We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a Forster resonance energy transfer (FRET) acceptor in ratiometric imaging.

mScarlet: a bright monomeric red fluorescent protein for cellular imaging

Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.

High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells

ATR Prohibits Replication Catastrophe by Preventing Global Exhaustion of RPA

Base editors enable targeted single-nucleotide conversions in genomic DNA. Here we show that expression levels are a bottleneck in base-editing efficiency. We optimize cytidine (BE4) and adenine (ABE7.10) base editors by modification of nuclear localization signals (NLS) and codon usage, and ancestral reconstruction of the deaminase component. The resulting BE4max, AncBE4max, and ABEmax editors correct pathogenic SNPs with substantially increased efficiency in a variety of mammalian cell types.

Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

/pdf/long-term-expanding-human-airway-organoids-for-disease-4pftu4njk4.pdf

Long-term expanding human airway organoids for disease modeling

When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.

/pdf/high-cleavage-efficiency-of-a-2a-peptide-derived-from-1tlp5x4a1c.pdf

High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.

Background Apatinib is established to be the standard of care as third-line therapy for patients with previously treated advanced gastric cancer (GC). Programmed cell death protein 1 (PD-1) blockades also exhibited promising efficacy and safety for patients with treatment-refractory advanced GC. Objective This study was to explore the feasibility and tolerance of apatinib plus PD-1 inhibitors for patients with previously treated advanced GC. Methods This study was performed as a real-world study; patients with advanced GC who were treated with previous systemic chemotherapy were screened retrospectively. Eligible patients were administered with apatinib combined with PD-1 blockade treatment. Efficacy of the patients was assessed with the change of target lesion using radiological evidence according to RECIST 1.1 criteria, and follow-up was carried out regularly. A safety profile was collected and documented during the combination treatment. Univariate analysis based on baseline characteristic subgroup was implemented in univariate analysis to identify the potential factor that might contribute to progression-free survival (PFS). Results Between August 2018 and October 2021, a total of 39 patients with advanced GC or gastroesophageal junction adenocarcinoma participated in this study consecutively and all the patients were available for efficacy and safety assessment. The best overall response during apatinib plus PD-1 blockade administration exhibited that PR was observed in 8 patients, SD was noted in 19 patients, and PD was found in 12 patients, which yielded an ORR of 20.5% (95% CI: 9.3%-36.5%), and DCR was 69.2% (95% CI: 52.4%-83.0%). Furthermore, the relatively enough follow-up had resulted in the mature PFS and overall survival (OS) data, suggesting that the median PFS of the 39 patients with advanced GC was 3.9 months (95% CI: 2.74-5.06). Additionally, the median OS of the 39 patients with advanced GC was 7.8 months (95% CI: 4.82-10.78). Furthermore, the most common adverse reactions of the 39 patients who received apatinib plus PD-1 blockades treatment were fatigue (61.5%), nausea and vomiting (56.4%), diarrhea (48.7%), hypertension (46.2%), hand-foot syndrome (38.5%), and rash (28.2%). Furthermore, performance status was independently associated with PFS of apatinib plus PD-1 inhibitor combination administration in baseline characteristic subgroup analysis. Conclusion Apatinib plus PD-1 inhibitors exhibited promising effectiveness and acceptable tolerance for previously treated advanced GC preliminarily. And this conclusion should be confirmed in clinical trials in the future.

Li Hua Li

Papers

High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.

Feasibility and Tolerance of Apatinib plus PD-1 Inhibitors for Previously Treated Advanced Gastric Cancer: A Real-World Exploratory Study