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Investigating gene function relies on the efficient manipulation of endogenous gene expression. Currently, a limited number of tools are available to robustly manipulate endogenous gene expression between "on" and "off" states. In this study, we insert a 63 bp coding sequence of T3H38 ribozyme into the 3' untranslated region (UTR) of C. elegans endogenous genes using the CRISPR/Cas9 technology, which reduces the endogenous gene expression to a nearly undetectable level and generated loss-of-function phenotypes similar to that of the genetic null animals. To achieve conditional knockout, a cassette of loxP-flanked transcriptional termination signal and ribozyme is inserted into the 3' UTR of endogenous genes, which eliminates gene expression spatially or temporally via the controllable expression of the Cre recombinase. Conditional endogenous gene turn-on can be achieved by either injecting morpholino, which blocks the ribozyme self-cleavage activity or using the Cre recombinase to remove the loxP-flanked ribozyme. Together, our results demonstrate that these ribozyme-based tools can efficiently manipulate endogenous gene expression both in space and time and expand the toolkit for studying the functions of endogenous genes. Manipulation of endogenous genes in space and time in C. elegans is accomplished by inserting a ribozyme sequence into the 3'UTR with CRISPR/Cas9, utilizing self-cleavage of the ribozyme and Cre/loxP system as an "off and "on" switch, respectively. [ABSTRACT FROM AUTHOR]

Bacillus subtilis is a well-characterized Gram-positive bacterium and a valuable host for recombinant protein production because of its efficient secretion ability, high yield, and non-toxicity. Here, we comprehensively review the recent studies on recombinant protein production in B. subtilis to update and supplement other previous reviews. We have focused on several aspects, including optimization of B. subtilis strains, enhancement and regulation of expression, improvement of secretion level, surface display of proteins, and fermentation optimization. Among them, optimization of B. subtilis strains mainly involves undirected chemical/physical mutagenesis and selection and genetic manipulation; enhancement and regulation of expression comprises autonomous plasmid and integrated expression, promoter regulation and engineering, and fine-tuning gene expression based on proteases and molecular chaperones; improvement of secretion level predominantly involves secretion pathway and signal peptide screening and optimization; surface display of proteins includes surface display of proteins on spores or vegetative cells; and fermentation optimization incorporates medium optimization, process condition optimization, and feeding strategy optimization. Furthermore, we propose some novel methods and future challenges for recombinant protein production in B. subtilis. Key points • A comprehensive review on recombinant protein production in Bacillus subtilis. • Novel techniques facilitate recombinant protein expression and secretion. • Surface display of proteins has significant potential for different applications. [ABSTRACT FROM AUTHOR]

Small cell lung cancer (SCLC) is classified as a high-grade neuroendocrine (NE) tumor, but a subset of SCLC has been termed "variant" due to the loss of NE characteristics. In this study, we computed NE scores for patient-derived SCLC cell lines and xenografts, as well as human tumors. We aligned NE properties with transcription factor-defined molecular subtypes. Then we investigated the different immune phenotypes associated with high and low NE scores. We found repression of immune response genes as a shared feature between classic SCLC and pulmonary neuroendocrine cells of the healthy lung. With loss of NE fate, variant SCLC tumors regain cell-autonomous immune gene expression and exhibit higher tumor-immune interactions. Pan-cancer analysis revealed this NE lineage-specific immune phenotype in other cancers. Additionally, we observed MHC I re-expression in SCLC upon development of chemoresistance. These findings may help guide the design of treatment regimens in SCLC. Ling Cai et al. used transcriptomic profiling data of healthy lung, patient-derived small cell lung cancer cell lines, xenografts, and primary tumors to examine a link between neuroendocrine (NE) signatures and immune gene expression. Their findings suggest that cell-autonomous immune gene repression is a shared feature between healthy and tumor cells of NE lineage and may influence tumor-immune cell interaction and response to immunotherapy. [ABSTRACT FROM AUTHOR]

Voltage-gated potassium (Kv) channels are involved in the proliferation and transformation of mammary epithelial cells. They are thought to be related to the development of breast carcinoma, although the exact role they play in this event remains unclear. In this study, we investigated whether the expression and function of Kv channels is associated with Caveolin-1 (Cav-1, a principal component of caveolae) in different cell lines. We found that expression of Cav-1 correlated with the expression of Kv channels in mammary epithelial cells (MCF10A, MCF10A-ST1, and MCF7), and silencing of Cav-1 inhibited the expression of KCNA5 (voltage-gated shaker-related subfamily A, member 5). Immunofluorescence analysis indicated the colocalization of KCNA5 with Cav-1, whereas immunoprecipitation suggested a possible interaction between the two proteins. Overall, our finding indicated that KCNA5 protein may interact with Cav-1, thereby contributing to the proliferation and early transformation of mammary cells. [ABSTRACT FROM AUTHOR]