Rapid Lysis Research Articles

Using Acanthamoeba spp. as a cell model to evaluate Leishmania infections.

Leishmaniasis represents a severe global health problem. In the last decades, there have been significant challenges in controlling this disease due to the unavailability of licensed vaccines, the high toxicity of the available drugs, and an unrestrained surge of drug-resistant parasites, and human immunodeficiency virus (HIV)-Leishmania co-infections. Leishmania spp. preferentially invade macrophage lineage cells of vertebrates for replication after subverting cellular functions of humans and other mammals. These early events in host-parasite interactions are likely to influence the future course of the disease. Thus, there is a continuing need to discover a simple cellular model that reproduces the in vivo pathogenesis. Acanthamoeba spp. are non-mammalian phagocytic amoeba with remarkable similarity to the cellular and functional aspects of macrophages. We aimed to assess whether the similarity reported between macrophages and Acanthamoeba spp. is sufficient to reproduce the infectivity of Leishmania spp. Herein, we analyzed co-cultures of Acanthamoeba castellanii or Acanthamoeba polyphaga with Leishmania infantum, Leishmania amazonensis, Leishmania major, and Leishmania braziliensis. Light and fluorescence microscopy revealed that the flagellated promastigotes attach to the A. castellanii and/or A. polyphaga in a bipolar and or random manner, which initiates their uptake via pseudopods. Once inside the cells, the promastigotes undergo significant changes, which result in the obligatory amastigote-like intracellular form. There was a productive infection with a continuous increase in intracellular parasites. However, we frequently observed intracellular amastigotes in vacuoles, phagolysosomes, and the cytosol of Acanthamoeba spp. Our findings corroborate that Leishmania spp. infects Acanthamoeba spp. and replicates in them but does not cause their rapid degeneration or lysis. Overall, the evidence presented here confirms that Acanthamoeba spp. have all prerequisites and can help elucidate how Leishmania spp. infect mammalian cells. Future work exposing the mechanisms of these interactions should yield novel insights into how these pathogens exploit amoebae.

Development and application of a bacteriophage cocktail for Shigella flexneri biofilm inhibition on the stainless steel surface

Food contamination and biofilm formation by Shigella in food processing facilities are major causes of acute gastrointestinal infection and mortality in humans. Bacteriophages (phages) are promising alternatives to antibiotics in controlling plankton and biofilms in food matrices. This study isolated two novel phages, S2_01 and S2_02, with lytic activity against various Shigella spp. From sewage samples. Transmission electron microscopy revealed that phages S2_01 and S2_02 belonged to the Caudovirales order. On characterizing their lytic ability, phage S2_01 initially exhibited relatively weak antibacterial activity, while phage S2_02 initially displayed rapid antibacterial activity after phage application. A combination of these phages in a 1:9 ratio was selected, as it has been suggested to elicit the most rapid and sustained lysis ability for up to 24 h. It demonstrated lytic activity against various foodborne pathogens, including six Shigella spp. The phage cocktail exhibited biofilm inhibition and disruption abilities of approximately 79.29% and 42.55%, respectively, after 24 h in a 96-well microplate. In addition, inhibition (up to 23.42%) and disruption (up to 19.89%) abilities were also observed on stainless steel surfaces, and plankton growth was also significantly suppressed. Therefore, the phage cocktail formulated in this study displays great potential as a biological control agent in improving food safety against biofilms and plankton.

Isolation and Characterization of a Novel Phage against Vibrio alginolyticus Belonging to a New Genus.

Vibrio alginolyticus causes substantial economic losses in the aquaculture industry. With the rise of multidrug-resistant Vibrio strains, phages present a promising solution. Here, a novel lytic Vibrio phage, vB_ValC_RH2G (RH2G), that efficiently infects the pathogenic strain V. alginolyticus ATCC 17749T, was isolated from mixed wastewater from an aquatic market in Xiamen, China. Transmission electron microscopy revealed that RH2G has the morphology of Siphoviruses, featuring an icosahedral head (73 ± 2 nm diameter) and long noncontractile tail (142 ± 4 nm). A one-step growth experiment showed that RH2G had a short latent period (10 min) and a burst size of 48 phage particles per infected cell. Additionally, RH2G was highly species-specific and was relatively stable at 4-55 °C and pH 4-10. A genomic analysis showed that RH2G has a 116,749 bp double-stranded DNA genome with 43.76% GC content. The intergenomic similarity between the genome sequence of RH2G and other phages recorded in the GenBank database was below 38.8%, suggesting that RH2G represents a new genus. RH2G did not exhibit any virulence or resistance genes. Its rapid lysis capacity, lytic activity, environmental resilience, and genetic safety suggested that RH2G may be a safe candidate for phage therapy in combatting vibriosis in aquaculture settings.

Detection and differentiation of low virulence and virulent Orthoavulavirus javaense using a molecular beacon with RT-LAMP

Newcastle disease (ND), an economically important disease in poultry, is caused by virulent strains of the genetically diverse Orthoavulavirus javaense (OAVJ). Laboratories rely on quantitative real-time reverse transcription PCR (qRT-PCR) to detect OAVJ and differentiate between OAVJ pathotypes. This study demonstrates that a fusion cleavage site based molecular beacon with reverse transcription loop mediated isothermal amplification (MB-RT-LAMP) assay can detect and differentiate OAVJ pathotypes in a single assay. Data show that the assay can rapidly identify diverse OAVJ genotypes with sensitivity only one log-fold lower than the current fusion qRT-PCR assay (104 copies), exhibits a high degree of specificity for OAVJ, and the molecular beacon can differentiate mesogenic/velogenic sequences from lentogenic sequences. Further, data show that a two-minute rapid lysis protocol preceding MB-RT-LAMP can detect and differentiate OAVJ RNA from both spiked samples and oropharyngeal swabs without the need for RNA isolation. As the MB-RT-LAMP assay can rapidly detect and discriminate between lentogenic and mesogenic/velogenic sequences of OAVJ within one assay, without the need for RNA isolation, and is adaptable to existing veterinary diagnostic laboratory workflow without additional equipment, this assay could be a rapid primary screening tool before qRT-PCR based validation in resource limited settings.

Competitive advantages of T-even phage lysis inhibition in response to secondary infection.

T-even bacteriophages are known to employ lysis inhibition (LIN), where the lysis of an infected host is delayed in response to secondary adsorptions. Upon the eventual burst of the host, significantly more phage progenies are released. Here, we analysed the competitive advantage of LIN using a mathematical model. In batch culture, LIN provides a bigger phage yield at the end of the growth where all the hosts are infected due to an exceeding number of phage particles and, in addition, gives a competitive advantage against LIN mutants with rapid lysis by letting them adsorb to already infected hosts in the LIN state. By simulating plaque formation in a spatially structured environment, we show that, while LIN phages will produce a smaller zone of clearance, the area over which the phages spread is actually comparable to those without LIN. The analysis suggests that LIN induced by secondary adsorption is favourable in terms of competition, both in spatially homogeneous and inhomogeneous environments.

Engineering EPS in Halomonas bluephagenesis leads to self-flocculation and filamentation for convenient downstream processing

The halophile Halomonas bluephagenesis has established itself as a cost-effective chassis in industrial bioproduction. However, downstream processing (DSP) expense is a persistent challenge. This study uses genetic knockout of exopolysaccharides (EPS-KO) in six industrially relevant H. bluephagenesis strains, each producing distinct polyhydroxyalkanoate (PHA), to evaluate their impact on reducing DSP costs. Remarkably, EPS-KO cells exhibited an unprecedented filamentous morphology, reaching lengths over 50 times (10–75 μm) that of standard cells. This unexpected behavior initially posed a challenge to industrial applicability due to rapid cell lysis under fermentation agitation. Systematic media supplementation of salt, carbon source, nitrogen, and essential minerals significantly influenced morphology, and tailored nutrient schemes that mitigated filamentation while maintaining production levels were designed. Scaled production in a 5000 L bioreactor demonstrated 85 % PHA and 96 g/L cell dry weight after 40 h fermentation, consistent with non-KO cells. During fermentation, EPS-KO enhanced cell-wall penetrability, reducing the need for high-cost γ-butyrolactone by 40 %. Additionally, a new innovative 5000 L FlipFlow bioreactor design cut rotation speed by 50 % and air input by 25 %. During DSP, centrifugation time decreased tenfold due to rapid cell self-flocculation (15–30 min), and the water and enzyme required for PHA purification was reduced by 30 % and 35 %, respectively. This study highlights the use of EPS-KO cells, a controlled media strategy, and low shear force FlipFlow bioreactor to successfully reduce DSP costs without sacrificing productivity. These novel findings represent a notable advancement in cost-saving strategies while presenting the intriguing discovery of media-controlled filamentous cell behavior.

Rapid, Point-of-Care Microwave Lysis and Electrochemical Detection of Clostridioides difficile Directly from Stool Samples.

The rapid detection of the spore form of Clostridioides difficile has remained a challenge for clinicians. To address this, we have developed a novel, precise, microwave-enhanced approach for near-spontaneous release of DNA from C. difficile spores via a bespoke microwave lysis platform. C. difficile spores were microwave-irradiated for 5 s in a pulsed microwave electric field at 2.45 GHz to lyse the spore and bacteria in each sample, which was then added to a screen-printed electrode and electrochemical DNA biosensor assay system to identify presence of the pathogen's two toxin genes. The microwave lysis method released both single-stranded and double-stranded genome DNA from the bacterium at quantifiable concentrations between 0.02 μg/mL to 250 μg/mL allowing for subsequent downstream detection in the biosensor. The electrochemical bench-top system comprises of oligonucleotide probes specific to conserved regions within tcdA and tcdB toxin genes of C. difficile and was able to detect 800 spores of C. difficile within 300 µL of unprocessed human stool samples in under 10 min. These results demonstrate the feasibility of using a solid-state power generated, pulsed microwave electric field to lyse and release DNA from human stool infected with C. difficile spores. This rapid microwave lysis method enhanced the rapidity of subsequent electrochemical detection in the development of a rapid point-of-care biosensor platform for C. difficile.

Enhancement of waterborne pathogen removal by functionalized biochar with ε-polylysine ″dynamic arms″: Potential application in ultrafiltration system

Widespread outbreaks of threatening infections caused by unknown pathogens and water transmission have spawned the development of adsorption methods for pathogen elimination. We proposed a biochar functionalization strategy involving ε-polylysine (PLL), a bio-macromolecular poly(amino acid)s with variable folding conformations, as a "pathogen gripper" on biochar. PLL was successfully bridged onto biochar via polydopamine (PDA) crosslinking. The extension of electropositive side chains within PLL enables the capture of both nanoscale viruses and micrometer-scale bacteria in water, achieving excellent removal performances. This functionalized biochar was tentatively incorporated into ultrafiltration (UF) system, to achieve effective and controllable adsorption and retention of pathogens, and to realize the transfer of pathogens from membrane surface/pore to biochar surface as well as flushing water. The biochar-amended UF systems presents complete retention (∼7 LRV) and hydraulic elution of pathogens into membrane flushing water. Improvements in removal of organics and anti-fouling capability were observed, indicating the broken trade-off in UF pathogen removal dependent on irreversible fouling. Chemical characterizations revealed adsorption mechanisms encompassing electrostatic/hydrophobic interactions, pore filling, electron transfer, chemical bonding and secondary structure transitions. Microscopic and mechanical analyses validated the mechanisms for rapid adsorption and pathogen lysis. Low-concentration alkaline solution for used biochar regeneration, facilitated the deprotonation and transformation of PLL side chain to folded structures (α-helix/β-sheet). Biochar regeneration process also promoted the effective detachment/inactivation of pathogens and protection of functional groups on biochar, corroborated by physicochemical inspection and molecular dynamics simulation. The foldability of poly(amino acid)s acting like dynamic arms, significantly contributed to pathogen capture/desorption/inactivation and biochar regeneration. This study also inspires future investigation for performances of UF systems amended by poly(amino acid)s-functionalized biochar under diverse pressure, temperature, reactive oxygen species of feeds and chemical cleaning solutions, with far-reaching implications for public health, environmental applications of biochar, and UF process improvement.

Identifying toxic effects and metabolic perturbations of Duttaphrynus melanostictus skin extracts in human erythrocytes.

Skin secretions of toads are widely used in medicine all over the world for their antiviral, anti-infective, and cardiotonic properties. Because these secretions are mostly employed to combat blood parasite infection, it is important to understand their potential toxic effects on human erythrocytes. Therefore, the objective of the current investigation was to elucidate the effects of Duttaphrynus melanostictus (Schneider) skin extracts on the physiology of human erythrocytes. Toads captured from their natural habitat were separated into three groups according to their body size. Hydroalcoholic extracts of toad skin were prepared by reflux heating. These extracts were then evaluated for their hemolytic and hemoglobin denaturation potential. The effects of the extracts on cytosolic and membrane-bound enzymes of human erythrocytes were assessed. The hemolysis and hemoglobin denaturation caused by these extracts correlated positively with the respective toad sizes. Extracts from medium and large toads led to increased osmotic fragility even at near iso-osmotic concentrations. Biochemical analysis of hemolysate showed that the treatment induced a shift of metabolic flux toward the glutathione pathway. Analysis of membrane-bound enzymes revealed a significant decrease in the activity of Na+/K+ ATPase and acetylcholinesterase. SDS-PAGE analysis of the erythrocyte membrane did not show the band of tropomodulin for the cells treated with 1000 𝜇g/ml extract from large toads. In conclusion, the present study demonstrates that the toxicity of toad skin secretions aggravates with the size of the animal and interferes with the physiology of human erythrocytes, leading to their membrane disruption and rapid lysis.

Magnetically modified bacteriophage-triggered ATP release activated EXPAR-CRISPR/Cas14a system for visual detection of Burkholderia pseudomallei

Burkholderia pseudomallei, widely distributed in tropical and subtropical ecosystems, is capable of causing the fatal zoonotic disease melioidosis and exhibiting a global trend of dissemination. Rapid and sensitive detection of B. pseudomallei is essential for environmental monitoring as well as infection control. Here, we developed an innovative biosensor for quantitatively detecting B. pseudomallei relies on ATP released triggered by bacteriophage-induced bacteria lysis. The lytic bacteriophage vB_BpP_HN01, with high specificity, is employed alongside magnetic nanoparticles assembly to create a biological receptor, facilitating the capture and enrichment of viable target bacteria. Following a brief extraction and incubation process, the captured target undergoes rapid lysis to release contents including ATP. The EXPAR-CRISPR cascade reaction provides an efficient signal transduction and dual amplification module that allowing the generated ATP to guide the signal output as an activator, ultimately converting the target bacterial amount into a detectable fluorescence signal. The proposed bacteriophage affinity strategy exhibited superior performance for B. pseudomallei detection with a dynamic range from 10^2 to 10^7 CFU mL−1, and a LOD of 45 CFU mL−1 within 80 min. Moreover, with the output signal compatible across various monitoring methods, this work offers a robust assurance for rapid diagnosis and on-site environmental monitoring of B. pseudomallei.

Antimicrobial peptide thanatin fused endolysin PA90 (Tha-PA90) for the control of Acinetobacter baumannii infection in mouse model

BackgroundThis study addresses the urgent need for infection control agents driven by the rise of drug-resistant pathogens such as Acinetobacter baumannii. Our primary aim was to develop and assess a novel endolysin, Tha-PA90, designed to combat these challenges.MethodsTha-PA90 incorporates an antimicrobial peptide (AMP) called thanatin at its N-terminus, enhancing bacterial outer membrane permeability and reducing host immune responses. PA90 was selected as the endolysin component. The antibacterial activity of the purified Tha-PA90 was evaluated using an in vitro colony-forming unit (CFU) reduction assay and a membrane permeability test. A549 cells were utilized to measure the penetration into the cytosol and the cytotoxicity of Tha-PA90. Finally, infection control was monitored in A. baumannii infected mice following the intraperitoneal administration of Tha-PA90.ResultsTha-PA90 demonstrated remarkable in vitro efficacy, completely eradicating A. baumannii strains, even drug-resistant variants, at a low concentration of 0.5 μM. Notably, it outperformed thanatin, achieving only a < 3-log reduction at 4 μM. Tha-PA90 exhibited 2–3 times higher membrane permeability than a PA90 and thanatin mixture or PA90 alone. Tha-PA90 was found within A549 cells' cytosol with no discernible cytotoxic effects. Furthermore, Tha-PA90 administration extended the lifespan of A. baumannii-infected mice, reducing bacterial loads in major organs by up to 3 logs. Additionally, it decreased proinflammatory cytokine levels (TNF-α and IL-6), reducing the risk of sepsis from rapid bacterial lysis. Our findings indicate that Tha-PA90 is a promising solution for combating drug-resistant A. baumannii. Its enhanced efficacy, low cytotoxicity, and reduction of proinflammatory responses render it a potential candidate for infection control.ConclusionsThis study underscores the significance of engineered endolysins in addressing the pressing challenge of drug-resistant pathogens and offers insights into improved infection management strategies.

Purine nucleosides replace cAMP in allosteric regulation of PKA in trypanosomatid pathogens.

Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signaling proteins, including PKA and PKG. PKA of phylogenetically distant Trypanosoma is the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al., 2019). Here, we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of two to three amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligand in vivo in T. brucei bloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signaling.

Purine nucleosides replace cAMP in allosteric regulation of PKA in trypanosomatid pathogens

Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signaling proteins, including PKA and PKG. PKA of phylogenetically distant Trypanosoma is the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al., 2019). Here, we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of two to three amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligand in vivo in T. brucei bloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signaling.

Inhibition of multiple staphylococcal growth states by a small molecule that disrupts membrane fluidity and voltage.

New molecular approaches to disrupting bacterial infections are needed. The bacterial cell membrane is an essential structure with diverse potential lipid and protein targets for antimicrobials. While rapid lysis of the bacterial cell membrane kills bacteria, lytic compounds are generally toxic to whole animals. In contrast, compounds that subtly damage the bacterial cell membrane could disable a microbe, facilitating pathogen clearance by the immune system with limited compound toxicity. A previously described small molecule, D66, terminates Salmonella enterica serotype Typhimurium (S. Typhimurium) infection of macrophages and reduces tissue colonization in mice. The compound dissipates bacterial inner membrane voltage without rapid cell lysis under broth conditions that permeabilize the outer membrane or disable efflux pumps. In standard media, the cell envelope protects Gram-negative bacteria from D66. We evaluated the activity of D66 in Gram-positive bacteria because their distinct envelope structure, specifically the absence of an outer membrane, could facilitate mechanism of action studies. We observed that D66 inhibited Gram-positive bacterial cell growth, rapidly increased Staphylococcus aureus membrane fluidity, and disrupted membrane voltage while barrier function remained intact. The compound also prevented planktonic staphylococcus from forming biofilms and a disturbed three-dimensional structure in 1-day-old biofilms. D66 furthermore reduced the survival of staphylococcal persister cells and of intracellular S. aureus. These data indicate that staphylococcal cells in multiple growth states germane to infection are susceptible to changes in lipid packing and membrane conductivity. Thus, agents that subtly damage bacterial cell membranes could have utility in preventing or treating disease.IMPORTANCEAn underutilized potential antibacterial target is the cell membrane, which supports or associates with approximately half of bacterial proteins and has a phospholipid makeup distinct from mammalian cell membranes. Previously, an experimental small molecule, D66, was shown to subtly damage Gram-negative bacterial cell membranes and to disrupt infection of mammalian cells. Here, we show that D66 increases the fluidity of Gram-positive bacterial cell membranes, dissipates membrane voltage, and inhibits the human pathogen Staphylococcus aureus in several infection-relevant growth states. Thus, compounds that cause membrane damage without lysing cells could be useful for mitigating infections caused by S. aureus.

Glycan strand cleavage by a lytic transglycosylase, MltD contributes to the expansion of peptidoglycan in Escherichia coli.

Peptidoglycan (PG) is a protective sac-like exoskeleton present in most bacterial cell walls. It is a large, covalently crosslinked mesh-like polymer made up of many glycan strands cross-bridged to each other by short peptide chains. Because PG forms a continuous mesh around the bacterial cytoplasmic membrane, opening the mesh is critical to generate space for the incorporation of new material during its expansion. In Escherichia coli, the 'space-making activity' is known to be achieved by cleavage of crosslinks between the glycan strands by a set of redundant PG endopeptidases whose absence leads to rapid lysis and cell death. Here, we demonstrate a hitherto unknown role of glycan strand cleavage in cell wall expansion in E. coli. We find that overexpression of a membrane-bound lytic transglycosylase, MltD that cuts the glycan polymers of the PG sacculus rescues the cell lysis caused by the absence of essential crosslink-specific endopeptidases, MepS, MepM and MepH. We find that cellular MltD levels are stringently controlled by two independent regulatory pathways; at the step of post-translational stability by a periplasmic adaptor-protease complex, NlpI-Prc, and post-transcriptionally by RpoS, a stationary-phase specific sigma factor. Further detailed genetic and biochemical analysis implicated a role for MltD in cleaving the nascent uncrosslinked glycan strands generated during the expansion of PG. Overall, our results show that the combined activity of PG endopeptidases and lytic transglycosylases is necessary for successful expansion of the cell wall during growth of a bacterium.

Field and laboratory studies of fluorescence-based technologies for real-time tracking of cyanobacterial cell lysis and potential microcystins release

Elevated levels of dissolved microcystins (MCs) in source water due to rapid cell lysis of harmful cyanobacterial blooms may pose serious challenges for drinking water treatment. Catastrophic cell lysis can result from outbreaks of naturally-occurring cyanophages – as documented in Lake Erie during the Toledo water crisis of 2014 and in 2019, or through the application of algaecides or water treatment chemicals. Real-time detection of cyanobacterial cell lysis in source water would provide a valuable tool for drinking water plant and reservoir managers. In this study we explored two real-time fluorescence-based devices, PhycoSens and PhycoLA, that can detect unbound phycocyanin (uPC) as a potential indication of cell lysis and MCs release. The PhycoSens was deployed at the Low Service pump station of the City of Toledo Lake Erie drinking water treatment plant from July 15 to October 19, 2022 during the annual cyanobacteria bloom season. It measured major algal groups and uPC in incoming lake water at 15-min intervals during cyanobacteria dominant and senescence periods. Intermittent uPC detections from the PhycoSens over a three-month period coincided with periods of increasing proportions of extracellular MCs relative to total (intracellular and extracellular) MCs, indicating potential for uPC use as an indicator of cyanobacterial cell integrity. Following exposures of laboratory-cultured MCs-producing Microcystis aeruginosa NIES-298 (120 μg chlorophyll/L) to cyanophage Ma-LMM01, copper sulfate (0.5 and 1 mg Cu/L), sodium carbonate peroxyhydrate (PAK® 27, 6.7 and 10 mg H2O2/L), and potassium permanganate (2.5 and 4 mg/L), appearance of uPC coincided with elevated fractions of extracellular MCs. The PhycoLA was used to monitor batch samples collected daily from Lake Erie water exposed to algaecides in the laboratory. Concurrence of uPC signal and surge of dissolved MCs was observed following 24-h exposures to copper sulfate and PAK 27. Overall results indicate the appearance of uPC is a useful indicator of the onset of cyanobacterial cell lysis and the release of MCs when MCs are present.

Chemical Adjustment of Fibrinolysis.

Fibrinolysis is the process of the fibrin-platelet clot dissolution initiated after bleeding has been stopped. It is regulated by a cascade of proteolytic enzymes with plasmin at its core. In pathological cases, the balance of normal clot formation and dissolution is replaced by a too rapid lysis, leading to bleeding, or an insufficient one, leading to an increased thrombotic risk. The only approved therapy for emergency thrombus lysis in ischemic stroke is recombinant tissue plasminogen activator, though streptokinase or urokinase-type plasminogen activators could be used for other conditions. Low molecular weight compounds are of great interest for long-term correction of fibrinolysis dysfunctions. Their areas of application might go beyond the hematology field because the regulation of fibrinolysis could be important in many conditions, such as fibrosis. They enhance or weaken fibrinolysis without significant effects on other components of hemostasis. Here we will describe and discuss the main classes of these substances and their mechanisms of action. We will also explore avenues of research for the development of new drugs, with a focus on the use of computational models in this field.

The effect of viral infection on the Black Sea microalgae Tetraselmis viridis: the role of nutrients and copper ions.

The TvV-SM2 virus, isolated from the coastal waters of the Black Sea, causes lysis of its host, the algae Tetraselmis viridis (Chlorophyta). Under optimal conditions for nutrients, an increase in the initial abundance of algae cells by four times caused a 3-fold reduction in the latent period of viral infection. During the period of the most rapid cell lysis of T. viridis , nitrogen deficiency leads to a decrease in the average daily rate of death of cells affected by the virus by 3.2times relative to the replete conditions, while in the case of phosphorus deficiency, this process slows down by up to 2.4times. Under deplete conditions, the rate of cell death was only 34% lower than under replete conditions. The effect of copper ions (100μgL-1 ) on the viral suspension for 6h led to the complete suppression of its activity. In the presence of the host of this virus, its activity is only partially suppressed. As a result, cell lysis under the influence of a viral infection occurred in two stages. The first stage was noted only during the first 6h of the experiment. The second main stage took place within 78-170h. This study showed that in conditions of nutrient deficiency and in the presence of copper ions in seawater, the impact of viruses on microalgae will be weaker.

Периферические язвы роговицы в ассоциации с ревматической патологией

Background: peripheral ulcerative keratitis (PUK) is a polyetiological disease. The pathogenesis of PUK is attributed to a combination of factors, including the anatomy of the peripheral cornea, the activation of autoimmune reactions, and the corneal destruction caused by autoan- tibodies and matrix metalloproteinases. Aim: to describe the clinical symptoms of PUK, its association with rheumatic diseases (RD), its course, and the efficacy of treatment options. Patients and Methods: а total of 32 patients (9 men and 23 women, mean age 59.3±9.4 years) with PUK associated with RD were under surveillance. The diagnosis was established based on the results of eye examinations, tear production test, and multidisciplinary examination. Treatment was performed in an outpatient or inpatient setting based on the severity of corneal lesions. Results: the most common association between PUK and RD was established with rheumatoid arthritis (56.2%). Clinical hallmarks of PUK included mild infiltration and perifocal corneal edema, accompanied by the appearance of an oval or sickle-shaped epithelial and stromal defect 2–4 mm from the limbus. Additionally, rapid corneal lysis and perforation (18.7%) and association with perilimbal vasculitis and scleritis (31.2%) were observed. Bilateral corneal lesions were observed in 56.2%. Tear production, as measured by Schirmer's test, was 6±2 mm, while tear film stability was 2±1 mm. Conservative management was found to be effective in 59.4% (outpatient treatment in 40.6% and inpatient treatment in 18.7%). Surgical interventions were performed for emergent indications, including tectonic keratoplasty (21.9%) and deep anterior lamellar keratoplasty (15.6%). Conclusion: PUK represents a severe clinical manifestation of visual disorders in RD. Treatment for PUK involves local and systemic steroids in the acute period, basic immunosup-pressive therapy, and, when necessary, keratoplasty. Systemic rheumatological therapy, regular use of tear substitutes and reparative preparations are prescribed to prevent recurrences. KEYWORDS: keratitis, corneal ulcer, limbus, autoimmune diseases, rheumatoid arthritis, keratoplasty. FOR CITATION: Drozdova E.A. Peripheral corneal ulcers in association with rheumatic diseases. Russian Journal of Clinical Ophthalmology. 2024;24(2):88–93 (in Russ.). DOI: 10.32364/2311-7729-2024-24-2-8.

An assessment of a biosensor system for the quantification of microcystins in freshwater cyanobacterial blooms

Microcystin-producing cyanobacterial blooms are a global issue threatening drinking water supplies and recreation on lakes and beaches. Direct measurement of microcystins is the only way to ensure waters have concentrations below guideline concentrations; however, analyzing water for microcystins takes several hours to days to obtain data. We tested LightDeck Diagnostics’ bead beater cell lysis and two versions of the quantification system designed to give microcystin concentrations within 20 min and compared it to the standard freeze-thaw cycle lysis method and ELISA quantification. The bead beater lyser was only 30 % effective at extracting microcystins compared to freeze-thaw. When considering freeze-thaw samples analyzed in 2021, there was good agreement between ELISA and LightDeck version 2 (n = 152; R2 = 0.868), but the LightDeck slightly underestimated microcystins (slope of 0.862). However, we found poor relationships between LightDeck version 2 and ELISA in 2022 (n = 49, slopes 0.60 to 1.6; R2 < 0.6) and LightDeck version 1 (slope = 1.77 but also a high number of less than quantifiable concentrations). After the quantification issues are resolved, combining the LightDeck system with an already-proven rapid lysis method (such as microwaving) will allow beach managers and water treatment operators to make quicker, well-informed decisions.