Impact of MPs on trypsin activity in simulated intestinal fluid

Mircoplastics (MPs) are an abundant contaminant in the environment with ingestion being the most common way of exposure for humans. Binding of protein to MPs is proposed to be multilayered with the formation of a soft and hard corona. It has been proven that MPs interact with enzymes present in the digestive system and impact their activity. The aim of this study is to investigate the impact of MPs on the activity of trypsin in simulated intestinal fluid (SIF). For this purpose, two sizes of polypropylene (large – 180-500 μm, small – 63-180 μm) and one size of polyethylene terephthalate (<80 μm) have been studied. Activity in bulk and soft corona was determined in SIF at 405 nm with N-α- Benzoyl-DL-arginine 4-nitroanilide hydrochloride after different times of incubation. Activity in hard corona was determined after 1 h of incubation with the MPs. Although specific activity in the control decreases through time, there is a tendency for all MPs to preserve activity in bulk and soft corona trypsin after 4 h of incubation. Trypsin remains active in the hard corona, with the activity being an order of magnitude lower than in the control, possibly due to significant changes in structure

Impact of MPs on trypsin activity in simulated intestinal fluid

Mircoplastics (MPs) are an abundant contaminant in the environment with ingestion being the most common way of exposure for humans. Binding of protein to MPs is proposed to be multilayered with the formation of a soft and hard corona1. It has been proven that MPs interact with enzymes present in the digestive system and impact their activity2. The aim of this study is to investigate the impact of MPs on the activity of trypsin in simulated intestinal fluid (SIF). For this purpose, two sizes of polypropylene (large – 180-500 μm, small – 63-180 μm) and one size of polyethylene terephthalate (<80 μm) have been studied. Activity in bulk and soft corona was determined in SIF at 405 nm with Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride after different times of incubation. Activity in hard corona was determined after 1 h of incubation with the MPs. Although specific activity in the control decreases through time, there is a tendency for all MPs to preserve activity in bulk and soft corona trypsin after 4 h of incubation. Trypsin remains active in the hard corona, with the activity being an order of magnitude lower than in the control, possibly due to significant changes in structure. Acknowledgements This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 965173. References 1. Monopoli MP, Åberg C, Salvati A, Dawson, KA. Biomolecular coronas provide the biological identity of nanosized materials. Nat Nanotechnol 2012;7:779-86. 2. de Guzman MK, et al. Small polystyrene microplastics interfere with the breakdown of milk proteins during static in vitro simulated human gastric digestion. Environ Pollut 2023;335:122282

Redox status of critical disulfides of SARS-CoV-2 receptor-binding-domain exposed to bioactive chomophore phycocyanobilin

The emergence of the novel coronavirus SARS-CoV-2 has attracted the attention of the whole scientific community. However, as there are significant concerns regarding the effectiveness of vaccines and drugs against novel SARS-CoV-2 variants, naturally derived broad-spectrum of antivirals seems to be precious adjuvant agents to assist in combat against this disease. Phycocyanobilin (PCB) is an open-chain tetrapyrrole chromophore of phycocyanin (PC), chromoprotein derived from Spirullina, with strong anti-oxidative action. The role of disulfide bonds and thiol-disulfide balance in RBD is considered to play a significant role in the binding of S protein to ACE2 receptor. In RBD, in contrast to C480–C488 disulfide, which is thermodynamically stable, C379–C432 and C391–C525 disulfides are in dynamic equilibrium with their thiol states and, thus these two pairs of disulfides are more sensitive to changes in redox poise. Our study aimed to investigate impact of PCB on disulfide balance of RBD by redox proteomics and to investigate structural changes in the protein exposed to PCB. The effect of PCB on RBD secondary structures was examined by far-UV CD spectroscopy after titration of RBD with increasing concentrations of PCB. The presence of PCB had a pronounced effect on the spectral shape. RBD is dominantly composed of random coils and β-sheets. In the presence of PCB a slight increase of α-helical and random coils content, while the content of β-sheets and β-turns is decreased. Mapping redox-active disulfides and reactive cysteines in recombinant SARS-CoV-2 RBD was done using redox proteomics on both recombinant RBD and PCB-exposed RBD. A mass shift caused by alkylation of free Cys residues was detected on three Cys residues demonstrating disulfides C379–C432 and 432-391 to be semi-stable in both RBD and PCB-exposed RBD. Our results demonstrate that RBD exposed to PCB undergo structural changes but does not change the redox state of its critical semi-stable disulfides. Acknowledgment: The authors acknowledge support of the special Research Fund (BOF) of Ghent University (grant number 01N01718) and the Ministry of Science, Innovation and technological development of the Republic of Serbia (Contract number: 451-03-68/2023-14/200168).Book of Abstract

Development of immuno-PCR for sensitive quantification of SARS-CoV-2 nucleocapsid protein

Accurately diagnosing people with suspected SARS-CoV-2 infection is essential to help manage COVID-19. Currently available SARS-CoV-2 diagnostics detect either RNA of the virus by RT-PCR or the presence of viral antigens in biological fluids by ELISA or similar techniques. Low sensitivity of antigen tests could lead to the risk of false negative results. Therefore, this study aimed to develop a highly sensitive immuno-PCR method for quantifying SARS-CoV-2 nucleocapsid (N) protein that combines the specificity of sandwich ELISA with the sensitivity of PCR. Recombinant N protein fragment was produced in E. coli as an expression system and purified using immobilized metal ion affinity chromatography. The antibodies against the N protein were raised in rabbits and mice. High-affinity polyclonal mice and rabbit N protein-specific antisera were purified using ammonium sulfate precipitation and used to develop sandwich ELISA for the quantification of N protein. Mice polyclonal serum was used as a capture for N protein. N protein bound to mice antibodies was detected with rabbit polyclonal sera. A double-stranded amino-DNA molecule of 77 base pairs was PCR-synthesized, covalently conjugated to a secondary goat anti-rabbit antibody and subsequently amplified and quantified by real-time PCR. The results were compared to analogous sandwich ELISA consisting of alkaline phosphatase-labeled goat anti-rabbit antibody. The sensitivity of immuno-PCR for quantification of N protein was increased by up to 7-fold compared to analogous ELISA, having a limit of detection of 92 pg/mL and a limit of quantification of 840 pg/mL. The developed immuno-PCR method thus has the potential to be used as a new antigen test for COVID-19 and beyond

Proizvodnja i prečišćavanje fragmenta N proteina SARS-Cov-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2) je u periodu od 2 godine uzrokovao smrt miliona ljudi. Brza dijagnostika je prvi front borbe protiv ovog virusa. Razvijanje ELISA tehnika za dijagnostifikovanje, praćenje toka bolesti, ali i efikasnosti vakcina, sada već uveliko dostupnih, su od velikog značaja. Ekspresija nukleokapsida - N proteina, jednog od strukturnih proteina SARS-CoV-2, u bakterijskom sistemu je brza, reproducibilna i jeftina u poređenju sa drugim sistemima, a daje odgovarajući protein kompatibilan za ELISA testove. U ovom radu je eksprimiran fragment N proteina jer je pokazano da ceo N protein daje lažno pozitivne rezultate zbog konzerviranog N - terminalnog N domena. N protein je proizveden u E. coli BL21, a zatim je prečišćen i proverena je njegova i reaktivnost u ELISA testu sa serumima pacijenata koji su preležali ovaj virus i razvili antitela na ovaj strukturni protein

Uvid u kovalentne interakcije Spike proteina SARS – CoV – 2 i fikocijanobilina

SARS-Cov-2 virus (eng. Severe Acute Respiratory Syndrome Coronavirus 2) je u periodu od 4 godine uzrokovao smrt više od 6 miliona ljudi. Jedna od strategija za borbu protiv ovog virusa jeste upotreba i razvijanje malih molekula koji bi mogli da spreče njegovu fuziju sa i ulazak u ćeliju domaćina. Ključni protein u procesu infekcije je protein „šiljka“ (eng. Spike, S). Ovaj transmembranski protein virusa interaguje sa angiotenzin konvertujućim enzimom 2 (eng. Angiotensin converting enzyme, ACE2) na ćeliji domaćina i katalizuju ulazak virusne RNA u ćeliju domaćina. Fikocijanobilin (eng. Phycocyanobiline, PCB) je hromofora izolovana iz alge Spironella pacifica. PCB poseduje antioksidativnu i antiviralnu sposobnost. U ovom radu je elektroforetskim metodama, testovima vezivanja za ACE2 receptor i masenom spektrometrijom pružen uvid u interakcije PCBna sa komercijalno dostupnim receptor vezujućim domenom (eng. Receptor Binding Domain, RBD) S proteina