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International Journal of Molecular... Jun 2024Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of...
Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.
Topics: Lipopolysaccharides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Animals; Mice; Macrophages; Lipid A; Cytokines; Endopeptidase K; Electrophoresis, Polyacrylamide Gel
PubMed: 38928052
DOI: 10.3390/ijms25126345 -
International Journal of Molecular... Jun 2024Mutations have driven the evolution and development of new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with potential implications for...
Mutations have driven the evolution and development of new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with potential implications for increased transmissibility, disease severity and vaccine escape among others. Genome sequencing is a technique that allows scientists to read the genetic code of an organism and has become a powerful tool for studying emerging infectious diseases. Here, we conducted a cross-sectional study in selected districts of the Eastern Province of Zambia, from November 2021 to February 2022. We analyzed SARS-CoV-2 samples ( = 76) using high-throughput sequencing. A total of 4097 mutations were identified in 69 SARS-CoV-2 genomes with 47% (1925/4097) of the mutations occurring in the spike protein. We identified 83 unique amino acid mutations in the spike protein of the seven Omicron sublineages (BA.1, BA.1.1, BA.1.14, BA.1.18, BA.1.21, BA.2, BA.2.23 and XT). Of these, 43.4% (36/83) were present in the receptor binding domain, while 14.5% (12/83) were in the receptor binding motif. While we identified a potential recombinant XT strain, the highly transmissible BA.2 sublineage was more predominant (40.8%). We observed the substitution of other variants with the Omicron strain in the Eastern Province. This work shows the importance of pandemic preparedness and the need to monitor disease in the general population.
Topics: Zambia; Humans; SARS-CoV-2; COVID-19; Genome, Viral; Spike Glycoprotein, Coronavirus; Mutation; Cross-Sectional Studies; Retrospective Studies; Phylogeny; Genomics; High-Throughput Nucleotide Sequencing
PubMed: 38928045
DOI: 10.3390/ijms25126338 -
Biomedicines May 2024The CRISPR-Cas9 system is a revolutionary tool in genetic engineering, offering unprecedented precision and efficiency in genome editing. Cas9, an enzyme derived from...
The CRISPR-Cas9 system is a revolutionary tool in genetic engineering, offering unprecedented precision and efficiency in genome editing. Cas9, an enzyme derived from bacteria, is guided by RNA to edit DNA sequences within cells precisely. However, while CRISPR-Cas9 presents notable benefits and encouraging outcomes as a molecular tool and a potential therapeutic agent, the process of producing and purifying recombinant Cas9 protein remains a formidable hurdle. In this study, we systematically investigated the expression of recombinant SpCas9-His in four distinct () strains (Rosetta2, BL21(DE3), BL21(DE3)-pLysS, and BL21(DE3)-Star). Through optimization of culture conditions, including temperature and post-induction time, the BL21(DE3)-pLysS strain demonstrated efficient SpCas9 protein expression. This study also presents a detailed protocol for the purification of recombinant SpCas9, along with detailed troubleshooting tips. Results indicate successful SpCas9 protein expression using BL21(DE3)-pLysS at 0.5 mM IPTG concentration. Furthermore, the findings suggest potential avenues for further enhancements, paving the way for large-scale Cas9 production. This research contributes valuable insights into optimizing strains and culture conditions for enhanced Cas9 expression, offering a step forward in the development of efficient genome editing tools and therapeutic proteins.
PubMed: 38927433
DOI: 10.3390/biomedicines12061226 -
Biomolecules May 2024Renal interstitial fibrosis (RIF) is a classic pathophysiological process of chronic kidney disease (CKD). However, the mechanisms underlying RIF remain unclear. The...
Renal interstitial fibrosis (RIF) is a classic pathophysiological process of chronic kidney disease (CKD). However, the mechanisms underlying RIF remain unclear. The present study found that a novel circular RNA, cirInpp5b, might be involved in RIF by high-throughput sequencing. Subsequent experiments revealed that circInpp5b was reduced in UUO mouse kidney tissues and TGF-β1-treated proximal tubular cells. The overexpression of circInpp5b inhibited RIF in UUO mice and prevented extracellular matrix (ECM) deposition in TGF-β1-treated proximal tubular cells. Furthermore, overexpression of circInpp5b down-regulated the protein level of DDX1. Mechanistically, circInpp5b bound to the DDX1 protein and promoted its lysosomal degradation. Collectively, the findings of our study demonstrate that circInpp5b ameliorates RIF by binding to the DDX1 protein and promoting its lysosomal degradation.
Topics: DEAD-box RNA Helicases; Animals; Mice; Lysosomes; Fibrosis; RNA, Circular; Proteolysis; Male; Mice, Inbred C57BL; Humans; Kidney; Kidney Diseases
PubMed: 38927017
DOI: 10.3390/biom14060613 -
Journal of Nanobiotechnology Jun 2024The use of stem cell-derived exosomes (Exos) as therapeutic vehicles is receiving increasing attention. Exosome administration has several advantages over cell...
BACKGROUND
The use of stem cell-derived exosomes (Exos) as therapeutic vehicles is receiving increasing attention. Exosome administration has several advantages over cell transplantation, thus making exosomes promising candidates for large-scale clinical implementation and commercialization. However, exosome extraction and purification efficiencies are relatively low, and therapeutic heterogeneity is high due to differences in culture conditions and cell viability. Therefore, in this study, we investigated a priming procedure to enhance the production and therapeutic effects of exosomes from human umbilical cord mesenchymal stem cells (hucMSCs). After preconditioning hucMSCs with agonists/inhibitors that target the Wnt/β-catenin pathway, we assessed both the production of exosomes and the therapeutic efficacy of the optimized exosomes in the context of diabetic wound healing, hoping to provide a safer, more stable and more effective option for clinical application.
RESULTS
The Wnt signalling pathway agonist CHIR99021 increased exosome production by 1.5-fold without causing obvious changes in the characteristics of the hucMSCs or the size of the exosome particles. Further studies showed that CHIR99021 promoted the production of exosomes by facilitating exocytosis. This process was partly mediated by SNAP25. To further explore whether CHIR99021 changed the cargo that was loaded into the exosomes and its therapeutic effects, we performed proteomic and transcriptomic analyses of exosomes from primed and control hucMSCs. The results showed that CHIR99021 significantly upregulated the expression of proteins that are associated with cell migration and wound healing. Animal experiments confirmed that, compared to control hucMSC-derived exosomes, CHIR99021-pretreated hucMSC-derived exosomes (CHIR-Exos) significantly accelerated wound healing in diabetic mice, enhanced local collagen deposition, promoted angiogenesis, and reduced chronic inflammation. Subsequent in vitro experiments confirmed that the CHIR-Exos promoted wound healing by facilitating cell migration, inhibiting oxidative stress-induced apoptosis, and preventing cell cycle arrest.
CONCLUSIONS
The Wnt agonist CHIR99021 significantly increased exosome secretion by hucMSCs, which was partly mediated by SNAP25. Notably, CHIR99021 treatment also significantly increased the exosomal levels of proteins that are associated with wound healing and cell migration, resulting in enhanced acceleration of wound healing. All of these results suggested that pretreatment of hucMSCs with CHIR99021 not only promoted exosome production but also improved the exosome therapeutic efficacy, thus providing a promising option for large-scale clinical implementation and commercialization.
Topics: Exosomes; Wound Healing; Mesenchymal Stem Cells; Humans; Animals; Wnt Signaling Pathway; Mice; Umbilical Cord; Pyridines; Diabetes Mellitus, Experimental; Pyrimidines; Male; Cells, Cultured; Cell Movement
PubMed: 38926800
DOI: 10.1186/s12951-024-02650-x -
BMC Microbiology Jun 2024The incidence of hospital-acquired infections in extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) has been increasing worldwide and is frequently associated...
BACKGROUND
The incidence of hospital-acquired infections in extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) has been increasing worldwide and is frequently associated with an increase in mortality and morbidity rates. The aim of this study was to characterize clinical XDR-PA isolates recovered during six months at three different hospitals in Egypt.
RESULTS
Seventy hospital-acquired clinical isolates of P. aeruginosa were classified into multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR), according to their antimicrobial resistance profile. In addition, the possession of genes associated with mobile genetic elements and genes encoding antimicrobial resistance determinants among isolates were detected using polymerase chain reaction. As a result, a significant percentage of the isolates (75.7%) were XDR, while 18.5% were MDR, however only 5.7% of the isolates were non-MDR. The phenotypic detection of carbapenemases, extended-spectrum β-lactamases (ESBLs) and metallo β-lactamase (MBL) enzymes showed that 73.6% of XDR-PA isolates were carbapenemases producers, whereas 75.5% and 88.7% of XDR-PA isolates produced ESBLs and MBL respectively. In addition, PCR screening showed that oxa gene was the most frequently detected gene of carbapenemases (91.4%), while aac(6')-lb gene was mostly detected (84.3%) among the screened aminoglycosides-resistance genes. Furthermore, the molecular detection of the colistin resistance gene showed that 12.9% of isolates harbored mcr-1 gene. Concerning mobile genetic element markers (intI, traA, tnp513, and merA), intI was the highest detected gene as it was amplified in 67 isolates (95.7%). Finally, phylogenetic and molecular typing of the isolates via ERIC-PCR analysis revealed 10 different ERIC fingerprints.
CONCLUSION
The present study revealed a high prevalence of XDR-PA in hospital settings which were resistant to a variety of antibiotics due to several mechanisms. In addition, 98% of the XDR-PA clinical isolates contained at least one gene associated with movable genetic elements, which could have aided the evolution of these XDR-PA strains. To reduce spread of drug resistance, judicious use of antimicrobial agents and strict infection control measures are therefore essential.
Topics: Pseudomonas aeruginosa; Humans; Pseudomonas Infections; Drug Resistance, Multiple, Bacterial; Cross Infection; Egypt; beta-Lactamases; Anti-Bacterial Agents; Microbial Sensitivity Tests; Bacterial Proteins; Hospitals; Interspersed Repetitive Sequences; Polymerase Chain Reaction
PubMed: 38926687
DOI: 10.1186/s12866-024-03321-5 -
Microbial Biotechnology Jun 2024Pharmaceuticals are of increasing environmental concern as they emerge and accumulate in surface- and groundwater systems around the world, endangering the overall... (Review)
Review
Pharmaceuticals are of increasing environmental concern as they emerge and accumulate in surface- and groundwater systems around the world, endangering the overall health of aquatic ecosystems. Municipal wastewater discharge is a significant vector for pharmaceuticals and their metabolites to enter surface waters as humans incompletely absorb prescription drugs and excrete up to 50% into wastewater, which are subsequently incompletely removed during wastewater treatment. Microalgae present a promising target for improving wastewater treatment due to their ability to remove some pollutants efficiently. However, their inherent metabolic pathways limit their capacity to degrade more recalcitrant organic compounds such as pharmaceuticals. The human liver employs enzymes to break down and absorb drugs, and these enzymes are extensively researched during drug development, meaning the cytochrome P450 enzymes responsible for metabolizing each approved drug are well studied. Thus, unlocking or increasing cytochrome P450 expression in endogenous wastewater microalgae could be a cost-effective strategy to reduce pharmaceutical loads in effluents. Here, we discuss the challenges and opportunities associated with introducing cytochrome P450 enzymes into microalgae. We anticipate that cytochrome P450-engineered microalgae can serve as a new drug removal method and a sustainable solution that can upgrade wastewater treatment facilities to function as "mega livers".
Topics: Microalgae; Cytochrome P-450 Enzyme System; Wastewater; Pharmaceutical Preparations; Water Purification; Water Pollutants, Chemical; Humans; Biodegradation, Environmental
PubMed: 38925623
DOI: 10.1111/1751-7915.14515 -
The Journal of Biological Chemistry Jun 2024Transthyretin (TTR) is a homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of...
Transthyretin (TTR) is a homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of them associated with familial amyloid cardiomyopathy (FAC). Recently, our group described a new variant of TTR in Brazil, namely A39D-TTR, which causes a severe cardiac condition. Position 39 is in the AB loop, a region of the protein that is located within the thyroxine-binding channels and is involved in tetramer formation. In the present study we solved the structure and characterize the thermodynamic stability of this new variant of TTR using urea and high hydrostatic pressure (HHP). Interestingly, during the process of purification, A39D-TTR turned out to be a dimer and not a tetramer, a variation that might be explained by the close contact of the four aspartic acids at position 39, where they face each other inside the thyroxine channel. In the presence of sub-denaturing concentrations of urea, bis-ANS binding and dynamic light scattering revealed A39D-TTR in the form of a molten-globule dimer. Co-expression of A39D and WT isoforms in the same bacterial cell did not produce heterodimers or heterotetramers, suggesting that somehow a negative charge at the AB loop precludes tetramer formation. A39D-TTR proved to be highly amyloidogenic, even at mildly acidic pH values where WT-TTR does not aggregate. Interestingly, despite being a dimer, aggregation of A39D-TTR was inhibited by diclofenac, which binds to the thyroxine channel in the tetramer, suggesting the existence of other pockets in A39D-TTR able to accommodate this molecule.
PubMed: 38925327
DOI: 10.1016/j.jbc.2024.107495 -
PloS One 2024Hepatitis B virus (HBV) constitutes a significant global health challenge, with more than 2 billion people infected globally and almost 291 million chronic cases. In...
Hepatitis B virus (HBV) constitutes a significant global health challenge, with more than 2 billion people infected globally and almost 291 million chronic cases. In Africa, coinfection of HBV with Human Immunodeficiency Virus (HIV) is high, yet the condition remains overlooked in many countries. While antiretroviral therapy (ART) has improved HIV survival, viral hepatitis continues to contribute to morbidity and mortality. Occult Hepatitis B infection (OBI), characterized by a low-level of HBV DNA in individuals with negative hepatitis B surface antigen (HBsAg), is an emerging concern among HIV seropositive individuals due to the risk of HBV reactivation and associated complications, especially hepatocellular carcinoma (HCC). Ghana has an estimated HBV/HIV coinfection prevalence of 13.6% making it important to also determine potential cases of OBI. This study aims to assess OBI prevalence in persons living with HIV (PLHIV). A cross-sectional study was conducted in five health facilities in the Cape Coast Metropolis. HBV-related serological markers were determined among 116 PLHIV using the Enzyme-Linked Immunosorbent Assay (ELISA) method. HBV DNA was extracted from 30 participants found to be HBsAg negative but positive for hepatitis B core antibody (HBcAb+). Nested PCR was employed in detecting HBV DNA and HBV viral load was performed using qPCR. The median age of the participants was 37 years (IQR 22-65). Serologically, 7.8% (n = 9, 95% CI: 3.5-22.7), 12.1% (n = 14), and 25.9% (n = 30) tested positive for solely HBsAg, HBsAb, and HBcAb respectively. OBI prevalence among HBsAg-/HBcAb+ participants was 16.7% (n = 5, 95% CI: 6.5-23.7) with a median HBV DNA level of 139.2 IU/ml (IQR, 96.7-142.0). The prevalence of OBI among HIV-positive participants in the Cape Coast Metropolis highlights the need to consider screening for HBV among HIV patients using nucleic acid amplification tests. This can inform medical management and reduce the risk of liver complications, including HCC.
Topics: Humans; Ghana; HIV Infections; Female; Male; Adult; Hepatitis B; Prevalence; Middle Aged; Cross-Sectional Studies; Hepatitis B virus; Hepatitis B Surface Antigens; Coinfection; DNA, Viral; Young Adult
PubMed: 38924017
DOI: 10.1371/journal.pone.0305862 -
Anais Da Academia Brasileira de Ciencias 2024Klebsiella pneumoniae (K. pneumoniae) is a major cause of healthcare-associated infections and plays a prominent role in the widespread antibiotic resistance crisis....
Klebsiella pneumoniae (K. pneumoniae) is a major cause of healthcare-associated infections and plays a prominent role in the widespread antibiotic resistance crisis. Accurate identification of carbapenemases is essential to facilitate effective antibiotic treatment and reduce transmission of K. pneumoniae. This study aimed to detect carbapenemase production in carbapenem-resistant K. pneumoniae strains using phenotypic and genotypic methods. A total of 67 carbapenem-resistant K. pneumoniae strains obtained from various clinical samples were utilized for identification and antimicrobial susceptibility by the Vitek 2 Compact system (Biomerieux, France). Carbapenemase production was determined by using the Polymerase chain reaction, Blue-carba test (BCT) and Carbapenem inactivation method (CIM). Out of the isolates, 59 (88.1%) were positive bla OXA-48, 16 (23.9%) bla IMP, and five (7.5%) were positive bla NDM. No bla KPC genes were detected. The CIM identified 62 (92.5%), BCT identified 63 (94%) of PCR-positive isolates. The sensitivity and specificity of the BCT and the CIM were determined to be 96.7%, 40%, and 96.7%, 25% respectively. The bla OXA-48 gene was found to be the most prevalent in K. pneumoniae isolates. Early identification of carbapenem resistance plays a vital role in designing effective infection control strategies and mitigating the emergence and transmission of carbapenem resistance, thus reducing healthcare-associated infections.
Topics: Klebsiella pneumoniae; Humans; Phenotype; Microbial Sensitivity Tests; Genotype; Anti-Bacterial Agents; Carbapenems; beta-Lactamases; Polymerase Chain Reaction; Bacterial Proteins; Klebsiella Infections; Carbapenem-Resistant Enterobacteriaceae
PubMed: 38922280
DOI: 10.1590/0001-3765202420231322