Detection and Quantification of Tp53 and p53-Anti-p53 Autoantibody Immune Complex: Promising Biomarkers in Early Stage Lung Cancer Diagnosis.

Lung cancer is a leading cause of death worldwide, claiming nearly 1.80 million lives in 2020. Screening with low-dose computed tomography (LDCT) reduces lung cancer mortality by about 20% compared to standard chest X-rays among current or heavy smokers. However, several reports indicate that LDCT has a high false-positive rate. In this regard, methods based on biomarker detection offer excellent potential for developing noninvasive cancer diagnostic tests to complement LDCT for detecting stage 0∼IV lung cancers. Herein, we have developed a method for detecting and quantifying a p53-anti-p53 autoantibody complex and the total p53 antigen (wild and mutant). The LOD for detecting Tp53 and PIC were 7.41 pg/mL and 5.74 pg/mL, respectively. The detection ranges for both biomarkers were 0-7500 pg/mL. The known interfering agents in immunoassays such as biotin, bilirubin, intra-lipid, and hemoglobin did not detect Tp53 and PIC, even at levels that were several folds higher levels than their normal levels. Furthermore, the present study provides a unique report on this preliminary investigation using the PIC/Tp53 ratio to detect stage I-IV lung cancers. The presented method detects lung cancers with 81.6% sensitivity and 93.3% specificity. These results indicate that the presented method has high applicability for the identification of lung cancer patients from the healthy population.

Artificial cell factory design for shikimate production in Escherichia coli.

Shikimate is a key intermediate in high demand for synthesizing valuable antiviral drugs, such as the anti-influenza drug and oseltamivir (Tamiflu®). Microbial-based shikimate production strategies have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. Although shikimate biosynthesis has been reported in several engineered bacterial species, the shikimate production yield is still unsatisfactory. This study designed an Escherichia coli cell factory and optimized the fed-batch culture process to achieve a high titer of shikimate production. Using the previously constructed dehydroshikimate (DHS)-overproducing E. coli strain, two genes (aroK and aroL) responsible for converting shikimate to the next step were disrupted to facilitate shikimate accumulation. The genes with negative effects on shikimate biosynthesis, including tyrR, ptsG, and pykA, were disrupted. In contrast, several shikimate biosynthetic pathway genes, including aroB, aroD, aroF, aroG, and aroE, were overexpressed to maximize the glucose uptake and intermediate flux. The shiA involved in shikimate transport was disrupted, and the tktA involved in the accumulation of both PEP and E4P was overexpressed. The rationally designed shikimate-overproducing E. coli strain grown in an optimized medium produced approximately 101 g/l of shikimate in 7-l fed-batch fermentation, which is the highest level of shikimate production reported thus far. Overall, rational cell factory design and culture process optimization for microbial-based shikimate production will play a key role in complementing traditional plant-derived shikimate production processes.

Zebrafish Crip2 plays a critical role in atrioventricular valve development by downregulating the expression of ECM genes in the endocardial cushion.

The initial step of atrioventricular (AV) valve development involves the deposition of extracellular matrix (ECM) components of the endocardial cushion and the endocardial-mesenchymal transition. While the appropriately regulated expression of the major ECM components, Versican and Hyaluronan, that form the endocardial cushion is important for heart valve development, the underlying mechanism that regulates ECM gene expression remains unclear. We found that zebrafish crip2 expression is restricted to a subset of cells in the AV canal (AVC) endocardium at 55 hours post-fertilization (hpf). Knockdown of crip2 induced a heart-looping defect in zebrafish embryos, although the development of cardiac chambers appeared to be normal. In the AVC of Crip2-deficient embryos, the expression of both versican a and hyaluronan synthase 2 (has2) was highly upregulated, but the expression of bone morphogenetic protein 4 (bmp4) and T-box 2b (tbx2b) in the myocardium and of notch1b in the endocardium in the AVC did not change. Taken together, these results indicate that crip2 plays an important role in AV valve development by downregulating the expression of ECM components in the endocardial cushion.