Engineering a synthetic gene circuit for high-performance inducible expression in mammalian systems.

Inducible gene expression systems can be used to control the expression of a gene of interest by means of a small-molecule. One of the most common designs involves engineering a small-molecule responsive transcription factor (TF) and its cognate promoter, which often results in a compromise between minimal uninduced background expression (leakiness) and maximal induced expression. Here, we focus on an alternative strategy using quantitative synthetic biology to mitigate leakiness while maintaining high expression, without modifying neither the TF nor the promoter. Through mathematical modelling and experimental validations, we design the CASwitch, a mammalian synthetic gene circuit based on combining two well-known network motifs: the Coherent Feed-Forward Loop (CFFL) and the Mutual Inhibition (MI). The CASwitch combines the CRISPR-Cas endoribonuclease CasRx with the state-of-the-art Tet-On3G inducible gene system to achieve high performances. To demonstrate the potentialities of the CASwitch, we apply it to three different scenarios: enhancing a whole-cell biosensor, controlling expression of a toxic gene and inducible production of Adeno-Associated Virus (AAV) vectors.

AXL receptor as an emerging molecular target in colorectal cancer.

AXL receptor tyrosine kinase (AXL) is a receptor tyrosine kinase whose aberrant expression has recently been associated with colorectal cancer (CRC), contributing to tumor growth, epithelial-mesenchymal transition (EMT), increased invasiveness, metastatic spreading, and the development of drug resistance. In this review we summarize preclinical data, the majority of which are limited to recent years, convincingly linking the AXL receptor to CRC. These findings support the value of targeting AXL with molecules in drug discovery, offering novel and advanced therapeutic or diagnostic tools for CRC management.

Cancer prevention in ulcerative colitis: long-term outcome following fluorescence-guided colonoscopy.

BACKGROUND: Patients with long-standing ulcerative colitis require repeated endoscopies for early detection of neoplasias, which, however, are frequently missed by standard colonoscopy. Fluorescence-guided colonoscopy is known to improve the detection rate but the long-term effects of fluorescence-guided colonoscopy are unknown. METHODS: Colitis patients with negative findings at index fluorescence-guided colonoscopy entered a prospective long-term study with conventional colonoscopies at 2-year intervals. Risk and time to progression were evaluated. The positive predictive value was assessed in patients with neoplasias at index fluorescence-guided colonoscopy who underwent immediate total colectomy. RESULTS: Thirty-one patients with negative fluorescence-guided colonoscopy were surveyed for a mean of 7.8 ± 0.9 years. Neoplasia was observed in only two of them (6%) after 7 and 8 years of follow-up, respectively. Neoplasia at index fluorescence-guided colonoscopy was observed in 10 patients. In all of them, multiple flat low-grade intraepithelial neoplasia was diagnosed. At immediate colectomy performed in eight of them, the diagnosis of flat low-grade intraepithelial neoplasia was confirmed, corresponding to a positive predictive value of 100%. However, synchronous more advanced neoplasia was detected in three of the eight patients (38%). All patients, those with and those without neoplasia, were alive at the end of the study. CONCLUSIONS: Fluorescence-guided colonoscopy misses, in contrast to standard colonoscopy, few, if any, patients with neoplasia. Most neoplasia-negative patients remain negative during prolonged follow-up. However, when low-grade dysplasia is diagnosed by fluorescence-guided colonoscopy, colectomy is recommended because more than a third of the patients harbor synchronous, more advanced neoplasia.

Time-gated fluorescence spectroscopy improves endoscopic detection of low-grade dysplasia in ulcerative colitis.

BACKGROUND: Dysplasia in ulcerative colitis is frequently missed with 4-quadrant biopsies. An experimental setup recording delayed fluorescence spectra simultaneously with white light endoscopy was recently developed. OBJECTIVE: We compared detection of invisible flat intraepithelial neoplasia with protoporphyrin IX fluorescence and standard 4-quadrant biopsies. DESIGN: Prospective, crossover design without randomization of the order of procedures. SETTING: Gastroenterology Department, Humboldt University, Charité, Berlin, Germany. PATIENTS: Forty-two patients with extensive ulcerative colitis of more than 10 years' duration were included. INTERVENTIONS: Colonoscopy with 4-quadrant biopsies and targeted biopsies of macroscopic lesions and time-gated fluorescence-guided colonoscopy were performed 2 weeks apart by 2 blinded endoscopists. Three independent pathologists examined the biopsy specimens. MAIN OUTCOME MEASUREMENTS: The primary outcome criterion was detection rate of invisible flat intraepithelial neoplasia. RESULTS: Invisible flat intraepithelial neoplasia was detected in 3 (7%) patients by white light 4-quadrant biopsies and in 10 (24%) patients by fluorescence-guided endoscopy (P = .02). The sensitivity and specificity for differentiating patients with and without dysplasia were 100% and 81%, respectively. Dysplastic and nondysplastic mucosa could be discriminated with a sensitivity and specificity of 73% and 81%, respectively. LIMITATIONS: The trial was not randomized. CONCLUSION: The detection rate of intraepithelial neoplasia in patients with ulcerative colitis can be improved by fluorescence-guided colonoscopy.