Comparative analysis of two timepoints on [18F]FAPI-42 PET/CT in various cancers.

PURPOSE: This study aimed to assess the biodistribution, detection rate, and uptake of the [18F]FAPI-42 at two distinct time intervals. METHODS: This prospective study enrolled 60 consecutive patients (median age 59; range 35-74) referred to [18F]FAPI-42 PET/CT. [18F]FAPI-42 PET/CT was performed early and late timepoint after tracer injection for staging or restaging. Positive lesions specified for anatomic locations (primary or recurrent tumor, LN metastasis and other metastasis) by visual analysis at both timepoints. Semiquantitative analysis of the tracer activity in lesions as well as normal tissues at both time points were measured and compared. In a subgroup analysis, eleven patients underwent 2-[18F]FDG PET/CT within 1 week, the detection rate and uptake of lesion were compared between early [18F]FAPI-42 and 2-[18F]FDG. RESULTS: Uptake of [18F]FAPI-42 in the late timepoint was significantly lower than the early timepoint in most organs (all p < 0.05), except for bone (SUVmean 0.88 vs. 0.85; p = 0.218). Tracer retention at biliary system showed less frequent at early timepoint than late timepoint. A total of 194 lesions were detected in 60 patients. One lesion was only seen at early timepoint but not at late timepoint. Lesions on early [18F]FAPI-42 PET/CT had higher visual score than that of late image(23 vs. 6). The uptake of lesion decreased significantly from early to late timepoint (all p < 0.05). In subgroup analysis, early [18F]FAPI-42 illustrated higher detection rate, visual score, and uptake of lesion than that of 2-[18F]FDG PET/CT. CONCLUSION: Early [18F]FAPI-42 PET/CT provided consistent detection rates and lesion uptake, but less tracer retention in the biliary system compared to late images. Therefore, acquisition at early timepoint could be a feasible strategy for improving acquisition protocols of [18F]FAPI-42 PET/CT. TRIAL REGISTRATION: ChiCTR2200063441. Registered 28 September 2022-Retrospectively registered, https://www.chictr.org.cn/bin/project/edit?pid=149714 .

Engineering of a fungal steroid 11α-hydroxylase and construction of recombinant yeast for improved production of 11α-hydroxyprogesterone.

Cytochrome P450 enzyme CYP68J5 from filamentous fungus Aspergillus ochraceus is industrially used for selective C11α-hydroxylation of canrenone and progesterone. To improve its selectivity of C11α-hydroxylation for relevant steroid substrates, a sequence-based targeted mutagenesis combined with saturation mutagenesis was conducted to search for variants with improved hydroxylation reaction specificity toward progesterone and D-ethylgonendione. Recombinant yeast expressing triple mutant V64F/E65G/N66T showed significantly increased C11α-hydroxylation selectivity (85 % VS WT 69.7 %). Saturation mutagenesis of V64, E65 and N66 resulted in the identification of single mutant V64K with greatly enhanced 11α-hydroxylation specificity toward progesterone (90.6 % VS WT 69.7 %). Furthermore, mutant N66D showed significant enhanced selectivity of C11α-hydroxylation toward D-ethylgonendione (70.8 % VS WT 58 %). Evaluation of recombinant yeast over-expressing V64K for progesterone transformation in 50 mL scale resulted in product 11α-OH progesterone concentrations of 432.5 mg/L, a 30.2 % increase compared with the CYP68J5 control. Our results also reveal that V64, E65 and N66 are key residues of CYP68J5 influencing its selectivity of C11α-hydroxylation, thus offering opportunities for further engineering of CYP68J5 for expanded industrial applications.

Grouper (Epinephelus coioides) MyD88 adaptor-like (Mal): Molecular cloning, expression, and functionality.

Initiation of the innate immune response requires recognition of pathogen-associated molecular patterns by pathogen recognition receptors such as Toll-like receptors (TLRs). MyD88 adaptor-like (Mal) is an adaptor that responds to TLR activation and acts as a bridging adaptor for MyD88. In the present study, the open reading frame of Mal was identified in orange-spotted grouper (Epinephelus coioides), and named EcMal. It contained 831 bp encoding 276 aa, and was encoded by a 1299 bp DNA sequence with three exons and two introns. EcMal and the Mal sequence of other species shared different degrees of sequence identity, and clustered into the same group. EcMal was distributed in all tissues tested in healthy grouper, with the highest expression level in the head kidney. After infection with Cryptocaryon irritans, the expression level of EcMal was up-regulated in the gill and spleen. In addition, EcMal exhibited global cytosolic and nucleus localization, and could significantly activate NF-κB activity in grouper spleen cells.

Molecular characterization and function analysis of grouper (Epinephelus coioides) Bruton's tyrosine kinase BTK.

Bruton's tyrosine kinase (BTK) is a Tec-family tyrosine kinase and plays a crucial role in B cell antigen receptor (BCR) signal pathway. Mutations in humans and mice BTK gene results in X-linked agammaglobulinemia (XLA) and X-linked immunodeficiency (XLD), respectively. To study the function of BTK in teleost, we cloned a BTK gene from orange-spotted grouper. Homology analysis showed that the grouper BTK (EcBTK) had a high amino acid identity with other vertebrates (63%-92%) and shared the highest amino acid identity with ballan wrasse Labrus bergylta BTK. EcBTK comprises a Bruton's tyrosine kinase pleckstrin homology (PH) domain, a Tec homology (TH) domain, a Src homology 3 (SH3) domain, a Src homology 2 (SH2) domain and a Protein Kinases, catalytic (PKc) domain. Tissue distribution analysis showed that EcBTK was mainly expressed in immune organs. EcBTK was uniform distributed throughout the cytoplasm of transfected HEK293T cells and overexpression of EcBTK slightly down-regulates NF-κB activity. Ibrutinib treatment can reduce the phosphorylation level of grouper's BTK. In groupers infected with Cryptocaryon irritans, up-regulation of EcBTK were not seen in the early stage of infected skin and gill until days 14-21. The phosphorylation level of grouper BTK was significantly increased in infected skin and gill.