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Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is used to edit eukaryotic genomes. Here, we show that CRISPRi can also be used for fine-tuning prokaryotic gene expression while simultaneously regulating multiple essential gene expression with less labor and time consumption. As a case study, CRISPRi was used to control polyhydroxyalkanoate (PHA) biosynthesis pathway flux and to adjust PHA composition. A pathway was constructed in Escherichia coli for the production of poly(3-hydroxybutyrate- co -4-hydroxybutyrate) [P(3HB- co -4HB)] from glucose. The native gene sad encoding E. coli succinate semi-aldehyde dehydrogenase was expressed under the control of CRISPRi using five specially designed single guide RNAs (sgRNAs) for regulating carbon flux to 4-hydroxybutyrate (4HB) biosynthesis. The system allowed formation of P(3HB- co -4HB) consisting of 1–9 mol% 4HB. Additionally, succinate, generated by succinyl-coA synthetase and succinate dehydrogenase (respectively encoded by genes sucC , sucD and sdhA , sdhB ) was channeled preferentially to the 4HB precursor by using selected sgRNAs such as sucC2, sucD2, sdhB2 and sdhA1 via CRISPRi. The resulting 4HB content in P(3HB- co -4HB) was found to range from 1.4 to 18.4 mol% depending on the expression levels of down-regulated genes. The results show that CRISPRi is a feasible method to simultaneously manipulate multiple genes in E. coli . [ABSTRACT FROM AUTHOR]

Abstract: The fusellovirus SSV2 and the integrative plasmid pSSVi, which constitute a unique helper-satellite virus system, replicate in Sulfolobus solfataricus P2. In this study, we investigated the interplay among SSV2, pSSVi and their host by transcriptomic analysis. Following infection of S. solfataricus P2, SSV2 activated its promoters in a temporal and distributive fashion, starting from the transcription of ORF305. Expression of several host genes encoding DNA replication and transcription proteins was up-regulated, suggesting that SSV2 depended heavily on the host replication machinery for its replication. SSV2 gene expression appeared to follow a similar pattern in S. solfataricus P2 harboring pSSVi to that in S. solfataricus P2 lacking the plasmid. Several early genes of the virus were transcribed earlier and more efficiently in the presence of pSSVi than in its absence. These results provide valuable clues to the understanding of the three-way interactions among SSV2, pSSVi and the host. [Copyright &y& Elsevier]

• BMDMϕ engulf spinal cord debris, transform into proinflammatory foamy Mϕ in SCI. • Foamy Mϕ contribute to neuroinflammation and SCI progression. • ABCA1 deficiency in foamy Mϕ increases lipid accumulation and inflammation in SCI. • Enhancing ABCA1 inhibits the inflammation and rescues phagocytotic ability of Mϕ. • Enhancement of ABCA1 expression promotes lipid efflux and functional recovery. Spinal cord injury (SCI) triggers a complex cascade of events, including myelin loss, neuronal damage, neuroinflammation, and the accumulation of damaged cells and debris at the injury site. Infiltrating bone marrow derived macrophages (BMDMϕ) migrate to the epicenter of the SCI lesion, where they engulf cell debris including abundant myelin debris to become pro-inflammatory foamy macrophages (foamy Mϕ), participate neuroinflammation, and facilitate the progression of SCI. This study aimed to elucidate the cellular and molecular mechanisms underlying the functional changes in foamy Mϕ and their potential implications for SCI. Contusion at T10 level of the spinal cord was induced using a New York University (NYU) impactor (5 g rod from a height of 6.25 mm) in male mice. ABCA1, an ATP-binding cassette transporter expressed by Mϕ, plays a crucial role in lipid efflux from foamy cells. We observed that foamy Mϕ lacking ABCA1 exhibited increased lipid accumulation and a higher presence of lipid-accumulated foamy Mϕ as well as elevated pro-inflammatory response in vitro and in injured spinal cord. We also found that both genetic and pharmacological enhancement of ABCA1 expression accelerated lipid efflux from foamy Mϕ, reduced lipid accumulation and inhibited the pro-inflammatory response of foamy Mϕ, and accelerated clearance of cell debris and necrotic cells, which resulted in functional recovery. Our study highlights the importance of understanding the pathologic role of foamy Mϕ in SCI progression and the potential of ABCA1 as a therapeutic target for modulating the inflammatory response, promoting lipid metabolism, and facilitating functional recovery in SCI. [ABSTRACT FROM AUTHOR]

The inflammation and immune hypothesis of major depressive disorder (MDD) explains the mechanism of neuroinflammatory response to promote depression-like behaviors and provides targets for immunotherapy. Previous studies revealed that the neuronal function of the entorhinal cortex (EC) was relative to the depression symptoms in MDD. However, it remains largely unknown what role of neuroinflammation plays in the EC. Hence, we used immunofluorescence to determine c-Fos expression in the EC of lipopolysaccharide (LPS)-treated mice. Mice model was constructed of 10-day LPS treatment, and depression-related behaviors were assessed. We used gene expression microarray to determine differentially expressed genes (DEGs) in the EC of LPS group comparing to control group, and molecular verification was performed by quantitative real-time PCR and Western blot. We found that c-Fos expression was significant reduced in the two layers (Lateral 3.25 mm and 3.00 mm) of the EC in LPS-treated mice compared to saline-treated mice. Mice in LPS group exhibited depression- and anxiety-like behaviors in chronic model. Gene expression analyses identified 339 DEGs in the EC between LPS and control group. The molecular verification showed activation of IL-1R1/NF-κB/CCL5 signaling and upregulation of markers of astrocyte (GFAP) and microglia (AIF1 and CD86) in the EC. Our results suggested that LPS-induced neuroinflammation inhibited neuronal activity in the EC of mice, and that activation of IL-1R1/NF-κB/CCL5 signaling could be involved in the neuroinflammation in the EC of LPS-treated depression model. • Lipopolysaccharide (LPS) treatment reduced c-Fos expression in the entorhinal cortex (EC) of mice. • LPS-treated mice showed depression- and anxiety-like behaviors. • Gene expression analyses revealed neuroinflammatory alterations in the EC. • IL-1R1/NF-κB/CCL5 signaling could be involved in inflammatory response in the EC. [ABSTRACT FROM AUTHOR]

Abstract: It has been reported that the plasma levels of VEGF in tumor patients decreased during dendritic cell (DC)-based immunotherapy, but the underlying mechanism remains unclear. Our current report demonstrates that VEGF levels were significantly decreased in the supernatants of DCs incubated with rhVEGF or tumor conditioned medium (TCM) while the intracellular VEGF in DCs was increased. The increased intracellular VEGF was not due to the de novo VEGF synthesis by DCs because exogenous VEGF inhibited the mRNA expression of VEGF in DCs. More direct evidence was provided to demonstrate that Cy3-labeled VEGF could be internalized by DCs specifically and efficiently. In addition, the activity of DCs to internalize VEGF was abolished by neutralizing antibody against VEGF receptor-1 (Flt-1) and inhibitors of endocytosis such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and genistein. This study highlights a novel function of DCs and allows a better understanding of the DC–VEGF interaction. [Copyright &y& Elsevier]