Abstract Transcriptional factor (TF)‐based metabolite‐responsive biosensors are important tools for screening engineered enzymes with desired properties and for the dynamic regulation of biosynthetic pathways. However, TF‐based biosensor construction is often constrained by undesired effects of TF‐binding site sequence insertion on gene expression and unpredictable optimal TF expression levels. In the present study, a stepwise TF‐based biosensor construction approach was developed using an N‐acetylneuraminic acid (NeuAc) biosensor for Bacillus subtilis , as a case study. Specifically, 12 promoters with various strengths were selected as the first promoter library. Next, binding site sequences for the NanR were inserted into various positions of the selected promoter sequences to develop the second promoter library, resulting in 6 engineered promoters containing TF‐binding site sequences (NanO), without major effects on promoter strength. NanR expression cassettes with different expression levels were further integrated to construct the biosensor library, yielding 9 NeuAc biosensors with efficient repression in the absence of NeuAc. Finally, biosensor activation was characterized by testing fold changes in expression levels of the green fluorescent protein reporter in the presence of NeuAc in vivo , which revealed 61‐fold activation when NeuAc was present. The NeuAc biosensor developed in this study can be used for screening engineered enzymes for enhanced NeuAc biosynthesis in B. subtilis .
<div>Abstract<p>No special-type breast cancer [NST; commonly known as invasive ductal carcinoma (IDC)] and invasive lobular carcinoma (ILC) are the two major histological subtypes of breast cancer with significant differences in clinicopathological and molecular characteristics. The defining pathognomonic feature of ILC is loss of cellular adhesion protein, E-cadherin (<i>CDH1</i>). We have previously shown that E-cadherin functions as a negative regulator of the IGF1R and propose that E-cadherin loss in ILC sensitizes cells to growth factor signaling that thus alters their sensitivity to growth factor–signaling inhibitors and their downstream activators. To investigate this potential therapeutic vulnerability, we generated CRISPR-mediated <i>CDH1</i> knockout (<i>CDH1</i> KO) IDC cell lines (MCF7, T47D, and ZR75.1) to uncover the mechanism by which loss of E-cadherin results in IGF pathway activation. <i>CDH1</i> KO cells demonstrated enhanced invasion and migration that was further elevated in response to IGF1, serum and collagen I. <i>CDH1</i> KO cells exhibited increased sensitivity to IGF resulting in elevated downstream signaling. Despite minimal differences in membranous IGF1R levels between wild-type (WT) and <i>CDH1</i> KO cells, significantly higher ligand–receptor interaction was observed in the <i>CDH1</i> KO cells, potentially conferring enhanced downstream signaling activation. Critically, increased sensitivity to IGF1R, PI3K, Akt, and MEK inhibitors was observed in <i>CDH1</i> KO cells and ILC patient-derived organoids.</p>Implications:<p>Overall, this suggests that these targets require further exploration in ILC treatment and that <i>CDH1</i> loss may be exploited as a biomarker of response for patient stratification.</p></div>
Colorectal cancer (CRC) is a type of malignant gastroenteric tumors associated with a high mortality rate worldwide. Calycosin, a natural phytoestrogen, possesses potent anti-cancer properties. We structurally modified calycosin to improve its physicochemical properties, and generated a novel small molecule termed CA028. By using network pharmacology, followed by gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis and molecular docking, we aimed to predict and disclose the biological functions and mechanism of CA028 in the treatment of CRC through bioinformatic analyses. By searching the online Swiss Target Prediction and TargetNet databases, we identified 150 genes shared by CA028 and CRC. Using the Search Tool for the Retrieval of Interacting Genes (STRING) database and Cytoscape software, we identified 14 hub-functional genes, namely the FYN proto-oncogene, a Src family tyrosine kinase (FYN), mitogen-activated protein kinase 1 (MAPK1), MAPK8, MAPK14, Rac family small GTPase 1 (RAC1), epidermal growth factor receptor (EGFR), protein tyrosine kinase 2 (PTK2), sphingosine-1-phosphate receptor 1 (S1PR1), S1PR2, Janus kinase 1 (JAK1), JAK2, the RELA proto-oncogene NF-κB subunit (RELA), bradykinin receptor B1 (BDKRB1), and BDKRB2. Additionally, biological docking analysis using the Autodock Vina software revealed that FYN and MAPK1 were the main pharmacological proteins of CA028 against CRC. The gene ontology analysis using R-language packages further revealed the anti-CRC functions of CA028, including biological processes, cell components, and molecular pathways. CA028 exhibits effective pharmacological activity against CRC by suppressing the proliferation of CRC cells and improving the tumor microenvironment. Importantly, certain predicted genes (e.g., FYN and MAPK1) may be the pharmacological targets of CA028 in the treatment of CRC.
Abstract The purpose of this study is to explore the application value of positioning based on the treatment couch height in radiotherapy for left-sided breast cancer. Sixty patients who had undergone radical mastectomy for left breast cancer were selected, with each patient undergoing positioning based on the treatment couch height (couch height group) and positioning based on the reference marking lines (reference line group), to obtain the corresponding positioning errors. Meanwhile, 20 of 60 patients were randomly selected, and the planning system was used to simulate the changes in radiation doses in planning target volume (PTV) and organs at risk (OAR) along with the changes in positioning errors in dorsal (increasing couch height) and ventral (decreasing couch height), respectively. Compared with the original plan, when the positioning error in the dorsal direction reached 3mm, Dmean, V30, and V20 in The ipsilateral lung were increased by 35.12%, 16.35%, and 10.6% respectively, and V50 in PTV was decreased by 0.99% (all p < 0.05); when the positioning error in the ventral direction reached 1.5 mm, V50, V48, and V45 were decreased by 2.07%, 0.58%, and 0.14% respectively. The homogeneity index (HI) was increased by 14.28% (all p < 0.05). There was a statistically significant difference in the positioning errors in the ventral-dorsal directions between the couch height group (0.16±0.14 cm) and reference line group (0.36±0.25 cm) (p < 0.05); the percentages of the absolute positioning errors within 1.5mm and 3mm were 52.4%, 88.7% respectively in the couch height group and 29.8%, 54.4% respectively in the reference line group, (all p < 0.05). Dorsal positioning errors greater than 3 mm significantly worsen the dose distribution for both the PTV and OAR, while positioning based on the treatment couch height keeps 88.7% of positioning errors within 3 mm; ventral positioning errors greater than 1.5 mm result in significant changes in the dose within the PTV. Compared to the reference line group, positioning based on the treatment couch height controls 52.4% of positioning errors within 1.5 mm. Therefore, couch height positioning demonstrates greater advantages in managing ventral-dorsal positioning errors. This study provides a reference for clinical positioning in postoperative adjuvant radiotherapy of breast cancer. Keywords: Breast cancer radiotherapy, Positioning error, Treatment couch height, Dose distribution
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