Enhancing the expression of chondroitin 4-O-sulfotransferase for one-pot enzymatic synthesis of chondroitin sulfate A.

Chondroitin sulfate (CS) stands as a pivotal compound in dietary supplements for osteoarthritis treatment, propelling significant interest in the biotechnological pursuit of environmentally friendly and safe CS production. Enzymatic synthesis of CS for instance CSA has been considered as one of the most promising methods. However, the bottleneck consistently encountered is the active expression of chondroitin 4-O-sulfotransferase (C4ST) during CSA biosynthesis. This study meticulously delved into optimizing C4ST expression through systematic enhancements in transcription, translation, and secretion mechanisms via modifications in the 5' untranslated region, the N-terminal encoding sequence, and the Komagataella phaffii chassis. Ultimately, the active C4ST expression escalated to 2713.1 U/L, representing a striking 43.7-fold increase. By applying the culture broth supernatant of C4ST and integrating the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis module, we constructed a one-pot enzymatic system for CSA biosynthesis, achieving a remarkable sulfonation degree of up to 97.0 %. The substantial enhancement in C4ST expression and the development of an engineered one-pot enzymatic synthesis system promises to expedite large-scale CSA biosynthesis with customizable sulfonation degrees.

Multi-phasic CT arterial portography and CT hepatic arteriography improving the accuracy of liver cancer detection.

AIM: To evaluate the value of multi-phasic CT arterial portography (CTAP) and CT hepatic arteriography (CTHA) in differential diagnosis of liver diseases, and to improve the specificity of CTAP and CTHA for liver cancer detection. METHODS: From January 1999 to December 2002, multi-phasic CTAP and CTHA were performed in 20 patients with suspected liver disease. CT scanning was begun 25 s, 60 s and 120 s for the early-, late- and delayed-phase CTAP examinations, and 6 sec, 40 s and 120 s for the early-, late- and delayed-phase CTHA examinations respectively, after a transcatheter arterial injection of non-ionic contrast material. If a lesion was diagnosed as a liver cancer, transcatheter hepatic arterial chemoembolization (TACE) treatment was performed, and the follow-up CT was performed three or four weeks later. RESULTS: All eighteen HCCs in 12 cases were shown as nodular enhancement on early-phasic CTHA. The density of the whole tumor decreased rapidly on late and delayed phases, and the edge of 12 tumors (12/18) remained relatively hyperdense compared with the surrounding liver tissue, and demonstrated as rim enhancement. All HCCs were shown as perfusion defect nodules on multi-phasic CTAP. Five tumors (5/18) were shown as rim enhancement on delayed-phasic CTAP. Rim enhancement was shown as 1 to 2-mm-wide irregular, uneven and discontinuous circumferential enhancement at late-, and delayed-phase of CTHA or CTAP. Five pseudolesions and 4 hemoangiomas were found in multi-phasic CTAP and CTHA. No pseudolesions and hemoangiomas were shown as rim enhancement on late- or delayed-phasic CTHA and CTAP. CONCLUSION: Multi-phasic CTAP and CTHA could help to recognize the false-positive findings in CTAP and CTHA images, and improve the accuracy of CTAP and CTHA of liver cancer detection.