time points. At 5 and 10 minutes, faint peptide bands >10kDa were also observed. These suggest that pepsin and some milk peptides were adsorbed on the surface of PS10. Zeta potential analysis of PS revealed a slightly negative surface charge which could explain the adsorption and disappearance of peptide bands. This adsorption of pepsin on PS did not seem to affect its overall protease activity. However, the interaction of milk peptides with PS may reduce the nutrients human could acquire from milk. Acknowledgements This study was supported by Ghent University Global Campus; Special Research Fund (BOF) of Ghent University (grant number 01N01718) and IMPTOX European Union’s Horizon 2020 research and innovation program (grant number 965173). References [1] S. Sharma, S. Basu, N. P. Shetti, M. N. Nadagouda, T. M. Aminabhavi (2021) Chem. Eng. J., 408, 127317. [2] K. D. Cox, G. A. Covernton, H. L. Davies, J. F. Dower, F. Juanes, S. E. Dudas (2019) Environ. Sci. Technol., 53(12), 7068–7074. [3] A. Brodkorb et al. (2019) Nat. Protoc., 14(4), 991– 1014

Detection and quantification of tropomyosin in differentially treated clams from Korea

Tropomyosin (TM) is known to be a major shrimp allergen (e.g., Pen m 1) and considered a cross-reacting panallergen among shellfish/invertebrates. The clam TM is also considered its major allergen but has not been widely studied. The food processing techniques can alter the TM allergenicity. Hence, the objective of this research is to detect and quantify TM in fresh and differentially treated clams collected in Korea via in-house developed sandwich ELISA protocol to evaluate the effect of various real-life processing techniques on TM stability. Freshly bought live clams (FC), 4 groups of randomly selected equal number of similarly sized clams were differentially treated. Fresh and packaged (FPC), fresh and frozen at -20◦C (FroC). The fresh clams boiled (BC) in boiling water and the marinated clams (MC) suspended in marinade solution for 5 days; soluble protein extracted overnight from 5 samples in PBS buffer with protease inhibitor; BCA assay determined the protein content; capture-detection-enzyme linked secondary antibody in-house ELISA. ELISA was validated with specific antibody based Western blot (WB). The total soluble protein content of raw clams (FC, FPC, FroC) was between 2.8-4.9 mg/ml. The cooked clams (BC, MC) lost total protein during the cooking and was determined FroC (290) >FC (75) >MC. It (and WB) showed that boiling has no effect on heat stable TM IgG binding, BW contained considerable amount of TM (with pronounced IgG binding). MC, however showed no TM epitope recognition in WB (no band in SDS PAGE) and was not quantified by ELISA nor in MS (<LLOQ). Marination might degrade the TM to significant extent possibly altering the allergenicity

Evaluation of in vitro digestion methods and starch structure components as determinants for predicting the glycemic index of rice

Mainstreaming the low glycemic index (GI) trait in breeding programs is constrained by low-throughput and high-cost clinical GI phenotyping. This study aimed to evaluate the potential of starch fine structure components and simulated digestion parameters in predicting GI in rice. Amylose (AM1 and AM2; r = −0.94 and r = −0.80, respectively, p < .05) and amylopectin fine structure (MCAP, SCAP, and SCAP1; r = 0.78-0.86, p < .05) measured through size-exclusion chromatography along with resistant starch (r = −0.81, p < .05) in seven (7) rice accessions showed high correlation with in vivo GI. Meanwhile, starch hydrolysis extent (SH) and the corresponding area under the digestion curve (AUC) obtained through in vitro digestion were found to be of higher correlation with GI, even within shorter digestion periods of 5 min or 30 min (r = 0.96, p < .01). These results highlight the potential use of these parameters as predictors of GI, with improved predictive capacity through a multiple regression model. Higher correlations of simulated digestion AUC with GI may be due to its ability to account for the overall food matrix native macro- and micro-structures, gaining an added advantage over SEC method as a predictive tool in studying rice GI variability. Validation in a larger population is an inevitable next step

MP-Net: Deep learning-based segmentation for fluorescence microscopy images of microplastics isolated from clams.

peer reviewedEnvironmental monitoring of microplastics (MP) contamination has become an area of great research interest, given potential hazards associated with human ingestion of MP. In this context, determination of MP concentration is essential. However, cheap, rapid, and accurate quantification of MP remains a challenge to this date. This study proposes a deep learning-based image segmentation method that properly distinguishes fluorescent MP from other elements in a given microscopy image. A total of nine different deep learning models, six of which are based on U-Net, were investigated. These models were trained using at least 20,000 patches sampled from 99 fluorescence microscopy images of MP and their corresponding binary masks. MP-Net, which is derived from U-Net, was found to be the best performing model, exhibiting the highest mean F1-score (0.736) and mean IoU value (0.617). Test-time augmentation (using brightness, contrast, and HSV) was applied to MP-Net for robust learning. However, compared to the results obtained without augmentation, no clear improvement in predictive performance could be observed. Recovery assessment for both spiked and real images showed that, compared to already existing tools for MP quantification, the MP quantities predicted by MP-Net are those closest to the ground truth. This observation suggests that MP-Net allows creating masks that more accurately reflect the quantitative presence of fluorescent MP in microscopy images. Finally, MAP (Microplastics Annotation Package) is introduced, an integrated software environment for automated MP quantification, offering support for MP-Net, already existing MP analysis tools like MP-VAT, manual annotation, and model fine-tuning

Ovalbumin interaction with polystyrene and polyethylene terephthalate microplastics alters its structural properties

Related to research data no. 1: [https://cherry.chem.bg.ac.rs/handle/123456789/6465]Related to research data no. 2: [https://cherry.chem.bg.ac.rs/handle/123456789/6469

Biocorona formation of hen egg white proteins onto the surface of polystyrene and polyethylene terephthalate

Ovalbumin (OVA), a main protein of egg white, has characteristic structural fold of a serpin-family of proteins, propensity to fibril formation and stability to digestion. Microplastics (MPs) contaminating our food can interact with food proteins in the food matrix and during digestion. In this study adsorption of OVA to polystyrene (PS) (110 μm and 260 μm), polyethylene terephthalate (PET) (140 μm) MPs were investigated in acidic (pH 3) and neutral (pH 7) conditions. Formations of corona on MPs were investigated using isolated OVA and egg white protein extract comparatively. OVA adsorption depends on MPs size, polymer chemistry and pH, being highest in acidic pH and higher for PS. Adsorption of OVA to PS and PET reaches dynamic equilibrium after 4h resulting in disruption of tertiary structure and formation of hard and soft corona around MPs. Shorter fragments of OVA populate hard corona, while soft corona exclusively consist of full length OVA, albeit in its non-native conformation. The conformational changes resemble those induced by heat treatment with re-arrangement of α-β secondary structures. Structural changes are striking for the OVA in corona around MPs. Soft corona OVA preserves thermal and proteolytic stability, but loses ability to form fibrils upon heating. OVA is abundantly present in corona around MPs also in the presence of other egg white proteins. MPs contaminating food may bind and change structure and functional properties of main egg white protein

Developing a Segmentation Model for Microscopic Images of Microplastics Isolated from Clams

peer reviewe

Small polystyrene microplastics interfere with the breakdown of milk proteins during static in vitro simulated human gastric digestion

Human ingestion of microplastics (MPs) is common and inevitable due to the widespread contamination of food items, but implications on the gastric digestion of food proteins are still unknown. In this study, the interactions between pepsin and polystyrene (PS) MPs were evaluated by investigating enzyme activity and conformation in a simulated human gastric environment in the presence or absence of PS MPs. The impact on food digestion was also assessed by monitoring the kinetics of protein hydrolysis through static in vitro gastric digestion of cow's milk contaminated with PS. The binding of pepsin to PS showed that the surface chemistry of MPs dictates binding affinity. The key contributor to pepsin adsorption seems to be π−π interactions between the aromatic residues and the PS phenyl rings. During quick exposure (10 min) of pepsin to increasing concentrations (222, 2219, 22188 particles/mL) of 10 μm PS (PS10) and 100 μm PS (PS100), total enzymatic activities were not affected remarkably. However, upon prolonged exposure at 1 and 2 h, preferential binding of pepsin to the small, low zeta-potential PS caused structural changes in the protein which led to a significant reduction of its activity. Digestion of cow's milk mixed with PS10 resulted in transient accumulation of larger peptides (10–35 kDa) and reduced bioavailability of short peptides (2–9 kDa) in the gastric phase. This, however, was only observed at extremely high PS10 concentration (0.3 mg/mL or 5.46E+05 particles/mL). The digestion of milk peptides, bound preferentially over pepsin within the hard corona on the PS10 surface, was delayed up to 15 min in comparison to bulk protein digestion. Intact caseins, otherwise rapidly digested, remained bound to PS10 in the hard corona for up to 15 min. This work presents valuable insights regarding the interaction of MPs, food proteins, and pepsin, and their dynamics during gastric digestion

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to &lt;90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], &gt;300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of &lt;15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P&lt;0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P&lt;0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Alcohol use and burden for 195 countries and territories, 1990-2016 : a systematic analysis for the Global Burden of Disease Study 2016

Background Alcohol use is a leading risk factor for death and disability, but its overall association with health remains complex given the possible protective effects of moderate alcohol consumption on some conditions. With our comprehensive approach to health accounting within the Global Burden of Diseases, Injuries, and Risk Factors Study 2016, we generated improved estimates of alcohol use and alcohol-attributable deaths and disability-adjusted life-years (DALYs) for 195 locations from 1990 to 2016, for both sexes and for 5-year age groups between the ages of 15 years and 95 years and older. Methods Using 694 data sources of individual and population-level alcohol consumption, along with 592 prospective and retrospective studies on the risk of alcohol use, we produced estimates of the prevalence of current drinking, abstention, the distribution of alcohol consumption among current drinkers in standard drinks daily (defined as 10 g of pure ethyl alcohol), and alcohol-attributable deaths and DALYs. We made several methodological improvements compared with previous estimates: first, we adjusted alcohol sales estimates to take into account tourist and unrecorded consumption; second, we did a new meta-analysis of relative risks for 23 health outcomes associated with alcohol use; and third, we developed a new method to quantify the level of alcohol consumption that minimises the overall risk to individual health. Findings Globally, alcohol use was the seventh leading risk factor for both deaths and DALYs in 2016, accounting for 2.2% (95% uncertainty interval [UI] 1.5-3.0) of age-standardised female deaths and 6.8% (5.8-8.0) of age-standardised male deaths. Among the population aged 15-49 years, alcohol use was the leading risk factor globally in 2016, with 3.8% (95% UI 3.2-4-3) of female deaths and 12.2% (10.8-13-6) of male deaths attributable to alcohol use. For the population aged 15-49 years, female attributable DALYs were 2.3% (95% UI 2.0-2.6) and male attributable DALYs were 8.9% (7.8-9.9). The three leading causes of attributable deaths in this age group were tuberculosis (1.4% [95% UI 1. 0-1. 7] of total deaths), road injuries (1.2% [0.7-1.9]), and self-harm (1.1% [0.6-1.5]). For populations aged 50 years and older, cancers accounted for a large proportion of total alcohol-attributable deaths in 2016, constituting 27.1% (95% UI 21.2-33.3) of total alcohol-attributable female deaths and 18.9% (15.3-22.6) of male deaths. The level of alcohol consumption that minimised harm across health outcomes was zero (95% UI 0.0-0.8) standard drinks per week. Interpretation Alcohol use is a leading risk factor for global disease burden and causes substantial health loss. We found that the risk of all-cause mortality, and of cancers specifically, rises with increasing levels of consumption, and the level of consumption that minimises health loss is zero. These results suggest that alcohol control policies might need to be revised worldwide, refocusing on efforts to lower overall population-level consumption.Peer reviewe