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CRISPRi-mediated gene regulation allows simultaneous control of many genes. However, highly specific sgRNA-promoter binding is, alone, insufficient to achieve independent transcriptional regulation of multiple targets. Indeed, due to competition for dCas9, the repression ability of one sgRNA changes significantly when another sgRNA becomes expressed. To solve this problem and decouple sgRNA-mediated regulatory paths, we create a dCas9 concentration regulator that implements negative feedback on dCas9 level. This allows any sgRNA to maintain an approximately constant dose-response curve, independent of other sgRNAs. We demonstrate the regulator performance on both single-stage and layered CRISPRi-based genetic circuits, zeroing competition effects of up to 15-fold changes in circuit I/O response encountered without the dCas9 regulator. The dCas9 regulator decouples sgRNA-mediated regulatory paths, enabling concurrent and independent regulation of multiple genes. This allows predictable composition of CRISPRi-based genetic modules, which is essential in the design of larger scale synthetic genetic circuits. CRISPRi allows for the simultaneous control of many genes, however the sgRNAs compete for binding to dCas9. Here the authors design a dCas9 concentration regulator to allow independent regulation of multiple genes.

The global race against antimicrobial resistance requires novel antimicrobials that are not only effective in killing specific bacteria, but also minimize the emergence of new resistances. Recently, CRISPR/Cas-based antimicrobials were proposed to address killing specificity with encouraging results. However, the emergence of target sequence mutations triggered by Cas-cleavage was identified as an escape strategy, posing the risk of generating new antibiotic-resistance gene (ARG) variants. Here, we evaluated an antibiotic re-sensitization strategy based on CRISPR interference (CRISPRi), which inhibits gene expression without damaging target DNA. The resistance to four antibiotics, including last resort drugs, was significantly reduced by individual and multi-gene targeting of ARGs in low- to high-copy numbers in recombinant E. coli . Escaper analysis confirmed the absence of mutations in target sequence, corroborating the harmless role of CRISPRi in the selection of new resistances. E. coli clinical isolates carrying ARGs of severe clinical concern were then used to assess the robustness of CRISPRi under different growth conditions. Meropenem, colistin and cefotaxime susceptibility was successfully increased in terms of MIC (up to > 4-fold) and growth delay (up to 11 h) in a medium-dependent fashion. ARG repression also worked in a pathogenic strain grown in human urine, as a demonstration of CRISPRi-mediated re-sensitization in host-mimicking media. This study laid the foundations for further leveraging CRISPRi as antimicrobial agent or research tool to selectively repress ARGs and investigate resistance mechanisms.

Bile salt hydrolase (BSH) enables microbial-mediated deconjugation of bile acids (BAs) in the gastrointestinal tract. BSH enzymes initiate bile acid metabolism by catalyzing the first, essential deconjugation step. Due to the strict connection between dysregulations of the BA pool and human or animal diseases, identification and characterization of strains with BSH activity are relevant for both healthcare and agroindustry. However, current methods are expensive, poorly sensitive, or require complex procedures. Here, a BSH screening assay for cultivated microbes is proposed, based on a bacterial biosensor that reports the concentration of different BA types via fluorescence. Although the biosensor is broadly responsive to various bile acids, the assay was designed to guarantee specificity by testing individual primary BAs within controlled concentration ranges. The assay was evaluated on two recombinant Escherichia coli strains bearing BSH genes from Lactobacillus johnsonii PF01 and a BSH-positive probiotic strain (Lactobacillus rhamnosus GG). Data showed a consistent activity pattern with previous assays on these enzymes. A crucial aspect addressed was the matrix effect, i.e., the impact of the growth media of the BSH-containing strains on biosensor output. This assay is expected to be a reproducible and accessible option, compatible with automated protocols.

The outbreak of Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, forced us to face a pandemic with unprecedented social, economic, and public health consequences. Several nations have launched campaigns to immunize millions of people using various vaccines to prevent infections. Meanwhile, therapeutic approaches and discoveries continuously arise; however, identifying infected patients that are going to experience the more severe outcomes of COVID-19 is still a major need, to focus therapeutic efforts, reducing hospitalization and mitigating drug adverse effects. Microbial communities colonizing the respiratory tract exert significant effects on host immune responses, influencing the susceptibility to infectious agents. Through 16S rDNAseq we characterized the upper airways’ microbiota of 192 subjects with nasopharyngeal swab positive for SARS-CoV-2. Patients were divided into groups based on the presence of symptoms, pneumonia severity, and need for oxygen therapy or intubation. Indeed, unlike most of the literature, our study focuses on identifying microbial signatures predictive of disease progression rather than on the probability of infection itself, for which a consensus is lacking. Diversity, differential abundance, and network analysis at different taxonomic levels were synergistically adopted, in a robust bioinformatic pipeline, highlighting novel possible taxa correlated with patients’ disease progression to intubation.

Background/Objectives: The rise in multidrug-resistant pathogens such as Pseudomonas aeruginosa (PA), coupled with declining antibiotic development, underscores the need for innovative therapeutic strategies. Repurposing approved drugs provides advantages of safety and rapid development. Since quorum sensing (QS) controls key virulence traits in PA, targeting this pathway represents a promising antivirulence approach. This study aimed to identify and repurpose existing drugs as QS inhibitors. Methods: An in silico docking screen of 3000 FDA-approved or clinically tested compounds was performed against the C4-HSL receptor RhlR. Seventeen candidates were tested in the laboratory strain PAO1 for lactone-dependent signaling inhibition. The most active compound, MK-8245, was further evaluated for effects on growth, cytotoxicity, lactone release, biofilm formation, pyocyanin, elastase, rhamnolipids, and swarming motility. Its activity was also assessed in 20 clinical PA isolates. Results: MK-8245 (40 µM) reduced QS-regulated gene expression by ~60% without affecting viability. In PAO1, it inhibited rhamnolipids (60%), pyocyanin (40%), elastase (25%), biofilm formation, and swarming motility (25%). MK-8245 also enhanced the efficacy of imipenem against biofilms. In clinical isolates, it consistently decreased lactone release (~60%), pyocyanin (~50%), rhamnolipids (~40%), biofilm formation (~30%), and swarming motility (~25%). Conclusions: MK-8245 emerges as a promising antivirulence candidate against P. aeruginosa. By disrupting QS signaling and impairing multiple virulence factors, it attenuates pathogenicity without bactericidal pressure. Its synergy with standard antibiotics and consistent activity in clinical isolates highlight its translational potential and warrant further preclinical evaluation.

Increasing antibiotic resistance and the decline in the pharmaceutical industry’s investments have amplified the need for novel treatments for multidrug-resistant bacteria. Quorum sensing (QS) inhibitors reduce pathogens’ virulence without selective pressure on bacteria and provide an alternative to conventional antibiotic-based therapies. P. aeruginosa uses complex QS signaling to control virulence and biofilm formation. We aimed to identify inhibitors of P. aeruginosa QS acting on acyl-homoserine lactones (AHL)-mediated circuits. Bioluminescence and qRT-PCR assays were employed to screen a library of 81 small phenolic derivatives to reduce AHL-dependent signaling. We identified GM-50 as the most active compound inhibiting the expression of AHL-regulated genes but devoid of cytotoxic activity in human epithelial cells and biocidal effects on bacteria. GM-50 reduces virulence factors such as rhamnolipids, pyocyanin, elastase secretion, and swarming motility in P. aeruginosa PAO1 laboratory strain. By molecular docking, we provide evidence that GM-50 highly interacts with RhlR. GM-50 significantly improved aztreonam-mediated biofilm disruption. Moreover, GM-50 prevents adhesion of PAO1 and inflammatory damage in the human A549 cell line and protects Galleria mellonella from PAO1-mediated killing. GM-50 significantly reduces virulence factors in 20 P. aeruginosa clinical isolates from patients with respiratory tract infections. In conclusion, GM-50 inhibits AHL-signaling, reduces virulence factors, enhances the anti-biofilm activity of aztreonam, and protects G. mellonella larvae from damage induced by P. aeruginosa . Since GM-50 is active on clinical strains, it represents a starting point for identifying and developing new phenolic derivatives acting as QS-inhibitors in P. aeruginosa infections.

MicroRNAs, highly-conserved small RNAs, act as key regulators of many biological functions in both plants and animals by post-transcriptionally regulating gene expression through interactions with their target mRNAs. The microRNA research is a dynamic field, in which new and unconventional aspects are emerging alongside well-established roles in development and stress adaptation. A recent hypothesis states that miRNAs can be transferred from one species to another and potentially target genes across distant species. Here, we propose to look into the -kingdom potential of miRNAs as a tool to bridge conserved pathways between plant and human cells. To this aim, a novel multi-faceted bioinformatic analysis pipeline was developed, enabling the investigation of common biological processes and genes targeted in plant and human transcriptome by a set of publicly available miRNAs. Multiple datasets, including miRNA, gene, transcript and protein sequences, expression profiles and genetic interactions, were used. Three different strategies were employed, namely a network-based pipeline, an alignment-based pipeline, and a network reconstruction approach, to study functional modules and to evaluate gene/protein similarities among miRNA targets. The results were compared in order to find common features, e.g., microRNAs targeting similar processes. Biological processes like exocytosis and response to viruses were common denominators in the investigated species. Since the involvement of miRNAs in the regulation of DNA damage response (DDR)-associated pathways is barely explored, especially in the plant kingdom, a special attention is given to this aspect. Hereby, miRNAs predicted to target genes involved in DNA repair, recombination and replication, chromatin remodeling, cell cycle and cell death were identified in both plants and humans, paving the way for future interdisciplinary advancements.

Cardiorespiratory fitness (CRF) is a powerful marker of cardiovascular health, especially in youth. Several field tests can provide accurate measurement of CRF, the Cooper Run Test (CRT) is generally preferred by physical education (PE) teachers and trainers. The CRT performance in adolescents has been compared to reference distance values, gender and age but the differences among the anthropometric characteristics of youth has not been evaluated. For these reasons, the aim of this study was to develop reference standards for CRT and evaluate possible correlations between biometric measurements and athletic performance. This cross-sectional study involved a total of 9,477 children (4,615 girls) aged 11-14 years, freely recruited from North Italian middle schools. Mass, height and CRT performances were assessed in the morning during PE classes as scheduled (mornings-Monday to Friday). The anthropometric measures were collected at least 20 min before the CRT run test. We found a better CRT result in boys ( < 0.001), however a smaller SD in girls suggested a more homogeneous aerobic performance for girls ( ., 371.12 m 282.00 m). In addition, the Shapiro-Wilk test showed a low -value ( < 0.001) but the effect size (0.031 for boys and 0.022 for girls) was small enough that the correction on this parameter allows a practical assumption of normality for the distributions. A visual homoskedastic distribution in both sexes is evident for both body mass index (BMI), mass and VO peak with respect to CRT results. In addition, there were low linear correlation coefficients for both BMI, mass and VO peak compared to the CRT results, with a R2 < 0.5 for every covariate. The only visual heteroskedastic distribution was observed in regression between distance in CRT and age at peak high velocity. Our findings suggested that anthropometric characteristics are not powerful markers to predict Cooper Run Test results in a well-mixed, unpolarized and unbiased pool of middle school boys and girls. PE teachers and trainers should prefer endurance tests over the use of indirect formulas to predict performance.

The side effects of antibiotic treatment directly correlate with intestinal dysbiosis. However, a balanced gut microbiota supports the integrity of the enteric nervous system (ENS), which controls gastrointestinal neuromuscular functions. In this study, we investigated the long-term effects of antibiotic-induced microbial dysbiosis on the ENS and the impact of the spontaneous re-establishment of the gut microbiota on gastrointestinal functions. C57BL/6J mice were treated daily for two weeks with antibiotics. After 0–6 weeks of antibiotics wash-out, we determined (a) gut microbiota composition, (b) gastrointestinal motility, (c) integrity of the ENS, (d) neurochemical code, and (e) inflammation. Two weeks of antibiotic treatment significantly altered gut microbial composition; the genera Clostridium, Lachnoclostridium, and Akkermansia did not regain their relative abundance following six weeks of antibiotic discontinuation. Mice treated with antibiotics experienced delayed gastrointestinal transit and altered expression of neuronal markers. The anomalies of the ENS persisted for up to 4 weeks after the antibiotic interruption; the expression of neuronal HuC/D, glial-derived neurotrophic factor (Gdnf), and nerve growth factor (Ngf) mRNA transcripts did not recover. In this study, we strengthened the idea that antibiotic-induced gastrointestinal dysmotility directly correlates with gut dysbiosis as well as structural and functional damage to the ENS.

CRISPR and CRISPRi systems have revolutionized our biological engineering capabilities by enabling the editing and regulation of virtually any gene, via customization of single guide RNA (sgRNA) sequences. CRISPRi modules can work as programmable logic inverters, in which the dCas9-sgRNA complex represses a target transcriptional unit. They have been successfully used in bacterial synthetic biology to engineer information processing tasks, as an alternative to the traditionally adopted transcriptional regulators. In this work, we investigated and modulated the transfer function of several model systems with specific focus on the cell load caused by the CRISPRi logic inverters. First, an optimal expression cassette for dCas9 was rationally designed to meet the low-burden high-repression trade-off. Then, a circuit collection was studied at varying levels of dCas9 and sgRNAs targeting three different promoters from the popular tet , lac and lux systems, placed at different DNA copy numbers. The CRISPRi NOT gates showed low-burden properties that were exploited to fix a high resource-consuming circuit previously exhibiting a non-functional input-output characteristic, and were also adopted to upgrade a transcriptional regulator-based NOT gate into a 2-input NOR gate. The obtained data demonstrate that CRISPRi-based modules can effectively act as low-burden components in different synthetic circuits for information processing.