Phycocyanobilin (PCB) is a kind of light-harvesting pigment which naturally exists in algae and plays important roles in absorbing and transferring energy. Based on its antioxidant and optical properties, PCB has been applied in food, medicine, and cosmetics. Currently, PCB is mainly extracted from Spirulina through complicated steps; thus, the biosynthesis of PCB in Escherichia coli has attracted more attention. However, due to the lower catalytic efficiency of synthetic enzymes and the deficiency of precursors and cofactors, the titer of PCB remains at a low level. Here, we report the efficient synthesis of PCB by the expression of heme oxygenase-1 from Thermosynechococcus elongatus and PCB: ferredoxin oxidoreductase (PcyA) from Synechocystis sp. using a high-copy number plasmid with an inducible T7lac promoter and the assembly of these two enzymes at a suitable ratio of 2:1 with DNA scaffolds. Additionally, the synthesis of PCB was further enhanced by direct supplementation of 5-aminolevulinic acid (ALA), moderate overexpression of key enzymes in the heme biosynthetic pathway (hemB and hemH), and accelerated cycle of cofactors (NADPH) through the expression of NAD + kinase and the addition of a reducing agent. Finally, based on the optimal conditions (Modified R medium with 200 mg/L ALA, 20 mg/L FeSO 4 •7H 2 O, and 5 g/L vitamin C induced by 0.8 mM isopropylthio-β-galactoside at 30 °C), the highest reported titer of PCB (28.32 mg/L) was obtained at the fermenter level by feeding glucose and FeSO 4 •7H 2 O. The strategies applied in this study will be useful for the synthesis of other natural pigments and PCB or heme derivatives in E. coli.
Background and Aims: Endoscopic ultrasound-guided gastroenterostomy (EUS-GE) is a minimally invasive treatment for gastric outlet obstruction that results in a low incidence of recurrence. This is the first study to investigate a suitable position for performing EUS-GE. Methods: We reviewed the clinical data from 20 upper gastrointestinal examinations and 20 computed tomography scans of the abdomen and hypothesized that the posterior wall of the stomach was a suitable place to perform EUS-GE. We verified this hypothesis by performing an animal study using 5 swine. Results: All retrievable anchor assisted EUS-GE were successfully performed at the intestinal region that was proximal to the head of the pancreas. The mean procedure time of EUS-GE was 28.4 ± 3.85 minutes (range of 22–32 minutes). There were no adverse events, such as bleeding. Necropsy of the animals showed that all of the stents were in the correct position. The mean distance between the site of gastroenterostomy and pylorus was 34.6 ± 3.58 cm (range of 30–38 cm). Conclusions: The intestinal region that was proximal to the head of the pancreas was a suitable place for performing endoscopic ultrasound-guided gastroenterostomy. The suitability of this location should be confirmed by further animal and clinical studies.
e15519 Background: In recent years, the etiology and molecular mechanism of early-onset colorectal cancers (EOCRC) has evoking more and more attention. Previous studies suggested that EOCRC had unique somatic mutational characteristics, such as frequent mutations in genes of WNT signaling pathway. However, the features of chromosome instability in this particular subgroup of colorectal cancer remain unclear. Somatic copy number alteration (SCNA) is the main manifestation of chromosome instability. We performed a systematic analysis in regard to SCNAs in a large cohort of Chinese colorectal. Since the microsatellite instable (MSI-H) cancers rarely occurred SCNAs, only microsatellite stable (MSS) patients were included. Methods: A total of 1696 Chinese MSS CRC patients diagnosed in 2018-2020 were included. Early-onset were defined as patients diagnosed under 50 year-olds and the others were late-onset (LO). Genomic alterations and SCNAs were elucidated in all patients’ tumor specimen using next-generation sequencing (NGS) testing. Results: All patients were stratified as EOCRC (n = 439) and LOCRC (n = 1214). 3.87% EOCRC harbored germline cancer susceptibility genes (mainly APC and ATM) mutations while in the LOCRC counterpart the proportion was only 2.06% (mainly MUTYH and BRCA2). Of note, MYC amplification was detected in 71.43% EOCRC with copy-number gain and presented a trend to correlated with APC germline mutations (Fisher’s exact test, p-value = 0.07). In the other CRC (n = 1654) with no germline cancer susceptibility genes, genomic amplifications were detected in 831 (50.2%) cases. The frequency of 8q and 13q amplification were equal in EOCRC and LOCRC. However, EOCRC had higher proportion of 8p and 17q amplification, lower proportion of 20q amplification than LOCRC. Moreover, a higher ratio of EOCRC had high-level amplifications, mainly concentrated in 17q ( ERBB2, CDK12 and RARA) and 13q ( FLT3, CDK8 and FLT1), than LOCRC (23.7% vs 19.1%). Other high-level amplified genes include MYC, CCND2 and EGFR. EGFR high-level amplifications occurred more in EOCRC than LOCRC (13.5 % 2.6%), while CCND2 high-level amplifications were only detected in LOCRC. Gene copy number losses were detected in 195 (11.8%) cases. Loss of SMAD4, CDKN2A, CDKN2B, MAP2K4, PTEN and AMER1 genes were most recurrent. The incidence of SMAD4 (43.1% vs 22.9%), CDKN2B (11.8% vs 6.2%), MAP2K4 (7.8% vs 4.2%) gene losses were higher in EOCRC while the incidence of PTEN (13.9% vs 7.8%) and SMAD3 (6.9% vs 1.9%) gene losses were higher in LOCRC. Conclusions: EOCRC present different SCNA characteristics from LOCRC, including unique focal amplifications, distinct recurrent genes with copy number losses, and more high-level amplifications events mainly distributed in 17q and 13q. It can be reasonably speculated that MYC gene gain was a critical event correlated with familial mutations in EOCRC.
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