-
Signal Transduction and Targeted Therapy Feb 2024Pyrogen, often as a contaminant, is a key indicator affecting the safety of almost all parenteral drugs (including biologicals, chemicals, traditional Chinese medicines...
Pyrogen, often as a contaminant, is a key indicator affecting the safety of almost all parenteral drugs (including biologicals, chemicals, traditional Chinese medicines and medical devices). It has become a goal to completely replace the in vivo rabbit pyrogen test by using the in vitro pyrogen test based on the promoted 'reduction, replacement and refinement' principle, which has been highly considered by regulatory agencies from different countries. We used NF-κB, a central signalling molecule mediating inflammatory responses, as a pyrogenic marker and the monocyte line THP-1 transfected with a luciferase reporter gene regulated by NF-κB as an in vitro model to detect pyrogens by measuring the intensity of a fluorescence signal. Here, we show that this test can quantitatively and sensitively detect endotoxin (lipopolysaccharide from different strains) and nonendotoxin (lipoteichoic acid, zymosan, peptidoglycan, lectin and glucan), has good stability in terms of NF-κB activity and cell phenotypes at 39 cell passages and can be applied to detect pyrogens in biologicals (group A & C meningococcal polysaccharide vaccine; basiliximab; rabies vaccine (Vero cells) for human use, freeze-dried; Japanese encephalitis vaccine (Vero cells), inactivated; insulin aspart injection; human albumin; recombinant human erythropoietin injection (CHO Cell)). The within-laboratory reproducibility of the test in three independent laboratories was 85%, 80% and 80% and the interlaboratory reproducibility among laboratories was 83.3%, 95.6% and 86.7%. The sensitivity (true positive rate) and specificity (true negative rate) of the test were 89.9% and 90.9%, respectively. In summary, the test provides a novel alternative for pyrogen detection.
Topics: Animals; Chlorocebus aethiops; Rabbits; Humans; Pyrogens; NF-kappa B; Vero Cells; Reproducibility of Results; Cell Line
PubMed: 38369543
DOI: 10.1038/s41392-024-01744-0 -
BMC Complementary Medicine and Therapies Feb 2024Modern medicine is not the choice of patients with "shimetere" in the Gurage community owing to their perception of 'parenteral medication use severely aggravates the...
BACKGROUND
Modern medicine is not the choice of patients with "shimetere" in the Gurage community owing to their perception of 'parenteral medication use severely aggravates the disease'. For this reason, the root part of Polygala sadebeckiana Gürke is commonly utilized as traditional medicine in the management of the disease. The aim of this study was to evaluate the antimicrobial activity of Polygala sadebeckiana Gürke extract on bacterial isolates from wound samples of patients with "Shimetere".
METHODS
The agar well diffusion method was used to evaluate antibacterial activity, and the agar dilution method was utilized to determine minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MICs). The crude extract was tested against isolated bacteria at concentrations of 25, 50, 75 and 100 mg/mL in triplicate (3x). The positive controls were azithromycin (15 µg) and cloxacillin disk (5 µg), and the negative control was dimethylsulfoxide (5%). The group mean comparisons were made using one-way ANOVA at a significance level of p < 0.05, and the results are presented as the mean ± standard deviation. The presence of secondary metabolites from crude extract was checked by standard testing procedures.
RESULTS
S. aureus and S. pyrogen were the two identified bacteria from 9 (60%) and 3 (20%) wound samples, respectively. All identified bacterial strains were susceptible to the reference antibiotics. Tannins and saponins were the most abundant secondary metabolites found in the crude extracts. The average inhibition zones of the plant extracts with 100, 75, 50 and 25 mg/mL concentrations were 27, 20.33, 15.25, and 11.96 mm (p < 0.000) for S. aureus and 30.02, 24.50, 19.07, and 15.77 mm (p < 0.000) for S. pyrogen bacteria, respectively. The MIC and MBC of the crude extract were 1.67 and 10 mg/mL for S. aureus and 0.98 and 4 mg/mL for S. pyrogen.
CONCLUSION
Polygala sadebeckiana Gürke contained significant tannins and saponins as secondary metabolites and had antibacterial activities against isolated bacteria (S. aureus and S. pyrogen) from "Shimetere". The potential mechanism of antibacterial action of the plant extract was cell wall synthesis inhibition.
Topics: Humans; Polygala; Tannins; Staphylococcus aureus; Agar; Pyrogens; Plant Extracts; Anti-Bacterial Agents; Bacteria; Phytochemicals; Saponins
PubMed: 38302996
DOI: 10.1186/s12906-024-04371-y -
Carbon Balance and Management Mar 2024Black carbon (BC) encompasses a range of carbonaceous materials--including soot, char, and charcoal--derived from the incomplete combustion of fossil fuels and biomass.... (Review)
Review
BACKGROUND
Black carbon (BC) encompasses a range of carbonaceous materials--including soot, char, and charcoal--derived from the incomplete combustion of fossil fuels and biomass. Urban soils can become enriched in BC due to proximity to these combustion sources. We conducted a literature review of BC in urban soils globally and found 26 studies reporting BC and total organic carbon (TOC) content collected to a maximum of 578 cm depth in urban soils across 35 cities and 10 countries. We recorded data on city, climate, and land use/land cover characteristics to examine drivers of BC content and contribution to TOC in soil.
RESULTS
All studies were conducted in the northern hemisphere, with 68% of the data points collected in China and the United States. Surface samples (0-20 cm) accounted for 62% of samples in the dataset. Therefore, we focused our analysis on 0-10 cm and 10-20 cm depths. Urban soil BC content ranged from 0-124 mg/g (median = 3 mg/g) at 0-10 cm and from 0-53 mg/g (median = 2.8 mg/g) at 10-20 cm depth. The median proportional contribution of BC to TOC was 23% and 15% at 0-10 cm and 10-20 cm, respectively. Surface soils sampled in industrial land use and near roads had the highest BC contents and proportions, whereas samples from residential sites had among the lowest. Soil BC content decreased with mean annual soil temperature.
CONCLUSIONS
Our review indicates that BC comprises a major fraction (nearly one quarter) of the TOC in urban surface soils, yet sampling bias towards the surface could hide the potential for BC storage at depth. Land use emerged as an importer driver of soil BC contents and proportions, whereas land cover effects remain uncertain. Warmer and wetter soils were found to have lower soil BC than cooler and drier soils, differences that likely reflect soil BC loss mechanisms. Additional research on urban soil BC at depth and from diverse climates is critical to better understand the role of cities in the global carbon cycle.
PubMed: 38429441
DOI: 10.1186/s13021-024-00255-3 -
Frontiers in Bioengineering and... 2024Clinical grade magnetic bead implants have important applications in interfacing with the human body, providing contactless mechanical attachment or wireless...
Clinical grade magnetic bead implants have important applications in interfacing with the human body, providing contactless mechanical attachment or wireless communication through human tissue. We recently developed a new strategy, magnetomicrometry, that uses magnetic bead implants as passive communication devices to wirelessly sense muscle tissue lengths. We manufactured clinical-grade magnetic bead implants and verified their biocompatibility via intramuscular implantation, cytotoxicity, sensitization, and intracutaneous irritation testing. In this work, we test the pyrogenicity of the magnetic bead implants via a lagomorph model, and we test the biocompatibility of the magnetic bead implants via a full chemical characterization and toxicological risk assessment. Further, we test the cleaning, sterilization, and dry time of the devices that are used to deploy these magnetic bead implants. We find that the magnetic bead implants are non-pyrogenic and biocompatible, with the insertion device determined to be safe to clean, sterilize, and dry in a healthcare setting. These results provide confidence for the safe use of these magnetic bead implants in humans.
PubMed: 38444650
DOI: 10.3389/fbioe.2024.1290453 -
The Science of the Total Environment Sep 2023Green infrastructure drainage systems are innovative treatment units that capture and treat stormwater. Unfortunately, highly polar contaminants remain challenging to...
Green infrastructure drainage systems are innovative treatment units that capture and treat stormwater. Unfortunately, highly polar contaminants remain challenging to remove in conventional biofilters. To overcome treatment limitations, we assessed the transport and removal of stormwater vehicle-related organic contaminants with persistent, mobile, and toxic (in short: PMTs) properties, such as 1H-benzotriazole, NN'-diphenylguanidine, and hexamethoxymethylmelamine (PMT precursor), using batch experiments and continuous-flow sand columns amended with pyrogenic carbonaceous materials, like granulated activated carbon (GAC) or wheat-straw derived biochar. Our results indicated that all investigated contaminants were subjected to nonequilibrium interactions in sand-only and geomedia-amended columns, with kinetic effects upon transport. Experimental breakthrough curves could be well described by a one-site kinetic transport model assuming saturation of sorption sites, which we inferred could occur due to dissolved organic matter fouling. Furthermore, from both batch and column experiments, we found that GAC could remove contaminants significantly better than biochar with higher sorption capacity and faster sorption kinetics. Hexamethoxymethylmelamine, with the lowest organic carbon-water partition coefficient (K) and largest molecular volume among target chemicals, exhibited the lowest affinity in both carbonaceous adsorbents based on estimated sorption parameters. Results suggest that sorption of investigated PMTs was likely driven by steric and hydrophobic effects, and coulombic and other weak intermolecular forces (e.g., London-van der Waals, H-bonding). Results from extrapolating our data to a 1-m depth geomedia-amended sand filter suggested that GAC and biochar could enhance the removal of organic contaminants in biofilters and last for more than one decade. Overall, our work is the first to study treatment alternatives for NN'-diphenylguanidine and hexamethoxymethylmelamine, and contributes to better PMT contaminant removal strategies in environmental applications.
Topics: Charcoal; Guanidines; Adsorption
PubMed: 37209730
DOI: 10.1016/j.scitotenv.2023.164264 -
Environmental Science and Ecotechnology Mar 2024Contaminants, such as polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), heavy metals, and per and polyfluoroalkyl substances (PFASs), primarily...
Contaminants, such as polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), heavy metals, and per and polyfluoroalkyl substances (PFASs), primarily reach the Arctic through long-range atmospheric and oceanic transport. However, local sources within the Arctic also contribute to the levels observed in the environment, including legacy sources and new sources that arise from activities associated with increasing commercial and industrial development. The City of Iqaluit in Frobisher Bay, Nunavut (Canada), has seen rapid population growth and associated development during recent decades yet remains a site of interest for ocean protection, where Inuit continue to harvest country food. In the present study, seven dated marine sediment cores collected in Koojesse Inlet near Iqaluit, and from sites in inner and outer Frobisher Bay, respectively, were analyzed for total mercury (THg), major and trace elements, PAHs, PCBs, and PFASs. The sedimentary record in Koojesse Inlet shows a period of Aroclor 1260-like PCB input concurrent with military site presence in the 1950-60s, followed by decades of input of pyrogenic PAHs, averaging about ten times background levels. Near-surface sediments in Koojesse Inlet also show evidence of transient local-source inputs of THg and PFASs, and recycling or continued slow release of PCBs from legacy land-based sources. Differences in PFAS congener composition clearly distinguish the local sources from long-range transport. Outside Koojesse Inlet but still in inner Frobisher Bay, 9.2 km from Iqaluit, sediments showed evidence of both local source (PCB) and long-range transport. In outer Frobisher Bay, an up-core increase in THg and PFASs in sediments may be explained by ongoing inputs of these contaminants from long-range transport. The context for ocean protection and country food harvesting in this region of the Arctic clearly involves both local sources and long-range transport, with past human activities leaving a long legacy insofar as levels of persistent organic pollutants are concerned.
PubMed: 37860827
DOI: 10.1016/j.ese.2023.100313 -
Archives of Microbiology Dec 2023Vibrio parahaemolyticus is a significant cause of seafood-associated gastroenteritis and pestilence in aquaculture worldwide. Despite extensive research, strategies for...
Vibrio parahaemolyticus is a significant cause of seafood-associated gastroenteritis and pestilence in aquaculture worldwide. Despite extensive research, strategies for protein depletion in this pathogen remain limited. Herein, we constructed a new CRISPR interference (CRISPRi) system for gene repression based on the combination of a shuttle vector pVv3 and the nuclease-null Cas9 variant (dead Cas9, or dCas9) from Streptococcus pyrogens. This CRISPRi is induced by adding both IPTG and arabinose. We showed that gene repression is scalable via the use of multiple sgRNAs. We also demonstrated that this gene repression can be precisely tuned by adjusting the amount of two different inducers and can be reversed by removing the inducers. This system provides a simple approach for selective gene repression on a genome-wide scale in V. parahaemolyticus. Application of this system will dramatically accelerate investigations of this bacterium, including studies of physiology, pathogenesis, and drug target discovery.
Topics: Vibrio parahaemolyticus; RNA, Guide, CRISPR-Cas Systems; Aquaculture; Arabinose; Drug Discovery
PubMed: 38147133
DOI: 10.1007/s00203-023-03770-y -
Microbial Genomics Dec 2023The standalone regulator RofA is a positive regulator of the pilus locus in . Found in only certain genotypes, RofA has been reported to regulate other virulence...
The standalone regulator RofA is a positive regulator of the pilus locus in . Found in only certain genotypes, RofA has been reported to regulate other virulence factors, although its role in the globally dominant 1 is unclear. Given the recent emergence of a new 1 (M1) toxigenic lineage that is distinguished by three non-synonymous SNPs in , we characterized the regulon in six 1 strains that are representative of the two contemporary major 1 lineages (M1 and M1) using RNAseq analysis, and then determined the specific role of the M1-specific SNPs. Deletion of in three M1 strains led to altered expression of 14 genes, including six non-pilus locus genes. In M1 strains, deletion of led to altered expression of 16 genes, including nine genes that were unique to M1. Only the pilus locus genes were common to the RofA regulons of both lineages, while transcriptomic changes varied between strains even within the same lineage. Although introduction of the three SNPs into did not impact gene expression in an M1 strain, reversal of three SNPs in an M1 strain led to an unexpected number of transcriptomic changes that in part recapitulated transcriptomic changes seen when deleting RofA in the same strain. Computational analysis predicted that interactions with a key histidine residue in the PRD domain of RofA would differ between M1 and M1. RofA is a positive regulator of the pilus locus in all 1 strains but effects on other genes are strain- and lineage-specific, with no clear, common DNA binding motif. The SNPs in that characterize M1 may impact regulation of RofA; whether they alter phosphorylation of the RofA PRD domain requires further investigation.
Topics: Streptococcus pyogenes; Regulon; Bacterial Proteins; Pandemics; United Kingdom
PubMed: 38117674
DOI: 10.1099/mgen.0.001159 -
Nucleic Acids Research Jun 2024CRISPR-Cas systems serve as adaptive immune systems in bacteria and archaea, protecting against phages and other mobile genetic elements. However, phages and archaeal...
CRISPR-Cas systems serve as adaptive immune systems in bacteria and archaea, protecting against phages and other mobile genetic elements. However, phages and archaeal viruses have developed countermeasures, employing anti-CRISPR (Acr) proteins to counteract CRISPR-Cas systems. Despite the revolutionary impact of CRISPR-Cas systems on genome editing, concerns persist regarding potential off-target effects. Therefore, understanding the structural and molecular intricacies of diverse Acrs is crucial for elucidating the fundamental mechanisms governing CRISPR-Cas regulation. In this study, we present the structure of AcrIIA28 from Streptococcus phage Javan 128 and analyze its structural and functional features to comprehend the mechanisms involved in its inhibition of Cas9. Our current study reveals that AcrIIA28 is a metalloprotein that contains Zn2+ and abolishes the cleavage activity of Cas9 only from Streptococcus pyrogen (SpyCas9) by directly interacting with the REC3 domain of SpyCas9. Furthermore, we demonstrate that the AcrIIA28 interaction prevents the target DNA from being loaded onto Cas9. These findings indicate the molecular mechanisms underlying AcrIIA28-mediated Cas9 inhibition and provide valuable insights into the ongoing evolutionary battle between bacteria and phages.
Topics: CRISPR-Associated Protein 9; CRISPR-Cas Systems; DNA; Gene Editing; Metalloproteins; Models, Molecular; Protein Binding; Protein Domains; Streptococcus; Streptococcus Phages; Viral Proteins; Zinc
PubMed: 38726868
DOI: 10.1093/nar/gkae357 -
International Journal of Molecular... Jul 2023The development of sensitive methods for the detection of endotoxin molecules, such as lipopolysaccharides (LPS), is essential for food safety and health control....
The development of sensitive methods for the detection of endotoxin molecules, such as lipopolysaccharides (LPS), is essential for food safety and health control. Conventional analytical methods used for LPS detection are based on the pyrogen test, plating and culture-based methods, and the limulus amoebocyte lysate method (LAL). Alternatively, the development of reliable biosensors for LPS detection would be highly desirable to solve some critical issues, such as high cost and a long turnaround time. In this work, we present a label-free Surface-Enhanced Raman Spectroscopy (SERS)-based method for LPS detection in its free form. The proposed method combines the benefits of plasmonic enhancement with the selectivity provided by a specific anti-lipid A antibody (Ab). A high-enhancing nanostructured silver substrate was coated with Ab. The presence of LPS was quantitatively monitored by analyzing the changes in the Ab spectra obtained in the absence and presence of LPS. A limit of detection (LOD) and quantification (LOQ) of 12 ng/mL and 41 ng/mL were estimated, respectively. Importantly, the proposed technology could be easily expanded for the determination of other biological macromolecules.
Topics: Animals; Endotoxins; Lipopolysaccharides; Spectrum Analysis, Raman; Horseshoe Crabs; Biosensing Techniques; Metal Nanoparticles
PubMed: 37569474
DOI: 10.3390/ijms241512099