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Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR–induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint.

Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Gu ide RNA A ssisted R eduction of D amage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design. Off-target editing remains a concern for therapeutic applications of CRISPR-Cas9. Here the authors present CRISPR GUARD, which uses very short non-cleaving gRNAs to prevent editing at off-target sites.

The ZFP36 family of RNA-binding proteins acts post-transcriptionally to repress translation and promote RNA decay. Studies of genes and pathways regulated by the ZFP36 family in CD4 + T cells have focussed largely on cytokines, but their impact on metabolic reprogramming and differentiation is unclear. Using CD4 + T cells lacking Zfp36 and Zfp36l1 , we combined the quantification of mRNA transcription, stability, abundance and translation with crosslinking immunoprecipitation and metabolic profiling to determine how they regulate T cell metabolism and differentiation. Our results suggest that ZFP36 and ZFP36L1 act directly to limit the expression of genes driving anabolic processes by two distinct routes: by targeting transcription factors and by targeting transcripts encoding rate-limiting enzymes. These enzymes span numerous metabolic pathways including glycolysis, one-carbon metabolism and glutaminolysis. Direct binding and repression of transcripts encoding glutamine transporter SLC38A2 correlated with increased cellular glutamine content in ZFP36/ZFP36L1-deficient T cells. Increased conversion of glutamine to α-ketoglutarate in these cells was consistent with direct binding of ZFP36/ZFP36L1 to Gls (encoding glutaminase) and Glud1 (encoding glutamate dehydrogenase). We propose that ZFP36 and ZFP36L1 as well as glutamine and α-ketoglutarate are limiting factors for the acquisition of the cytotoxic CD4 + T cell fate. Our data implicate ZFP36 and ZFP36L1 in limiting glutamine anaplerosis and differentiation of activated CD4 + T cells, likely mediated by direct binding to transcripts of critical genes that drive these processes.

Genome-wide CRISPR sgRNA libraries have emerged as transformative tools to systematically probe gene function. While these libraries have been iterated over time to be more efficient, their large size limits their use in some applications. Here, we benchmarked publicly available genome-wide single-targeting sgRNA libraries and evaluated dual targeting as a strategy for pooled CRISPR loss-of-function screens. We leveraged this data to design two minimal genome-wide human CRISPR-Cas9 libraries that are 50% smaller than other libraries and that preserve specificity and sensitivity, thus enabling broader deployment at scale.

The ubiquitin-like modifier FAT10 targets proteins for degradation by the proteasome and is activated by the E1 enzyme UBA6. In this study, we identify the UBA6-specific E2 enzyme (USE1) as an interaction partner of FAT10. Activated FAT10 can be transferred from UBA6 onto USE1 in vitro , and endogenous USE1 and FAT10 can be coimmunoprecipitated from intact cells. Small interfering RNA-mediated downregulation of USE1 mRNA resulted in a strong reduction of FAT10 conjugate formation under endogenous conditions, suggesting that USE1 is a major E2 enzyme in the FAT10 conjugation cascade. Interestingly, USE1 is not only the first E2 enzyme but also the first known substrate of FAT10 conjugation, as it was efficiently auto-FAT10ylated in cis but not in trans . The ubiquitin-like modifier FAT10 targets proteins for proteolytic degradation. Here the ubiquitin-conjugating enzyme USE1 is identified as a component of the FAT10 conjugation cascade.

Genome-wide CRISPR sgRNA libraries have emerged as transformative tools to systematically probe gene function. While these libraries have been iterated over time to be more efficient, their large size limits their use in some applications. Here, we benchmarked publicly available genome-wide single-targeting sgRNA libraries and evaluated dual targeting as a strategy for pooled CRISPR loss-of-function screens. We leveraged this data to design two minimal genome-wide human CRISPR-Cas9 libraries that are 50% smaller than other libraries and that preserve specificity and sensitivity, thus enabling broader deployment at scale.

The article reviews issues associated with the operation of stationary and non-stationary electronic fire alarm systems (FASs). These systems are employed for the fire protection of selected buildings (stationary) or to monitor vast areas, e.g., forests, airports, logistics hubs, etc. (non-stationary). An FAS is operated under various environmental conditions, indoor and outdoor, favourable or unfavourable to the operation process. Therefore, an FAS has to exhibit a reliable structure in terms of power supply and operation. To this end, the paper discusses a representative FAS monitoring a facility and presents basic tactical and technical assumptions for a non-stationary system. The authors reviewed fire detection methods in terms of fire characteristic values (FCVs) impacting detector sensors. Another part of the article focuses on false alarm causes. Assumptions behind the use of unmanned aerial vehicles (UAVs) with visible-range cameras (e.g., Aviotec) and thermal imaging were presented for non-stationary FASs. The FAS operation process model was defined and a computer simulation related to its operation was conducted. Analysing the FAS operation process in the form of models and graphs, and the conducted computer simulation enabled conclusions to be drawn. They may be applied for the design, ongoing maintenance and operation of an FAS. As part of the paper, the authors conducted a reliability analysis of a selected FAS based on the original performance tests of an actual system in operation. They formulated basic technical and tactical requirements applicable to stationary and mobile FASs detecting the so-called vast fires.

This article presents selected issues related to the reliability of the power supply for electronic security systems (ESSs) used in smart buildings (SBs). ESSs operate in diverse environmental conditions and are responsible for the safety of lives, property and the natural environment of SB users. The operational tasks of ESSs in SBs require a continuous power supply from various sources, including renewable energy sources. The authors conducted an analysis of the power supply for selected ESSs used in SBs, which enabled the development of a power supply model. For the proposed model, the authors designed a proprietary graph of the ESS operational process, taking into account power supply implementation. Considering the operational indicators for the analysed ESSs, such as repair and failure rates, a computer simulation was performed. The simulation allowed the determination of the reliability of the ESS power supply within the considered redundancy configuration of additional energy sources, which can be utilised during the design phase. The reliability analysis of the power supply and the determination of rational parameters conducted in the article are crucial for achieving all the functionalities of ESSs in SBs, as envisioned during the design process. The article is divided into six chapters, structured to address the topics sequentially: an introduction to the state of the issue, a critical literature review, an analysis of the power supply for selected ESSs, implementation of renewable energy sources, the development of a proprietary model and operational graph, a computer simulation and conclusions.

Complex studies were carried out to recognize the fen structure and peat properties in the Całowanie Fen area, belonging to the Natura 2000 network. The studies were conducted in two study areas that differ significantly in terms of peat thickness. Electrical resistivity imaging (ERI) was used to identify the properties of the peat and its substrate, such as thickness and electrical resistivity. Comparison of the field studies with the laboratory tests has shown that the ash content rises electrical resistivity in peat. In addition, the study has shown that the application of non-invasive geophysical methods in protected areas is justified. The fen, as a medium containing mostly water, was a proper test area for the ERI measurements.

Complex studies were carried out to recognize the fen structure and peat properties in the Caowanie Fen area, belonging to the Natura 2000 network. The studies were conducted in two study areas that differ significantly in terms of peat thickness. Electrical resistivity imaging (ERI) was used to identify the properties of the peat and its substrate, such as thickness and electrical resistivity. Comparison of the field studies with the laboratory tests has shown that the ash content rises electrical resistivity in peat. In addition, the study has shown that the application of non-invasive geophysical methods in protected areas is justified. The fen, as a medium containing mostly water, was a proper test area for the ERI measurements.