Search Results (74)

Fullerenes and fullerenols exhibit antioxidant and anti-inflammatory properties, making them promising candidates for Alzheimer’s disease (AD) therapy. Unlike conventional anti-inflammatory drugs, these compounds have multitargeted effects, including their ability to inhibit amyloid fibril formation. However, few studies have explored their efficacy in high-validity AD models. Female APPswe/PS1E9 (APP/PS1) mice and their wild-type (WT) littermates were orally administered with fullerene C₆₀ (0.1 mg/kg/day) or fullerenol C₆₀(OH)₂₄ (0.15 mg/kg/day) for 10 months starting at 2 months of age. Behavioral assessments were performed at 12 months of age. Amyloid plaque density and size were analyzed in the brain regions using Congo red staining. The expression of genes related to inflammation and plasticity was examined, and an in vitro assay was used to test the toxicity of fullerenol and its effect on amyloid β peptide 42 (Aβ42)-induced reactive oxygen species (ROS) production. Fullerenol reduced the maximum plaque size in the cortex and hippocampus, decreased the small plaque density in the hippocampus and thalamus, and prevented an increase in glial fibrillary acidic protein (GFAP) positive cell density in the mutants. Both treatments improved cognitive and emotional behaviors and reduced Il1β and increased Sirt1 expression. In vitro, fullerenol was non-toxic across a range of concentrations and reduced Aβ42-induced ROS production in brain endothelial cells and astrocytes. Long-term administration of fullerene or fullerenol improved behavioral and molecular markers of AD in APP/PS1 mice, with fullerenol showing additional benefits in reducing amyloid burden. Full article

Small heat shock proteins (sHsps) are ubiquitous low-molecular-weight chaperones that prevent protein aggregation under cellular stress conditions. In the absence of stress, they assemble into large oligomers. In response to stress, such as elevated temperatures, they undergo conformational changes that expose hydrophobic surfaces, allowing them to interact with denatured proteins. At heat shock temperatures in bacteria, large sHsp oligomers disassemble into smaller oligomeric forms. Methanogens are a diverse group of microorganisms, ranging from thermophilic to psychrophilic and halophilic species. Accordingly, their sHsps exhibit markedly different temperature dependencies based on their optimal growth temperatures. In this study, we characterized sHsps from both hyperthermophilic and mesophilic methanogens to investigate the mechanisms underlying their temperature-dependent behavior. Using analytical ultracentrifugation, we observed the dissociation of sHsps from a mesophilic methanogen into dimers. The dissociation equilibrium of these oligomers was found to be dependent not only on temperature but also on protein concentration. Furthermore, by generating various mutants, we identified the specific amino acid residues responsible for the temperature dependency observed. The C-terminal region containing the IXI/V motif and the α-crystallin domain were found to be the primary determinants of oligomer stability and its temperature dependence. Full article

PDE11A is a little-studied phosphodiesterase sub-family that breaks down cAMP/cGMP, with the PDE11A4 isoform enriched in the memory-related hippocampal formation. Age-related increases in PDE11A expression occur in human and rodent hippocampus and cause age-related cognitive decline of social memories. Interestingly, age-related increases in PDE11A4 protein ectopically accumulate in spherical clusters that group together in the brain to form linear filamentous patterns termed “PDE11A4 ghost axons”. The biophysical/physiochemical mechanisms underlying this age-related clustering are not known. Here, we determine if age-related clustering of PDE11A4 reflects liquid–liquid phase separation (LLPS; biomolecular condensation), and if PDE11A inhibitors can reverse this LLPS. We show human and mouse PDE11A4 exhibit several LLPS-promoting sequence features, including intrinsically disordered regions, non-covalent pi–pi interactions, and prion-like domains that were particularly enriched in the N-terminal regulatory region. Further, multiple bioinformatic tools predict PDE11A4 undergoes LLPS. Consistent with these predictions, aging-like PDE11A4 clusters in HT22 hippocampal neuronal cells were membraneless spherical droplets that progressively fuse over time in a concentration-dependent manner. Deletion of the N-terminal intrinsically disordered region prevented PDE11A4 LLPS despite equal protein expression between WT and mutant constructs. 1,6-hexanediol, along with tadalafil and BC11-38 that inhibit PDE11A4, reversed PDE11A4 LLPS in HT22 hippocampal neuronal cells. Interestingly, PDE11A4 inhibitors reverse PDE11A4 LLPS independently of increasing cAMP/cGMP levels via catalytic inhibition. Importantly, orally dosed tadalafil reduced PDE11A4 ghost axons in old mouse ventral hippocampus by 50%. Thus, PDE11A4 exhibits the four defining criteria of LLPS, and PDE11A inhibitors reverse this age-related phenotype both in vitro and in vivo. Full article

Background: The FDA-cleared antimicrobial quaternary ammonium silane K21 is recognized for its antimicrobial properties. This study explored potential applications of the K21 molecule in human health protection, disease prevention, and treatment using the zebrafish model. Method: A multi-dimensional approach was utilized to assess the toxicity, tolerance, and optimal dosage of K21 through serial dilutions at various concentrations. Acute and chronic exposure studies were performed at different developmental stages (embryonic, larval, juvenile, and adult) to evaluate its efficacy and toxicity in wild-type (WT), Casper (transparent skin mutant), and transgenic zebrafish lines. Results: Significant weight gain was observed in the F1 generation following K21 treatment, a trend that continued into the F2 and F3 generations. The effects of K21 on lipopolysaccharide-induced inflammation were also examined in Casper NFkB:GFP transgenic lines. Treatment with K21 reduced inflammation, indicating anti-inflammatory properties. Improved hatching rates, accelerated larval development, an increased adult mass, and modest reductions in embryonic motility (less than 20%) suggested positive developmental influences. Single-cell RNA sequencing further validated the biological impacts of K21, revealing the potential activation of a novel pathway that accelerates zebrafish growth. Summary and Conclusions: These findings position K21 as a promising candidate for biomedical applications and aquaculture, warranting further investigation into its underlying molecular mechanisms. Our additional study on the effect of K21 on the artemia (brine shrimp) hatching process provide strong evidence of better hatching ratio of 90% for brine shrimp in the group with K21 drug treatment as compared to 70% in the group without the K21 drug at 24 h of treatment; the K21 drug helps the early hatching process, as observed the 90% hatching rate in 20 h K21 treatment group hatching while in the group without K21, only 40% of brine shrimps hatched. Full article

Bacterial killing is important for recovering from infection. Pasteurella multocida is a key bacterial pathogen causing swine respiratory disease and is associated with substantial mortality. Antimicrobial therapy remains an important therapeutic intervention for treating infected animals. Pradofloxacin (fluoroquinolone) is the most recently approved antimicrobial agent for treating pigs with swine respiratory disease. We compared in vitro killing of swine P. multocida strains by pradofloxacin in comparison to ceftiofur, enrofloxacin, florfenicol, marbofloxacin, tildipirosin, tilmicosin, and tulathromycin over a range of bacterial densities and four clinically relevant drug concentrations. Pradofloxacin killed 92–96.9% of cells across 10⁶–10⁸ cfu/mL densities at the mutant prevention drug concentration following 2–24 h of drug exposure, 96.9–98.9% of cells across 10⁶–10⁹ cfu/mL at the maximum serum drug concentration following 30 min of drug exposure, increasing to 99.9–100% kill following 12–24 h of drug exposure. At the maximum tissue drug concentration and against bacterial densities of 10⁶–10⁹ cfu/mL, pradofloxacin killed 91.3–99.8% of cells following 2 h of drug exposure, which increased to 99.9–100% kill following 12–24 h of drug exposure. Pradofloxacin was rapidly bactericidal across a range of bacterial densities and at clinically relevant drug concentrations. Pradofloxacin will be an important antibiotic for treating pigs with swine respiratory disease and where clinically indicated. Full article

Background/Objectives: Acinetobacter baumannii is a globally emerging pathogen with widespread antimicrobial resistance driven by multiple mechanisms, such as altered expression of efflux pumps like AdeABC, placing it as a priority for research. Driven by the lack of new treatments, alternative approaches are being explored to combat its infections, among which efficacy-enhancing adjuvants can be found. This study presents and characterizes MV6, a synthetic cyclic peptide that boosts aminoglycoside efficacy. Methods: MV6’s activity was assessed through antimicrobial susceptibility testing in combination with different antibiotic classes against A. baumannii strains characterized by PCR and RT-qPCR. PAβN served as a reference efflux pump inhibitor. Synergy was evaluated using checkerboard assays, and spontaneous mutants were generated with netilmicin with/without MV6 (100 mg/L). Whole-genome sequencing and variant calling analysis were then performed. Results: MV6 presented low antimicrobial activity in A. baumannii with MICs higher than 2048 mg/L. MV6 showed a better boosting effect for aminoglycosides, especially netilmicin, exceeding that of PAβN. Checkerboard assays confirmed a strong synergy between netilmicin and MV6, and a significant correlation was found between netilmicin MIC and adeB overexpression, which was mitigated by the presence of MV6. MV6 reduced, by 16-fold, the mutant prevention concentration of netilmicin. Mutations in a TetR-family regulator and ABC-binding proteins were found in both groups, suggesting a direct or indirect implication of these proteins in the resistance acquisition process. Conclusions: MV6 lacks intrinsic antimicrobial activity, minimizing selective pressure, yet enhances netilmicin’s effectiveness except for strain 210, which lacks the AdeABC efflux pump. Resistant mutants indicate specific aminoglycoside resistance mechanisms involving efflux pump mutations, suggesting synergistic interactions. Further research, including transcriptomic analysis, is essential to elucidate MV6’s role in enhancing netilmicin efficacy and its resistance mechanisms. Full article

Pradofloxacin is a dual targeting, bactericidal fluoroquinolone recently approved for treating bacteria causing swine respiratory disease. Currently, an abundance of in vitro data does not exist for pradofloxacin. We determined the minimum inhibitory concentration (MIC) and mutant prevention concentrations (MPC) of pradofloxacin compared to ceftiofur, enrofloxacin, florfenicol, marbofloxacin, tildipirosin, tilmicosin and tulathromycin against swine isolates of Actinobacillus pleuropneumoniae and Pasteurella multocida. Overall, pradofloxacin had the lowest MIC and MPC values as compared to the other agents tested. For example, pradofloxacin MIC values for 50%, 90% and 100% of A. pleuropneumoniae strains were ≤0.016 µg/mL, ≤0.016 µg/mL and ≤0.016 µg/mL and for P. multocida were ≤0.016 µg/mL, ≤0.016 µg/mL and 0.031 µg/mL, respectively. The MPC values for 50%, 90% and 100% of A. pleuropneumoniae strains were 0.031 µg/mL, 0.063 µg/mL and 0.125 µg/mL and for P. multocida were ≤0.016 µg/mL, 0.031 µg/mL and 0.0.063 µg/mL, respectively. By MPC testing, all strains were at or below the susceptibility breakpoint. Based on MPC testing, pradofloxacin appears to have a low likelihood for resistance selection. This study represents the most comprehensive in vitro comparison of the above noted drugs and the first report for pradofloxacin and tildipirosin. Full article

Background and Objectives: Rivaroxaban is a direct-acting anticoagulant used to prevent stroke in patients with atrial fibrillation. Rivaroxaban is a substrate for P-glycoprotein, which is encoded by the ABCB1 gene. Rivaroxaban is also metabolized by the CYP3A5 gene. Therefore, the current study is carried out to study the effects of polymorphisms in the ABCB1 and CYP3A5 genes, which may affect the plasma levels of rivaroxaban, with subsequent clinical outcomes (bleeding events) associated with the therapy. Materials and Methods: The study was conducted on 66 naive patients with atrial fibrillation treated with rivaroxaban. Blood samples of rivaroxaban were taken at 3 h and after 1 month following the administration of the drug to measure plasma levels. The blood level of rivaroxaban was measured with an HPLC-UV detector. Sanger sequencing was used to find polymorphisms in the targeted genes. Coagulation parameters were measured at 3 h and after 1 month of administration of rivaroxaban. Frequencies of bleeding events were recorded throughout the one-month course of drug therapy. Results: The heterozygous and homozygous mutant genotypes of ABCB1 (rs2032582, rs1045642, rs1128503, and rs4148738) and CYP3A5 (rs776746) showed lower plasma concentrations as compared to the wild-type genotype. ABCB1 (rs2032582, rs1045642, rs1128503, and rs4148738) and CYP3A5 (rs776746) gene polymorphisms had a statistically significant impact on the plasma concentration of rivaroxaban among the heterozygous and homozygous mutant genotypes compared to the wild-type genotype. The heterozygous variant of ABCB1 and homozygous variant of CYP3A5 suffered more events of bleeding. Conclusions: It was concluded that ABCB1 (rs2032582, rs1045642, rs1128503, and rs4148738) and CYP3A5 (rs776746) gene polymorphisms had a significant impact on the plasma levels of rivaroxaban in patients treated for atrial fibrillation on day three as well as after one month of the therapy. The lowest plasma levels were observed in patients with a homozygous variant of ABCB1 (rs2032582, rs1045642, or rs4148738) along with the CYP3A5*1/*3 allele. The heterozygous variant of ABCB1 SNPs and homozygous variant of CYP3A5 SNPs suffered more events of bleeding. Full article

DNA topoisomerase II (TOP2) is an enzyme that resolves DNA topological problems arising in various nuclear processes, such as transcription. Aclarubicin, a member of the anthracyclines, is known to prevent the association of TOP2 with DNA, inhibiting the early step of TOP2 catalytic reactions. During our research on the subnuclear distribution of human TOP2B, we found that aclarubicin affects the mobility of TOP2B in the nucleus. FRAP analysis demonstrated that aclarubicin decreased the nuclear mobility of EGFP-tagged TOP2B in a concentration-dependent manner. Aclarubicin exerted its inhibitory effects independently of TOP2B enzymatic activities: TOP2B mutants defective for either ATPase or topoisomerase activity also exhibited reduced nuclear mobility in the presence of aclarubicin. Immunofluorescence analysis showed that aclarubicin antagonized the induction of DNA damage by etoposide. Although the prevention of the TOP2-DNA association is generally considered a primary action of aclarubicin in TOP2 inhibition, our findings highlight a previously unanticipated effect of aclarubicin on TOP2B in the cellular environment. Full article

Despite the urgent need for new antibiotics, very few innovative antibiotics have recently entered clinics or clinical trials. To provide a constant supply of new drug candidates optimized in terms of their potential to select for resistance in natural settings, in vitro resistance-predicting studies need to be improved and scaled up. In this review, the following in vitro parameters are presented: frequency of spontaneous mutant selection (FSMS), mutant prevention concentration (MPC), dominant mutant prevention concentration (MPC-D), inferior-mutant prevention concentration (MPC-F), and minimal selective concentration (MSC). The utility of various adaptive laboratory evolution (ALE) approaches (serial transfer, continuous culture, and evolution in spatiotemporal microenvironments) for comparing hits in terms of the level and time required for multistep resistance to emerge is discussed. We also consider how the hit-to-lead stage can benefit from high-throughput ALE setups based on robotic workstations, do-it-yourself (DIY) continuous cultivation systems, microbial evolution and growth arena (MEGA) plates, soft agar gradient evolution (SAGE) plates, microfluidic chips, or microdroplet technology. Finally, approaches for evaluating the fitness of in vitro-generated resistant mutants are presented. This review aims to draw attention to newly emerged ideas on how to improve the in vitro forecasting of the potential of compounds to select for resistance in natural settings. Full article

Clostridioides difficile is a widely distributed anaerobic pathogen. C. difficile infection is a serious problem in healthcare. Its biofilms have been found to exhibit biocorrosivity, albeit very little, but sufficient for it to correlate with biofilm growth/health. This work demonstrated the use of a disposable electrochemical biofilm test kit using two solid-state electrodes (a 304 stainless steel working electrode, and a graphite counter electrode, which also served as the reference electrode) in a 10 mL serum vial. It was found that the C. difficile 630∆erm Adp-4 mutant had a minimum inhibitory concentration (MIC) for vancomycin twice that of the 630∆erm wild type strain in biofilm prevention (2 ppm vs. 1 ppm by mass) on 304 stainless steel. Glutaraldehyde, a commonly used hospital disinfectant, was found ineffective at 2% (w/w) for the prevention of C. difficile 630∆erm wild type biofilm formation, while tetrakis(hydroxymethyl)phosphonium sulfate (THPS) disinfectant was very effective at 100 ppm for both biofilm prevention and biofilm killing. These antimicrobial efficacy data were consistent with sessile cell count and biofilm imaging results. Furthermore, the test kit provided additional transient biocide treatment information. It showed that vancomycin killed C. difficile 630∆erm wild type biofilms in 2 d, while THPS only required minutes. Full article

Fosfomycin is a bactericidal drug recommended as an alternative treatment for canine bacterial cystitis, particularly in cases involving multidrug-resistant (MDR) infections when no other options are available. In this study, minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) of fosfomycin were determined against 79 clinical E. coli isolates using the agar dilution method. The susceptibility rate of E. coli to fosfomycin was 86.06%, with MIC₅₀ and MIC₉₀ values of 4 mg/L and 96 mg/L, respectively. MPC₅₀ and MPC₉₀ values were 64 mg/L and 192 mg/L. Using pharmacokinetic (PK) data from dogs given a single 80 mg/kg oral dose of fosfomycin, the area under the curve per MIC₅₀ (AUC_0–24/MIC₅₀) was 85.79 with time above MIC₅₀ (T > MIC₅₀) exceeding 50%. In urine, the AUC_0–24/MIC₅₀ was 10,694.78, and the AUC_0–24/MPC₉₀ was 222.81, with T > MPC₉₀ extending beyond 24 h. Therefore, fosfomycin exhibited significant antibacterial activity against canine uropathogenic E. coli, including MDR strains, at concentrations below the susceptible MIC breakpoint. However, the high MPC values, especially the MPC₉₀, indicate the critical importance of performing susceptibility testing for fosfomycin and maintaining ongoing resistance monitoring. Full article

Pradofloxacin is the newest of the veterinary fluoroquinolones to be approved for use in animals—initially companion animals and most recently food animals. It has a broad spectrum of in vitro activity, working actively against Gram-positive/negative, atypical and some anaerobic microorganisms. It simultaneously targets DNA gyrase (topoisomerase type II) and topoisomerase type IV, suggesting a lower propensity to select for antimicrobial resistance. The purpose of this study was to determine the rate and extent of bacterial killing by pradofloxacin against bovine strains of Mannheimia haemolytica and Pasteurella multocida, in comparison with several other agents (ceftiofur, enrofloxacin, florfenicol, marbofloxacin, tildipirosin, tilmicosin and tulathromycin) using four clinically relevant drug concentrations: minimum inhibitory and mutant prevention drug concentration, maximum serum and maximum tissue drug concentrations. At the maximum serum and tissue drug concentrations, pradofloxacin killed 99.99% of M. haemolytica cells following 5 min of drug exposure (versus growth to 76% kill rate for the other agents) and 94.1–98.6% of P. multocida following 60–120 min of drug exposure (versus growth to 98.6% kill rate for the other agents). Statistically significant differences in kill rates were seen between the various drugs tested depending on drug concentration and time of sampling after drug exposure. Full article

Pradofloxacin—a dual-targeting fluoroquinolone—is the most recent approved for use in food animals. Minimum inhibitory and mutant prevention concentration values were determined for pradofloxacin, ceftiofur, enrofloxacin, florfenicol, marbofloxacin, tildipirosin, tilmicosin, and tulathromycin. For M. haemolytica strains, MIC_50/90/100 values were ≤0.016/≤0.016/≤0.016 and MPC_50/90/100 values were 0.031/0.063/0.063; for P. multocida strains, the MIC_50/90/100 values ≤0.016/≤0.016/0.031 and MPC_50/90/100 ≤ 0.016/0.031/0.063 for pradofloxacin. The pradofloxacin C_max/MIC₉₀ and C_max/MPC₉₀ values for M. haemolytica and P. multocida strains, respectively, were 212.5 and 53.9 and 212.5 and 109.7. Similarly, AUC₂₄/MIC₉₀ and AUC₂₄/MPC₉₀ for M. haemolytica were 825 and 209.5, and for P. multocida, they were 825 and 425.8. Pradofloxacin would exceed the mutant selection window for >12–16 h. Pradofloxacin appears to have a low likelihood for resistance selection against key bovine respiratory disease bacterial pathogens based on low MIC and MPC values. Full article

Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder impairing cortisol synthesis due to reduced enzymatic activity. This leads to persistent adrenocortical overstimulation and the accumulation of precursors before the blocked enzymatic step. The predominant form of CAH arises from mutations in CYP21A2, causing 21-hydroxylase deficiency (21-OHD). Despite emerging treatment options for CAH, it is not always possible to physiologically replace cortisol levels and counteract hyperandrogenism. Moreover, there is a notable absence of an effective in vivo model for pre-clinical testing. In this work, we developed an animal model for CAH with the clinically relevant point mutation p.R484Q in the previously humanized CYP21A2 mouse strain. Mutant mice showed hyperplastic adrenals and exhibited reduced levels of corticosterone and 11-deoxycorticosterone and an increase in progesterone. Female mutants presented with higher aldosterone concentrations, but blood pressure remained similar between wildtype and mutant mice in both sexes. Male mutant mice have normal fertility with a typical testicular appearance, whereas female mutants are infertile, exhibit an abnormal ovarian structure, and remain in a consistent diestrus phase. Conclusively, we show that the animal model has the potential to contribute to testing new treatment options and to prevent comorbidities that result from hormone-related derangements and treatment-related side effects in CAH patients. Full article