HDR-based CRISPR/Cas9-mediated Knockout of PD-L1 in C57BL/6 Mice

The immune-inhibitory molecule programmed cell death ligand 1 (PD-L1) has been shown to play a role in pathologies such as autoimmunity, infections, and cancer. The expression of PD-L1 not only on cancer cells but also on non-transformed host cells is known to be associated with cancer progression. Generation of PD-L1 deficiency in the murine system enables us to specifically study the role of PD-L1 in physiological processes and diseases. One of the most versatile and easy to use site-specific gene editing tools is the CRISPR/Cas9 system, which is based on an RNA-guided nuclease system. Similar to its predecessors, the Zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 catalyzes double-strand DNA breaks, which can result in frameshift mutations due to random nucleotide insertions or deletions via non-homologous end joining (NHEJ). Furthermore, although less frequently, CRISPR/Cas9 can lead to insertion of defined sequences due to homology-directed repair (HDR) in the presence of a suitable template. Here, we describe a protocol for the knockout of PD-L1 in the murine C57BL/6 background using CRISPR/Cas9. Targeting of exon 3 coupled with the insertion of a HindIII restriction site leads to a premature stop codon and a loss-of-function phenotype. We describe the targeting strategy as well as founder screening, genotyping, and phenotyping. In comparison to NHEJ-based strategy, the presented approach results in a defined stop codon with comparable efficiency and timelines as NHEJ, generates convenient founder screening and genotyping options, and can be swiftly adapted to other targets

Genome-wide mapping of the RNA targets of the Pseudomonas aeruginosa riboregulatory protein RsmN

Pseudomonads typically carry multiple non-identical alleles of the post-transcriptional regulator rsmA. In P. aeruginosa, RsmN is notable in that its structural rearrangement confers distinct and overlapping functions with RsmA. However, little is known about the specificities of RsmN for its target RNAs and overall impact on the biology of this pathogen. We purified and mapped 503 transcripts directly bound by RsmN in P. aeruginosa. About 200 of the mRNAs identified encode proteins of demonstrated function including some determining acute and chronic virulence traits. For example, RsmN reduces biofilm development both directly and indirectly via multiple pathways, involving control of Pel exopolysaccharide biosynthesis and c-di-GMP levels. The RsmN targets identified are also shared with RsmA, although deletion of rsmN generally results in less pronounced phenotypes than those observed for ΔrsmA or ΔrsmArsmNind mutants, probably as a consequence of different binding affinities. Targets newly identified for the Rsm system include the small non-coding RNA CrcZ involved in carbon catabolite repression, for which differential binding of RsmN and RsmA to specific CrcZ regions is demonstrated. The results presented here provide new insights into the intricacy of riboregulatory networks involving multiple but distinct RsmA homologues

Cereal processing at Early Neolithic Göbekli Tepe, southeastern Turkey

We analyze the processing of cereals and its role at Early Neolithic Göbekli Tepe, southeastern Anatolia (10th / 9th millennium BC), a site that has aroused much debate in archaeological discourse

Genome-wide analysis of targets for post-transcriptional regulation by Rsm proteins in Pseudomonas putida

Post-transcriptional regulation is an important step in the control of bacterial gene expression in response to environmental and cellular signals. Pseudomonas putida KT2440 harbors three known members of the CsrA/RsmA family of post-transcriptional regulators: RsmA, RsmE and RsmI. We have carried out a global analysis to identify RNA sequences bound in vivo by each of these proteins. Affinity purification and sequencing of RNA molecules associated with Rsm proteins were used to discover direct binding targets, corresponding to 437 unique RNA molecules, 75 of them being common to the three proteins. Relevant targets include genes encoding proteins involved in signal transduction and regulation, metabolism, transport and secretion, stress responses, and the turnover of the intracellular second messenger c-di-GMP. To our knowledge, this is the first combined global analysis in a bacterium harboring three Rsm homologs. It offers a broad overview of the network of processes subjected to this type of regulation and opens the way to define what are the sequence and structure determinants that define common or differential recognition of specific RNA molecules by these proteins.This work was supported by grants BFU2013-43469-P, BFU2016-80122-P and PID2019-109372GB-I00 from the Plan Estatal de I+D+I (Agencia Estatal de Investigación, Spanish Ministry of Science and Innovation and FEDER funds). Funding from the Biotechnology and Biological Sciences Research Council, United Kingdom (BB/R012415/1), and the University of Malaya (FRGS grant FP022-2018A and HIR grant H-50001-00-A000027) are also gratefully acknowledged

Genome-Wide Analysis of Targets for Post-Transcriptional Regulation by Rsm Proteins in Pseudomonas putida

© Copyright © 2021 Huertas-Rosales, Romero, Chan, Hong, Cámara, Heeb, Barrientos-Moreno, Molina-Henares, Travieso, Ramos-González and Espinosa-Urgel. Post-transcriptional regulation is an important step in the control of bacterial gene expression in response to environmental and cellular signals. Pseudomonas putida KT2440 harbors three known members of the CsrA/RsmA family of post-transcriptional regulators: RsmA, RsmE and RsmI. We have carried out a global analysis to identify RNA sequences bound in vivo by each of these proteins. Affinity purification and sequencing of RNA molecules associated with Rsm proteins were used to discover direct binding targets, corresponding to 437 unique RNA molecules, 75 of them being common to the three proteins. Relevant targets include genes encoding proteins involved in signal transduction and regulation, metabolism, transport and secretion, stress responses, and the turnover of the intracellular second messenger c-di-GMP. To our knowledge, this is the first combined global analysis in a bacterium harboring three Rsm homologs. It offers a broad overview of the network of processes subjected to this type of regulation and opens the way to define what are the sequence and structure determinants that define common or differential recognition of specific RNA molecules by these proteins

2-Tridecanone impacts surface-associated bacterial behaviours and hinders plant-bacteria interactions

Surface motility and biofilm formation are behaviours which enable bacteria to infect their hosts and are controlled by different chemical signals. In the plant symbiotic alpha‐proteobacterium Sinorhizobium meliloti, the lack of long‐chain fatty acyl‐coenzyme A synthetase activity (FadD) leads to increased surface motility, defects in biofilm development, and impaired root colonization. In this study, analyses of lipid extracts and volatiles revealed that a fadD mutant accumulates 2‐tridecanone (2‐TDC), a methylketone known as a natural insecticide. Application of pure 2‐TDC to the wild‐type strain phenocopies the free‐living and symbiotic behaviours of the fadD mutant. Structural features of the methylketone determine its ability to promote S. meliloti surface translocation, which is mainly mediated by a flagella‐independent motility. Transcriptomic analyses showed that 2‐TDC induces differential expression of iron uptake, redox, and stress‐related genes. Interestingly, this methylketone also influences surface motility and impairs biofilm formation in plant and animal pathogenic bacteria. Moreover, 2‐TDC not only hampers alfalfa nodulation but also the development of tomato bacterial speck disease. This work assigns a new role to 2‐TDC as an infochemical that affects important bacterial traits and hampers plant‐bacteria interactions by interfering with microbial colonization of plant tissues

Assessment of iron nanoparticle distribution in mouse models using ultrashort echo-time magnetic resonance imaging

Microscopic magnetic field inhomogeneities caused by iron deposition or tissue-air interfaces may result in rapid decay of transverse magnetization in magnetic resonance imaging (MRI). The aim of this study is to detect and quantify the distribution of iron-based nanoparticles in mouse models applying ultrashort echo-time (UTE) sequences in tissues exhibiting extremely fast transverse relaxation. In 24 C57BL/6 mice (2 controls), suspensions containing either non-oxidic Fe or AuFeOx nanoparticles were injected into the tail vein at two doses (200 μg and 600 μg per mouse). Mice underwent MRI using a UTE sequence at 4.7T field strength with five different echo-times between 100 μs and 5000 μs. Transverse relaxation times T2* were computed for the lung, liver, and spleen by mono-exponential fitting. In UTE imaging, the MRI signal could reliably be detected even in liver parenchyma exhibiting the highest deposition of nanoparticles. In animals treated with Fe nanoparticles (600 μg per mouse), the relaxation time substantially decreased in the liver (3418±1534 μs (control) vs. 228±67 μs), the spleen (2170±728 μs vs. 299±97 μs), and the lungs (663±101 μs vs. 413±99 μs). The change in transverse relaxation was dependent on the amount and composition of the nanoparticles. By pixel-wise curve fitting, T2* maps were calculated showing nanoparticle distribution. In conclusion, UTE sequences may be used to assess and quantify nanoparticle distribution in tissues exhibiting ultra-fast signal decay in MRI

Assessment of iron nanoparticle distribution in mouse models using ultrashort-echo-time MRI

Microscopic magnetic field inhomogeneities caused by iron deposition or tissue-air interfaces may result in rapid decay of transverse magnetization in MRI. The aim of this study is to detect and quantify the distribution of iron-based nanoparticles in mouse models by applying ultrashort-echo-time (UTE) sequences in tissues exhibiting extremely fast transverse relaxation. In 24 C57BL/6 mice (two controls), suspensions containing either non-oxidic Fe or AuFeOx nanoparticles were injected into the tail vein at two doses (200 μg and 600 μg per mouse). Mice underwent MRI using a UTE sequence at 4.7 T field strength with five different echo times between 100 μs and 5000 μs. Transverse relaxation times T2* were computed for the lung, liver, and spleen by mono-exponential fitting. In UTE imaging, the MRI signal could reliably be detected even in liver parenchyma exhibiting the highest deposition of nanoparticles. In animals treated with Fe nanoparticles (600 μg per mouse), the relaxation time substantially decreased in the liver (3418 ± 1534 μs (control) versus 228 ± 67 μs), the spleen (2170 ± 728 μs versus 299 ± 97 μs), and the lungs (663 ± 101 μs versus 413 ± 99 μs). The change in transverse relaxation was dependent on the number and composition of the nanoparticles. By pixel-wise curve fitting, T2* maps were calculated showing nanoparticle distribution. In conclusion, UTE sequences may be used to assess and quantify nanoparticle distribution in tissues exhibiting ultrafast signal decay in MRI.ISSN:0952-3480ISSN:1099-149

Multiscale Multimodal Investigation of the Intratissural Biodistribution of Iron Nanotherapeutics with Single Cell Resolution Reveals Co-Localization with Endogenous Iron in Splenic Macrophages

Imaging of iron-based nanoparticles (NPs) remains challenging because of the presence of endogenous iron in tissues that is difficult to distinguish from exogenous iron originating from the NPs. Here, an analytical cascade for characterizing the biodistribution of biomedically relevant iron-based NPs from the organ scale to the cellular and subcellular scales is introduced. The biodistribution on an organ level is assessed by elemental analysis and quantification of magnetic iron by electron paramagnetic resonance, which allowed differentiation of exogenous and endogenous iron. Complementary to these bulk analysis techniques, correlative whole-slide optical and electron microscopy provided spatially resolved insight into the biodistribution of endo- and exogenous iron accumulation in macrophages, with single-cell and single-particle resolution, revealing coaccumulation of iron NPs with endogenous iron in splenic macrophages. Subsequent transmission electron microscopy revealed two types of morphologically distinct iron-containing structures (exogenous nanoparticles and endogenous ferritin) within membrane-bound vesicles in the cytoplasm, hinting at an attempt of splenic macrophages to extract and recycle iron from exogenous nanoparticles. Overall, this strategy enables the distinction of endo- and exogenous iron across scales (from cm to nm, based on the analysis of thousands of cells) and illustrates distribution on organ, cell, and organelle levels.ISSN:2366-960

Conventional NK cells and tissue-resident ILC1s join forces to control liver metastasis

The liver is a major metastatic target organ, and little is known about the role of immunity in controlling hepatic metastases. Here, we discovered that the concerted and nonredundant action of two innate lymphocyte subpopulations, conventional natural killer cells (cNKs) and tissue-resident type I innate lymphoid cells (trILC1s), is essential for antimetastatic defense. Using different preclinical models for liver metastasis, we found that trILC1 controls metastatic seeding, whereas cNKs restrain outgrowth. Whereas the killing capacity of trILC1s was not affected by the metastatic microenvironment, the phenotype and function of cNK cells were affected in a cancer type-specific fashion. Thus, individual cancer cell lines orchestrate the emergence of unique cNK subsets, which respond differently to tumor-derived factors. Our findings will contribute to the development of therapies for liver metastasis involving hepatic innate cells. Keywords: conventional NK cells; innate lymphocytes; metastatic surveillance; tissue-resident ILC1s