Search Results (37)

This study aimed to characterize and evaluate the fibrinolytic, thrombolytic, hematological, and toxicological aspects of a serine protease (AsKSP) from Aspergillus tamarii Kita UCP 1279. The enzyme was purified using a two-phase aqueous system and assessed for optimal pH (7.0) and temperature (50 °C), stability, and effects of metal ions, inhibitors, and surfactants. AsKSP exhibited stability for up to 120 min at 50 °C and 36 h at pH 7.0. Enzymatic activity was enhanced by Na⁺ and Zn²⁺ and non-ionic surfactants (Tween-80) but inhibited by Cu²⁺, Fe³⁺, Triton X-100, and SDS, reducing activity by up to 62.35%. The highest amidolytic activity was observed for the substrate N-succinyl-Gly–Gly–Phe-p-nitroanilide. SDS-PAGE analysis indicated an approximate molecular mass of 90 kDa. The enzyme showed fibrinolytic activity, degrading 38.81% of fibrin clots in vitro after 90 min, without affecting fibrinogen. Cytotoxicity assays indicated no toxicity (cell viability > 80%). Coagulation assays showed slight prolongation of prothrombin time (PT) and activated partial thromboplastin time (aPTT), with no effect on thrombin time. No red blood cell lysis was observed, and albumin increased enzymatic activity by 31.70%. These findings demonstrate that Aspergillus tamarii Kita UCP 1279 produces a fibrinolytic protease with potential for thrombus treatment, providing a promising foundation for drug development. Full article

Antimicrobial peptides (AMPs) have emerged as promising candidates in the fight against multidrug-resistant pathogens due to their broad-spectrum antimicrobial activity and low potential for resistance development. However, their clinical application is limited by poor stability and susceptibility to enzymatic degradation. This study aims to address these limitations by synthesizing a series of cyclic hexapeptides using the hydrazide method and evaluating their antimicrobial activity and stability. The hydrazide method facilitated the synthesis of 11 cyclic peptides through a reaction between C-terminal hydrazides and cysteine-containing peptides. Antimicrobial assays showed that Cy-f2 and Cy-f4 exhibited potent inhibitory effects against different kinds of bacteria, including E. coli, Staphylococcus aureus, and S. aureus. Hemolysis assays revealed minimal red blood cell lysis at effective antimicrobial concentrations, indicating good biocompatibility. Stability tests demonstrated improved stability of the cyclic peptides compared to linear counterparts in SGF and 80 °C. In conclusion, the cyclic hexapeptides synthesized in this study demonstrate excellent antimicrobial activity, enhanced stability, and low toxicity, suggesting their potential as new candidates for treating drug-resistant bacterial infections. Full article

A powerful strategy to accelerate bioprocess development is to complement parallel bioreactor systems with an automated approach, often achieved using liquid handling stations. The benefit of such high-throughput experiments is determined by the employed monitoring procedures. To gain a molecular understanding of the microbial production strains in miniaturized parallel single-use bioreactors, we extended the at-line monitoring procedures to transcriptome analysis in a parallel approach using RNA-Seq. To perform automated RNA-Seq experiments, we developed a sample preparation workflow consisting of at-line cell disruption by enzymatic cell lysis, total RNA extraction, nucleic acid concentration normalization, and Nanopore cDNA Library preparation. The pH-controlled aerobic batch growth of Saccharomyces cerevisiae was studied with six different carbon sources (glucose, pyruvate, fructose, galactose, sucrose, and mannose) on a 11 mL scale using 24 parallel stirred tank bioreactors integrated into a liquid handling station while performing at-line sample preparation for RNA-Seq on the same deck. With four biological replicates per condition, 24 cDNA libraries were prepared over 11.5 h. Off-line Nanopore sequencing yielded 20.97 M classified reads with a Q-score > 9. Differential gene expression analysis revealed significant differences in transcriptomic profiles when comparing growth with glucose (exponential growth) to growth with pyruvate (stress conditions), allowing identification of 674 downregulated and 709 upregulated genes. Insignificant changes in gene expression patterns were measured when comparing growth with glucose and fructose, yielding only 64 differentially expressed genes. The expected differences in cellular responses identified in this study show a promising approach for transcriptomic profiling of bioreactor cultures, providing valuable insights on a molecular level at-line in a high-throughput fashion. Full article

Fish scales, an abundant yet underutilized by-product of fish processing, are rich in proteins and thus hold significant potential for value-added applications. This study aimed to develop a sustainable method for converting grass carp (Ctenopharyngodon idella) scales into bioactive protein hydrolysates and evaluate their potential as natural antimicrobial agents. Fish scale protein hydrolysates (FSPHs) were prepared through citric acid extraction followed by pepsin enzymatic hydrolysis. Antimicrobial activity and stability were systematically assessed against Escherichia coli and Staphylococcus aureus, alongside mechanistic investigations. Results demonstrated the potent inhibitory effects of FSPHs against both pathogens, with minimum inhibitory concentrations (MICs) of 4.2 μg∙mL⁻¹ and minimum bactericidal concentrations (MBCs) of 67.5 μg∙mL⁻¹ for E. coli and 33.7 μg∙mL⁻¹ for S. aureus. FSPHs exhibited exceptional thermal stability (<100 °C) and retained functionality over 10 freeze–thaw cycles. Mechanistic studies have revealed enhanced bacterial membrane permeability upon FSPH treatment, with microscopic evidence of cell aggregation and lysis after 16 h of exposure. This work validates grass carp scales as a viable source of antimicrobial peptides through optimized extraction protocols, offering a circular economy solution for fishery waste. The findings provide actionable insights for policymakers to promote eco-friendly alternatives to synthetic antibiotics while advancing methodologies for bioactive peptide research. Full article

Background: Membranoproliferative glomerulonephritis (MPGN) is an umbrella term for chronic disorders affecting the glomeruli. MPGN is often accompanied by the presence of autoantibodies against complement components. However, the actual pathogenic effects of such autoantibodies, if any, are rarely studied. In this work, we investigated the role of anti-complement autoantibodies in an IC-MPGN patient. Methods: The presence of autoantibodies, their binding site, isotype, and titer were analyzed in ELISA. Antibody–antigen complexes were detected in the patient’s serum using Western blot. Autoantibodies were studied in functional assays to analyze their effects on C3 convertase, complement deposition, cofactor activity, C3b binding, and hemolysis. Results: We identified autoantibodies against factor B (FB) and factor H (FH) in the patient’s serum. Both FB-, and FH-autoantibodies were of IgG2, IgG3, IgG4, and IgGκ, IgGλ isotypes. FB-autoantibodies bound to the Ba and the enzymatically active Bb part of FB. FH-autoantibodies bound to the N- and C-termini of FH and cross-reacted with FHL-1 and FHR-1 proteins. In vivo formed complexes of the autoantibodies with both FB and FH were detected in the IgG fraction isolated from the serum. The autoantibodies did not influence solid-phase C3 convertase assembly and its FH-mediated decay. The free autoantibodies had no effect on complement deposition and on FH cofactor activity but slightly reduced C3b binding to FH. The IgG fraction of the patient dose-dependently inhibited complement-mediated rabbit red blood cell lysis, and the free autoantibodies decreased the solid phase C3 convertase activity. Conclusions: This case highlights that FB- and FH-autoantibodies are not necessarily pathogenic in IC-MPGN. Full article

Background: This study presents a comprehensive analysis of cholesteatoma of the middle ear, focusing on its clinical presentation, diagnostic imaging, and treatment outcomes. Cholesteatomas are defined by the keratinized squamous epithelium within the middle ear, leading to significant bone erosion, often affecting the ossicular chain and surrounding structures. Methods: The study explores various mechanisms involved in cholesteatoma progression, including enzymatic lysis, inflammatory responses, and neurotrophic disturbances. The study conducted a retrospective clinical and statistical review of 580 patients over a 20-year period (2003–2023), highlighting the role of advanced imaging, including computed tomography (CT) and diffusion-weighted magnetic resonance imaging (DWI), in preoperative planning and postoperative follow-up. Results: Findings revealed that early detection and intervention are crucial in preventing severe complications such as intracranial infection and hearing loss. Surgical treatment primarily involved tympanoplasty and mastoidectomy, with a recurrence rate of 1.55% within two years. The study underscores the importance of integrating imaging advancements into clinical decision-making to enhance patient outcomes and suggests further investigation into molecular mechanisms underlying cholesteatoma progression and recurrence. Histopathological and microbiological analysis was performed to identify pathological patterns and microbial agents. Conclusions: The study highlights the importance of early diagnosis and intervention to prevent complications such as intracranial infections and permanent hearing loss, while also emphasizing the role of advanced imaging techniques in the management and long-term monitoring of cholesteatoma patients. Full article

Acinetobacter baumannii (A. baumannii) has emerged as a prominent multidrug-resistant (MDR) pathogen, significantly complicating treatment strategies due to its formidable resistance mechanisms, particularly against carbapenems. Reduced membrane permeability, active antibiotic efflux, and enzymatic hydrolysis via different β-lactamases are the main resistance mechanisms displayed by A. baumannii, and they are all effective against successful treatment approaches. This means that alternate treatment approaches, such as combination therapy that incorporates beta-lactams, β-lactamase inhibitors, and novel antibiotics like cefiderocol, must be investigated immediately. Cefiderocol, a new catechol-substituted siderophore cephalosporin, improves antibacterial activity by allowing for better bacterial membrane penetration. Due to its unique structure, cefiderocol can more efficiently target and destroy resistant bacteria by using iron transport systems. Through its inhibition of peptidoglycan formation through binding to penicillin-binding proteins (PBPs), cefiderocol avoids conventional resistance pathways and induces bacterial cell lysis. The possibility of resistance development due to β-lactamase synthesis and mutations in PBPs, however, emphasizes the need for continued investigation into cefiderocol’s efficacy in combination treatment regimes. Cefiderocol’s siderophore mimic mechanism is especially important in iron-limited conditions because it can use ferric-siderophore transporters to enter cells. Additionally, its passive diffusion through bacterial porins increases its intracellular concentrations, making it a good option for treating carbapenem-resistant A. baumannii, especially in cases of severe infections and ventilator-associated diseases (IVACs). Cefiderocol may reduce MDR infection morbidity and mortality when combined with customized antimicrobial treatments, but further investigation is needed to improve patient outcomes and address A. baumannii resistance issues. Full article

The enzymatic dissociation of human solid tissues is a critical process for disaggregating extracellular matrix and the isolation of individual cells for various applications, including the immortalizing primary cells, creating novel cell lines, and performing flow cytometry and its specialized type, FACS, as well as conducting scRNA-seq studies. Tissue dissociation procedures should yield intact, highly viable single cells that preserve morphology and cell surface markers. However, endocrine tissues, such as adrenal gland tumors, thyroid carcinomas, and pituitary neuroendocrine tumors, present unique challenges due to their complex tissue organization and morphological features. Our study conducted a morphological examination of these tissues, highlighting the intricate structures and secondary degenerative changes that complicate the dissociation process. We investigated the effects of various dissociation parameters, including the types of enzymes, incubation duration, and post-dissociation purification procedures, such as debris removal and nontarget blood cell lysis, on the viability of cells derived from different tumor types. The findings emphasize the importance of optimizing tissue digestion protocols to preserve cell viability and integrity, ensuring reliable outcomes for downstream analyses. Full article

The ammonia/ammonium (NH₃/NH₄⁺, AM) concentration in human erythrocytes (RBCs) is significantly higher than in plasma. Two main possible mechanisms for AM transport, including simple and facilitated diffusion, are described; however, the driving force for AM transport is not yet fully characterized. Since the erythroid ammonium channel RhAG forms a structural unit with anion exchanger 1 (eAE1) within the ankyrin core complex, we hypothesized the involvement of eAE1 in AM transport. To evaluate the functional interaction between eAE1 and RhAG, we used a unique feature of RBCs to swell and lyse in isotonic NH₄⁺ buffer. The kinetics of cell swelling and lysis were analyzed by flow cytometry and an original laser diffraction method, adapted for accurate volume sensing. The eAE1 role was revealed according to (i) the changes in cell swelling and lysis kinetics, and (ii) changes in intracellular pH, triggered by eAE1 inhibition or the modulation of eAE1 main ligand concentrations (Cl⁻ and HCO₃⁻). Additionally, the AM import kinetics was analyzed enzymatically and colorimetrically. In NH₄⁺ buffer, RBCs concentration-dependently swelled and lysed when [NH₄⁺] exceeded 100 mM. Cell swelling and hemolysis were tightly regulated by chloride concentration. The complete substitution of chloride with glutamate prevented NH₄⁺-induced cell swelling and hemolysis, and the restoration of [Cl⁻] dose-dependently amplified the rates of RBC swelling and lysis and the percentage of hemolyzed cells. Similarly, eAE1 inhibition impeded cell swelling and completely prevented hemolysis. Accordingly, eAE1 inhibition, or a lack of chloride anions in the buffer, significantly decreased NH₄⁺ import. Our data indicate that the eAE1-mediated chloride gradient is required for AM transport. Taken together, our data reveal a new player in AM transport in RBCs. Full article

Next-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. Although all microbiome sequencing methods depend on recovering the DNA from a sample as a first critical step, lysis methods can be a major determinant of microbiome profile bias. Gentle enzyme-based DNA preparation methods preserve DNA quality but can bias the results by failing to open difficult-to-lyse bacteria. Mechanical methods like bead beating can also bias DNA recovery because the mechanical energy required to break tougher cell walls may shear the DNA of the more easily lysed microbes, and shearing can vary depending on the time and intensity of beating, influencing reproducibility. We introduce a non-mechanical, non-enzymatic, novel rapid microbial DNA extraction procedure suitable for 16S rRNA gene-based microbiome profiling applications that eliminates bead beating. The simultaneous application of alkaline, heat, and detergent (‘Rapid’ protocol) to milligram quantity samples provided consistent representation across the population of difficult and easily lysed bacteria equal to or better than existing protocols, producing sufficient high-quality DNA for full-length 16S rRNA gene PCR. The novel ‘Rapid’ method was evaluated using mock bacterial communities containing both difficult and easily lysed bacteria. Human fecal sample testing compared the novel Rapid method with a standard Human Microbiome Project (HMP) protocol for samples from lung cancer patients and controls. DNA recovered from both methods was analyzed using 16S rRNA gene sequencing of the V1V3 and V4 regions on the Illumina platform and the V1V9 region on the PacBio platform. Our findings indicate that the ‘Rapid’ protocol consistently yielded higher levels of Firmicutes species, which reflected the profile of the bacterial community structure more accurately, which was confirmed by mock community evaluation. The novel ‘Rapid’ DNA lysis protocol reduces population bias common to bead beating and enzymatic lysis methods, presenting opportunities for improved microbial community profiling, combined with the reduction in sample input to 10 milligrams or less, and it enables rapid transfer and simultaneous lysis of 96 samples in a standard plate format. This results in a 20-fold reduction in sample handling time and an overall 2-fold time advantage when compared to widely used commercial methods. We conclude that the novel ‘Rapid’ DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing. Full article

Nucleic acid amplification testing facilitates the detection of disease through specific genomic sequences and is attractive for point-of-need testing (PONT); in particular, the early detection of microorganisms can alert early response systems to protect the public and ecosystems from widespread outbreaks of biological threats, including infectious diseases. Prior to nucleic acid amplification and detection, extensive sample preparation techniques are required to free nucleic acids and extract them from the sample matrix. Sample preparation is critical to maximize the sensitivity and reliability of testing. As the enzymatic amplification reactions can be sensitive to inhibitors from the sample, as well as from chemicals used for lysis and extraction, avoiding inhibition is a significant challenge, particularly when minimising liquid handling steps is also desirable for the translation of the assay to a portable format for PONT. The reagents used in sample preparation for nucleic acid testing, covering lysis and NA extraction (binding, washing, and elution), are reviewed with a focus on their suitability for use in PONT. Full article

Torularhodin is a fungus-derived carotenoid, and the lack of downstream processing of torularhodin is still a challenge for its large-scale production. To support the industrial production of torularhodin, this work initially evaluated the efficiency of carotenoid release from Rhodotorula mucilaginosa using thermal acid treatment, saponification and ultrasound-assisted enzymatic lysis. Based on the polarity, torularhodin was then purified using methanol/acetone/hexane (2/2/1, v/v/v) solution eluting from a silica cartridge. Thermal acid treatment was considered the most appropriate method for total carotenoid release and torularhodin recovery. The highest carotenoid content was 121.3 ± 7.0 μg/g dry cell weight and 63.0 ± 6.1% of torularhodin (50.5 ± 3.0 μg/g dry cell weight in total) was recovered after purification. To fast quantify the content of torularhodin extracted from yeast, the absorption coefficient ($E_{1\mathrm{c}\mathrm{m}}^{1\%}$ = 3342) of torularhodin dissolved in chloroform was assayed. With the developed strategy for torularhodin recovery, purification and quantification, the potential of this yeast to produce torularhodin using xylose and glycerol was further evaluated. It was found that carbon sources may influence the proportion of carotenoids in this yeast, but torularhodin was still the dominant pigment. The results obtained in this study identified the feasibility of sustainable production of torularhodin from Rhodotorula mucilaginosa with high efficiency and purity. Full article

The putative phospholipase Atg15 is required for the intravacuolar lysis of autophagic bodies and MVB vesicles. Intracellular membrane lysis is a highly sophisticated mechanism that is not fully understood. The amino-terminal transmembrane domain of Atg15 contains the sorting signal for entry into the MVB pathway. By replacing this domain, we generated chimeras located in the cytosol, the vacuole membrane, and the lumen. The variants at the vacuole membrane and in the lumen were highly active. Together with the absence of Atg15 from the phagophore and autophagic bodies, this suggests that, within the vacuole, Atg15 can lyse vesicles where it is not embedded. In-depth topological analyses showed that Atg15 is a single membrane-spanning protein with the amino-terminus in the cytosol and the rest, including the active site motif, in the ER lumen. Remarkably, only membrane-embedded Atg15 variants affected growth when overexpressed. The growth defects depended on its active site serine 332, showing that it was linked to the enzymatic activity of Atg15. Interestingly, the growth defects were independent of vacuolar proteinase A and vacuolar acidification. Full article

The spreading of microbial pathogens with more and more resistance to traditional low-molecular antibiotic agents demands new approaches to antibacterial therapy. The employment of bacteriophage enzymes capable of breaking bacterial cell walls has attracted much interest within this context. The specific features of the morphology of Gram-negative bacteria prevent the effective direct usage of lytic enzymes and require assistance from additional helpers to facilitate cell lysis. The current work is devoted to the study of boosting the lysis of Escherichia coli (E. coli) JM 109 and MH 1 strains induced by Lys394 bacteriophage endolysin by means of rod-like (56 × 13 nm) magnetic nanoparticles (MNPs) activated by a non-heating low-frequency magnetic field (LF MF) with a frequency of 50 Hz and a flux density of 68.5 mT in a pulse–pause mode (1 s on and 0.3 s off). According to theoretical assumptions, the mechanism of MNP assistance is presumably based upon the disordering of the outer membrane that facilitates enzyme permeation into peptidoglycans to its substrate. It is found that the effect of the LF MF reaches an almost a twofold acceleration of the enzyme reaction, resulting in almost 80 and 70%, respectively, of lysed E. coli JM 109 and MH 1 cells in 21 min. An increase in the membrane permeability was proven by two independent experiments employing β-lactamase periplasmic enzyme leakage and Nile Red (NR) hydrophobic dye fluorescence. It is shown that the outer membrane disordering of E. coli caused by exposure to LF MF nanoparticle movement leads to almost complete (more than 80%) β-lactamase release out of the cells’ periplasm to the buffer suspension. Experiments with NR (displaying fluorescence in a non-polar medium only) reveal a drastic reduction in NR fluorescence intensity, reaching a change of an order of magnitude when exposed to LF MF. The data obtained provide evidence of changes in the bacterial cell wall structure. The result shown open up the prospects of non-heating LF MF application in enhancing enzyme activity against Gram-negative pathogens. Full article

American foulbrood is caused by the spore-forming Paenibacillus larvae. Although the disease effects honey bee larvae, it threatens the entire colony. Clinical signs of the disease are seen at a very late stage of the disease and bee colonies are often beyond saving. Therefore, through active monitoring based on screening, an infection can be detected early and bee colonies can be protected with hygiene measures. As a result, the pressure to spread in an area remains low. The cultural and molecular biological detection of P. larvae is usually preceded by spore germination before detection. In this study, we compared the results of two methods, the culture detection and RT-PCR detection of DNA directly isolated from spores. Samples of honey and cells with honey surrounding the brood were used in a five-year voluntary monitoring program in a western part of Lower Austria. DNA-extraction from spores to speed up detection involved one chemical and two enzymatic steps before mechanical bashing-beat separation and additional lysis. The results are comparable to culture-based methods, but with a large time advantage. Within the voluntary monitoring program, the proportion of bee colonies without the detection of P. larvae was high (2018: 91.9%, 2019: 72.09%, 2020: 74.6%, 2021: 81.35%, 2022: 84.5%), and in most P. larvae-positive bee colonies, only a very low spore content was detected. Nevertheless, two bee colonies in one apiary with clinical signs of disease had to be eradicated. Full article