RESUMEN
The current investigation focused on separating Cerastes cerastes venom to produce the first Kunitz-type peptide. Based on its anti-trypsin effect, Cerastokunin, a 7.75 kDa peptide, was purified until homogenity by three steps of chromatography. Cerastokunin was found to include 67 amino acid residues that were obtained by de novo sequencing using LC-MALDI-MSMS. Upon alignment with Kunitz-type peptides, there was a high degree of similarity. Cerastokunin's 3D structure had 12% α-helices and 21% ß-strands with pI 8.48. Cerastokunin showed a potent anticoagulant effect by inhibiting the protease activity of thrombin and trypsin as well as blocking the intrinsic and extrinsic coagulation pathways. In both PT and aPPT, Cerastokunin increased the blood clotting time in a dose-dependent way. Using Lys48 and Gln192 for direct binding, Cerastokunin inhibited thrombin, Factor Xa and trypsin as shown by molecular docking. Cerastokunin exhibited a dose-response blockade of PARs-dependent pathway platelet once stimulated by thrombin. An increased concentration of Cerastokunin resulted in a larger decrease of tail thrombus in the mice-carrageenan model in an in vivo investigation when compared to the effects of antithrombotic medications. At all Cerastokunin doses up to 6 mg/kg, no in vivo toxicity was seen in challenged mice over the trial's duration.
RESUMEN
Antibiotic residues in milk are a major health threat for the consumer and a hazard to the dairy industry, causing significant economic losses. This study aims to assess the presence of antibiotic residues in raw milk comparatively by a rapid screening test (BetaStar® Combo) and Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS). A total of 445 samples were collected from 3 dairy companies of north-central Algeria (Algiers, Blida, Boumerdes), and they were rapidly screened for ß-lactams and tetracyclines; 52 samples, comprising 34 positive tanker-truck milk and 18 negative bulk-tank milk were tested by LC-MS/MS, which revealed 90.4% were contaminated (n = 47) and 55.3% exceeded the Maximum Residue Limit (MRL). The ß-lactams as parent compounds and their metabolites were the most frequently detected with maximum value for cloxacillin (1231 µg/kg) and penicillin G (2062 µg/kg). Under field condition, the false-positive results, particularly for tetracyclines, seems to be related to milk samples displaying extreme acidity values (≥19°D) or fat-level fluctuations (2.7 g/100 mL and 5.6-6.2 g/100 mL). Despite a relatively low prevalence (7.64%) of residues using the rapid test, the detection by LC-MS/MS of flumequine (52 µg/kg), cefaclor (maximum 220 µg/kg) and metabolites of ß-lactams at high levels should lead to reflections on the control of their human and environmental toxicological effects.