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Methods in Molecular Biology (Clifton,... 2024Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their...
Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their lives in 2021. Antimicrobial resistance (AMR) has been an ongoing crisis for decades; 4.95 million deaths were associated with antibiotic resistance in 2019. While AMR is a multi-faceted problem, drug discovery is an urgent part of the solution and is at the forefront of modern research.The landscape of drug discovery for TB has undoubtedly been transformed by the development of high-throughput gene-silencing techniques that enable interrogation of every gene in the genome, and their relative contribution to fitness, virulence, and AMR. A recent advance in this area is CRISPR interference (CRISPRi). The application of this technique to antimicrobial susceptibility testing (AST) is the subject of ongoing research in basic science.CRISPRi technology can be used in conjunction with the high-throughput SPOT-culture growth inhibition assay (HT-SPOTi) to rapidly evaluate and assess gene essentiality including non-essential, conditionally essential (by using appropriate culture conditions), and essential genes. In addition, the HT-SPOTi method can develop drug susceptibility and drug resistance profiles.This technology is further useful for drug discovery groups who have designed target-based inhibitors rationally and wish to validate the primary mechanisms of their novel compounds' antibiotic action against the proposed target.
Topics: Microbial Sensitivity Tests; Mycobacterium tuberculosis; Drug Discovery; Gene Silencing; Humans; CRISPR-Cas Systems; Antitubercular Agents; Anti-Bacterial Agents; High-Throughput Screening Assays; Drug Resistance, Bacterial; Tuberculosis
PubMed: 38949697
DOI: 10.1007/978-1-0716-3981-8_3 -
Methods in Molecular Biology (Clifton,... 2024There is an increasing need for new treatment regimens to combat antibiotic-resistant strains of bacteria. Staphylococcus aureus is a clinically important, opportunist...
There is an increasing need for new treatment regimens to combat antibiotic-resistant strains of bacteria. Staphylococcus aureus is a clinically important, opportunist pathogen that has developed resistance to a range of antibiotics. The zebrafish larval model of systemic disease has been increasingly utilized to elucidate S. aureus virulence mechanisms and host-pathogen interactions. Here, we outline how this model can be used to investigate the effects of different antibiotics alone and in combination against S. aureus.
Topics: Animals; Zebrafish; Anti-Bacterial Agents; Staphylococcus aureus; Larva; Staphylococcal Infections; Disease Models, Animal; Drug Therapy, Combination; Host-Pathogen Interactions; Microbial Sensitivity Tests
PubMed: 38949695
DOI: 10.1007/978-1-0716-3981-8_1 -
AMB Express Jun 2024Pseudomonas aeruginosa is a commonly found Gram-negative bacterium in healthcare facilities and is renowned for its ability to form biofilms and its virulence factors...
Pseudomonas aeruginosa is a commonly found Gram-negative bacterium in healthcare facilities and is renowned for its ability to form biofilms and its virulence factors that are controlled by quorum sensing (QS) systems. The increasing prevalence of multidrug-resistant strains of this bacterium poses a significant challenge in the field of medicine. Consequently, the exploration of novel antimicrobial agents has become a top priority. This research aims to optimize chitosan derived from white shrimp (Metapenaeus affinis) using the Response Surface Methodology (RSM) computational approach. The objective is to investigate chitosan's potential as a solution for inhibiting QS activity and biofilm formation in P. aeruginosa ATCC 10,145. Under optimized conditions, chitin was treated with NaOH (1.41 M) for 15.75 h, HCl (7.49% vol) for 2.01 h, and at a deacetylation temperature of 81.15 °C. The resulting chitosan exhibited a degree of deacetylation (DD%) exceeding 93.98%, as confirmed by Fourier-transform infrared (FTIR) spectral analysis, indicating its high purity. The extracted chitosan demonstrated a significant synergistic antibiotic effect against P. aeruginosa when combined with ceftazidime, enhancing its bactericidal activity by up to 15-fold. In addition, sub-MIC (minimum inhibitory concentration) concentrations of extracted chitosan (10 and 100 µg/mL) successfully reduced the production of pyocyanin and rhamnolipid, as well as the swimming motility, protease activity and biofilm formation ability in comparison to the control group (P < 0.05). Moreover, chitosan treatment downregulated the RhlR and LasR genes in P. aeruginosa when compared to the control group (P < 0.05). The optimized chitosan extract shows significant potential as a coating agent for surgical equipment, effectively preventing nosocomial infections caused by P. aeruginosa pathogens.
PubMed: 38949677
DOI: 10.1186/s13568-024-01732-1 -
Mikrochimica Acta Jun 2024A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for...
A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for the ultrasensitive and quantitative identification of pathogens, leveraging the sequence-specific detection capabilities of MBs. The microfluidic device contained three distinct functional units including droplet generation, pico-injection, and droplet counting. Utilizing a pico-injector, MBs are introduced into each droplet to specifically identify LAMP amplification products, thereby overcoming issues related to temperature incompatibility. Our methodology has been validated through the quantitative detection of Escherichia coli, achieving a detection limit as low as 9 copies/μL in a model plasmid containing the malB gene and 3 CFU/μL in a spiked milk sample. The total analysis time was less than 1.5 h. The sensitivity and robustness of this platform further demonstrated the potential for rapid pathogen detection and diagnosis, particularly when integrated with cutting-edge microfluidic technologies.
Topics: Nucleic Acid Amplification Techniques; Escherichia coli; Limit of Detection; Milk; Animals; Molecular Diagnostic Techniques; Microfluidic Analytical Techniques; DNA, Bacterial
PubMed: 38949666
DOI: 10.1007/s00604-024-06509-8 -
ELife Jul 2024Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation;...
Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.
Topics: Chemokine CCL5; Animals; Heparitin Sulfate; Humans; Cricetulus; CHO Cells; Mice; Chemotaxis; Heparin; Phase Separation
PubMed: 38949655
DOI: 10.7554/eLife.93871 -
The Journal of Experimental Medicine Sep 2024Children resist COVID-19, and previous studies reported increased innate immunity in their upper airways. A new paper by Watkins et al....
Children resist COVID-19, and previous studies reported increased innate immunity in their upper airways. A new paper by Watkins et al. (https://doi.org/10.1084/jem.20230911) shows that the nasal mucosa of children is characterized by often asymptomatic viral and/or bacterial infections that dynamically regulate distinct innate immune programs.
Topics: Humans; COVID-19; Child; Immunity, Innate; SARS-CoV-2; Nasal Mucosa
PubMed: 38949639
DOI: 10.1084/jem.20241027 -
The Journal of Experimental Medicine Sep 2024Studies during the COVID-19 pandemic showed that children had heightened nasal innate immune responses compared with adults. To evaluate the role of nasal viruses and...
Studies during the COVID-19 pandemic showed that children had heightened nasal innate immune responses compared with adults. To evaluate the role of nasal viruses and bacteria in driving these responses, we performed cytokine profiling and comprehensive, symptom-agnostic testing for respiratory viruses and bacterial pathobionts in nasopharyngeal samples from children tested for SARS-CoV-2 in 2021-22 (n = 467). Respiratory viruses and/or pathobionts were highly prevalent (82% of symptomatic and 30% asymptomatic children; 90 and 49% for children <5 years). Virus detection and load correlated with the nasal interferon response biomarker CXCL10, and the previously reported discrepancy between SARS-CoV-2 viral load and nasal interferon response was explained by viral coinfections. Bacterial pathobionts correlated with a distinct proinflammatory response with elevated IL-1β and TNF but not CXCL10. Furthermore, paired samples from healthy 1-year-olds collected 1-2 wk apart revealed frequent respiratory virus acquisition or clearance, with mucosal immunophenotype changing in parallel. These findings reveal that frequent, dynamic host-pathogen interactions drive nasal innate immune activation in children.
Topics: Humans; Immunity, Innate; Child, Preschool; Infant; COVID-19; Child; SARS-CoV-2; Female; Male; Nasopharynx; Viral Load; Nasal Mucosa; Cytokines; Host-Pathogen Interactions; Adolescent; Nose; Coinfection
PubMed: 38949638
DOI: 10.1084/jem.20230911 -
Molecular Plant-microbe Interactions :... Jul 2024The emergence of plant pathogens is often associated with waves of unique evolutionary and epidemiological events. pv. is one of the major pathogens causing bacterial...
The emergence of plant pathogens is often associated with waves of unique evolutionary and epidemiological events. pv. is one of the major pathogens causing bacterial spot disease of tomatoes. After its first report in the 1950s, there were no formal reports on this pathogen until the 1990s, despite active global research on the pathogens that cause tomato and pepper bacterial spot disease. Given the recently documented global distribution of pv. , our objective was to examine genomic diversification associated with its emergence. We sequenced the genomes of pv. strains collected in eight countries to examine global population structure and pathways of emergence using phylodynamic analysis. We found that strains isolated post-1990 group by region of collection and show minimal impact of recombination on genetic variation. A period of rapid geographic expansion in pv. is associated with acquisition of a large plasmid conferring copper tolerance by horizontal transfer and coincides with the burgeoning hybrid tomato seed industry through the 1980s. The ancestry of pv. is consistent with introduction to hybrid tomato seed production and dissemination during the rapid increase in trade of hybrid seeds.
PubMed: 38949619
DOI: 10.1094/MPMI-04-24-0035-R -
ACS Macro Letters Jul 2024The frequent mutations of influenza A virus (IAV) have led to an urgent need for the development of innovative antiviral drugs. Glycopolymers offer significant...
The frequent mutations of influenza A virus (IAV) have led to an urgent need for the development of innovative antiviral drugs. Glycopolymers offer significant advantages in biomedical applications owing to their biocompatibility and structural diversity. However, the primary challenge lies in the design and synthesis of well-defined glycopolymers to precisely control their biological functionalities. In this study, functional glycopolymers with sulfated fucose and 6'-sialyllactose were successfully synthesized through ring-opening metathesis polymerization and a postmodification strategy. The optimized heteropolymer exhibited simultaneous targeting of hemagglutinin and neuraminidase on the surface of IAV, as evidenced by MU-NANA assay and hemagglutination inhibition data. Antiviral experiments demonstrated that the glycopolymer displayed broad and efficient inhibitory activity against wild-type and mutant strains of H1N1 and H3N2 subtypes , thereby establishing its potential as a dual-targeted inhibitor for combating IAV resistance.
PubMed: 38949618
DOI: 10.1021/acsmacrolett.4c00221 -
Journal of Immunology (Baltimore, Md. :... Jul 2024Aberrant activity of NLRP3 has been shown associations with severe diseases. Palmitoylation is a kind of protein post-translational modification, which has been shown to...
Aberrant activity of NLRP3 has been shown associations with severe diseases. Palmitoylation is a kind of protein post-translational modification, which has been shown to regulate cancer development and the innate immune system. Here, we showed that NLRP3 is palmitoylated at Cys419 and that palmitoyltransferase ZDHHC17 is the predominant enzyme that mediates NLRP3 palmitoylation and promotes NLRP3 activation by interacting with NLRP3 and facilitating NIMA-related kinase 7 (NEK7)-NLRP3 interactions. Blockade of NLRP3 palmitoylation by a palmitoylation inhibitor, 2-bromopalmitate, effectively inhibited NLRP3 activation in vitro. Also, in a dextran sulfate sodium-induced colitis model in mice, 2-bromopalmitate application could attenuate weight loss, improve the survival rate, and rescue pathological changes in the colon of mice. Overall, our study reveals that palmitoylation of NLPR3 modulates inflammasome activation and inflammatory bowel disease development. We propose that drugs targeting NLRP3 palmitoylation could be promising candidates in the treatment of NLRP3-mediated inflammatory diseases.
PubMed: 38949555
DOI: 10.4049/jimmunol.2300241