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Journal of Medicinal Chemistry Dec 2023Developing drugs for brain infection by is an unmet medical need. We used a combination of cheminformatics, target-, and phenotypic-based drug discovery methods to...
Developing drugs for brain infection by is an unmet medical need. We used a combination of cheminformatics, target-, and phenotypic-based drug discovery methods to identify inhibitors that target an essential enzyme, sterol 14-demethylase (NfCYP51). A total of 124 compounds preselected were tested against . Nine primary hits with EC ≤ 10 μM were phenotypically identified. Cocrystallization with NfCYP51 focused attention on one primary hit, miconazole-like compound . The -enantiomer of produced a 1.74 Å cocrystal structure. A set of analogues was then synthesized and evaluated to confirm the superiority of the -configuration over the -configuration and the advantage of an ether linkage over an ester linkage. The two compounds, - and -, had an improved EC and compared to . Importantly, both were readily taken up into the brain. The brain-to-plasma distribution coefficient of - was 1.02 ± 0.12, suggesting further evaluation as a lead for primary amoebic meningoencephalitis.
Topics: Miconazole; 14-alpha Demethylase Inhibitors; Naegleria fowleri; Drug Discovery
PubMed: 38085955
DOI: 10.1021/acs.jmedchem.3c01898 -
BMJ Open Ophthalmology Apr 2024Microbial keratitis (MK) is a significant cause of blindness in sub-Saharan Africa. We investigated the feasibility of using a novel corneal impression membrane (CIM)...
OBJECTIVE
Microbial keratitis (MK) is a significant cause of blindness in sub-Saharan Africa. We investigated the feasibility of using a novel corneal impression membrane (CIM) for obtaining and processing samples by culture, PCR and whole-genome sequencing (WGS) in patients presenting with suspected MK in Malawi.
METHODS AND ANALYSIS
Samples were collected from patients presenting with suspected MK using a 12 mm diameter polytetrafluoroethylene CIM disc. Samples were processed using culture and PCR for , herpes simplex virus type 1 (HSV-1) and the bacterial 16S rRNA gene. Minimum inhibitory concentrations of isolates to eight antimicrobials were measured using susceptibility strips. WGS was used to characterise isolates.
RESULTS
71 eyes of 71 patients were included. The overall CIM isolation rate was 81.7% (58 positive samples from 71 participants). 69 (81.2%) of isolates were Gram-positive cocci. Coagulase-negative 31.8% and species 14.1% were the most isolated bacteria. Seven (9.9%) participants were positive for HSV-1. Fungi and were not detected. Moxifloxacin and chloramphenicol offered the best coverage for both Gram-positive and Gram-negative isolates when susceptibility was determined using known antimicrobial first quartile concentrations and European Committee on Antimicrobial Susceptibility Testing breakpoints, respectively. WGS identified known virulence genes associated with keratitis.
CONCLUSIONS
In a resource-poor setting, a CIM can be used to safely sample the cornea in patients presenting with suspected MK, enabling identification of causative microorganisms by culture and PCR. Although the microbiological spectrum found was limited to the dry season, these preliminary results could be used to guide empirical treatment.
Topics: Humans; Pilot Projects; Malawi; Male; Female; Adult; Middle Aged; Eye Infections, Bacterial; Young Adult; Bacteria; Microbial Sensitivity Tests; Cornea; Keratitis; Anti-Bacterial Agents; Aged; Polymerase Chain Reaction; Adolescent; Acanthamoeba; RNA, Ribosomal, 16S
PubMed: 38653537
DOI: 10.1136/bmjophth-2024-001682 -
Journal of Cell Science Jul 2023During developmental and immune responses, cells move towards or away from some signals. Although much is known about chemoattraction, chemorepulsion (the movement of...
During developmental and immune responses, cells move towards or away from some signals. Although much is known about chemoattraction, chemorepulsion (the movement of cells away from a stimulus) remains poorly understood. Proliferating Dictyostelium discoideum cells secrete a chemorepellent protein called AprA. Examining existing knockout strains, we previously identified proteins required for AprA-induced chemorepulsion, and a genetic screen suggested that the enzyme phosphatidylinositol phosphate kinase A (PIPkinA, also known as Pik6) might also be needed for chemorepulsion. Here, we show that cells lacking PIPkinA are not repelled by AprA, and that this phenotype is rescued by expression of PIPkinA. To bias cell movement, AprA inhibits Ras activation at the side of the cell closest to the source of AprA, and we find that PIPkinA is required for AprA to inhibit Ras activation. PIPkinA decreases levels of phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], and possibly because of these effects, potentiates phagocytosis and inhibits cell proliferation. Cells lacking PIPkinA show normal AprA binding, suggesting that PIPkinA regulates chemorepulsion at a step between the AprA receptor and AprA inhibition of Ras activation.
Topics: Dictyostelium; Phosphates; Protozoan Proteins; Cell Proliferation; Genetic Testing
PubMed: 37259831
DOI: 10.1242/jcs.260541 -
Biophysical Journal Aug 2023Identifying the directionality of signaling sources from noisy input to membrane receptors is an essential task performed by many cell types. A variety of models have...
Identifying the directionality of signaling sources from noisy input to membrane receptors is an essential task performed by many cell types. A variety of models have been proposed to explain directional sensing in cells. However, many of these require significant computational and memory capacities for the cell. We propose and analyze a simple mechanism in which a cell adopts the direction associated with the first few membrane binding events. This model yields an accurate angular estimate to the source long before steady state is reached in biologically relevant scenarios. Our proposed mechanism allows for reliable estimates of the directionality of external signals using temporal information and assumes minimal computational capacities of the cell.
Topics: Signal Transduction; Dictyostelium
PubMed: 37355773
DOI: 10.1016/j.bpj.2023.06.015 -
Cells Feb 2024The cell membrane is frequently subjected to damage, either through physical or chemical means. The swift restoration of the cell membrane's integrity is crucial to... (Review)
Review
The cell membrane is frequently subjected to damage, either through physical or chemical means. The swift restoration of the cell membrane's integrity is crucial to prevent the leakage of intracellular materials and the uncontrolled influx of extracellular ions. Consequently, wound repair plays a vital role in cell survival, akin to the importance of DNA repair. The mechanisms involved in wound repair encompass a series of events, including ion influx, membrane patch formation, endocytosis, exocytosis, recruitment of the actin cytoskeleton, and the elimination of damaged membrane sections. Despite the absence of a universally accepted general model, diverse molecular models have been proposed for wound repair in different organisms. Traditional wound methods not only damage the cell membrane but also impact intracellular structures, including the underlying cortical actin networks, microtubules, and organelles. In contrast, the more recent improved laserporation selectively targets the cell membrane. Studies on cells utilizing this method have introduced a novel perspective on the wound repair mechanism. This review commences by detailing methods for inducing wounds and subsequently reviews recent developments in the field.
Topics: Dictyostelium; Cell Membrane; Actins; Microtubules; Actin Cytoskeleton
PubMed: 38391954
DOI: 10.3390/cells13040341 -
Microbiology Spectrum Aug 2023The diatom Phaeodactylum tricornutum is regarded as a prospective "cell factory" for the high-value products fucoxanthin and eicosapentaenoic acid (EPA). However,...
The diatom Phaeodactylum tricornutum is regarded as a prospective "cell factory" for the high-value products fucoxanthin and eicosapentaenoic acid (EPA). However, contamination with grazing protozoa is a significant barrier to its commercial cultivation. Here, we describe a new species of heterolobosean amoeba, Euplaesiobystra perlucida, which caused the loss of in pilot-scale cultures. Morphological and molecular characteristics distinguish from the other species in the genus . is 1.4 to 3.2 times larger than other species in terms of average length/width and maximum length/width of the trophozoites. Unlike Euplaesiobystra salpumilio, has no cytostome; lacks a flagellate stage, whereas Euplaesiobystra hypersalinica and both display a flagellate stage in their life cycle. The small-subunit rRNA gene sequence of shared only 88.02% homology with that of its closest relative, Euplaesiobystra dzianiensis, and had two distinctive regions. Its phylogenetic branch was clustered with one uncultured heterolobosean clone (bootstrap support/posterior probability = 100%/1.00). Results of feeding experiments demonstrated that could graze on various unicellular and filamentous eukaryotic microalgae (chlorophytes, chrysophytes, euglenids, and diatoms) and cyanobacteria. 's ingestion rate declined exponentially with increasing size of unicellular prey, and attained the highest growth rates on . On the basis of its strong ability to graze on microalgae, capacity to form large populations in a short period of time, and capacity to form resistant resting cysts, this contaminant has the potential to cause severe problems in large-scale microalgal culture and merits further attention. Heteroloboseans have garnered considerable interest because of their extraordinary ecological, morphological, and physiological diversity. Many heteroloboseans have adapted to various extensive habitats, including halophilic, acidophilic, thermophilic, psychrophilic, and anaerobic habitats. Most heteroloboseans are bacterivores, with a few algivorous species reported. In this study, a new species of algivorous heterolobosean amoeba, , is described as a significant grazer that causes losses in outdoor industrial cultures. This study provides phenotypic, feeding, and genetic information on a previously unknown heterolobosean, emphasizes the impact of contaminating amoebae in commercial microalgal cultures, and will contribute to the management strategies for predicting this kind of contaminant in large-scale microalgal cultivation.
Topics: Amoeba; Diatoms; Phylogeny; Prospective Studies; Eukaryota
PubMed: 37378530
DOI: 10.1128/spectrum.00817-23 -
Anais Da Academia Brasileira de Ciencias 2024The family Physaraceae (Physarales, Myxomycetes) is represented in Brazil by eight genera and 75 species. Based on data obtained from the GBIF, SpeciesLink, Flora and...
The family Physaraceae (Physarales, Myxomycetes) is represented in Brazil by eight genera and 75 species. Based on data obtained from the GBIF, SpeciesLink, Flora and Funga do Brasil platforms, collections from the IPA and URM Herbaria and material collected since 1960 deposited in the UFP Herbarium, the microhabitats and distribution of Badhamiopsis (1sp.) and Badhamia (10 spp.) in Brazilian biomes are commented. An identification key for the species and the first report of B. melanospora from the state of Paraíba, B. panicea from the state of Paraná and B. ovispora from Brazil are presented.
Topics: Myxomycetes; Brazil; Ecosystem
PubMed: 38451623
DOI: 10.1590/0001-3765202420220698 -
European Journal of Protistology Feb 2024The shells of testate amoebae are morphologically diverse and persistent in the environment. Accordingly, the examination of the morphology and composition of shells...
The shells of testate amoebae are morphologically diverse and persistent in the environment. Accordingly, the examination of the morphology and composition of shells became a standard tool in ecological, palaeoecological, and evolutionary studies. However, so far the function of the shell remains poorly understood and, although based on limited evidence, the shell was considered as a defense mechanism. Based on recent evidence, we propose that the shell of arcellinid testate amoebae is a crucial component facilitating the amoebae's attack of large prey. Accordingly, the shell is not purely protective, but must be considered also as a weapon. This change in perspective opens up numerous new avenues in protistology and will lead to a substantial change in ecological, palaeoecological, and evolutionary research.
Topics: Phylogeny; Lobosea; Amoeba; Biological Evolution
PubMed: 38194835
DOI: 10.1016/j.ejop.2024.126051 -
Turkiye Parazitolojii Dergisi Sep 2023, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. keratitis and granulomatous amoebic encephalitis are...
OBJECTIVE
, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. keratitis and granulomatous amoebic encephalitis are among the most important clinical manifestations caused by . In this study, it was aimed to determine the sensitivity of the rapid loop mediated isothermal amplification (LAMP) test designed with primers specific to 18S rRNA gene to detect more rapidly the presence of in clinical and environmental samples.
METHODS
strain grown in culture was diluted in 200 μL as 1x106 trophozoites and DNA was isolated, and the amount of DNA was determined by Nano-Drop Spectrophotometer. The purified DNAs were diluted from 1000 pg to 0.001 pg and used in colorimetric and fluorescence-based LAMP reactions. The LAMP reaction mixture was incubated for 60 minutes at 63 °C in a total volume of 25 μL.
RESULTS
To determine the sensitivity of the test, positivity of genomic DNA was observed at 1, 10, 100 and 1000 pg/reaction in both colorimetric and fluorescence-based LAMP tests. The lowest analytical sensitivity of both calorimetric and fluorescent LAMP assay was determined as 1 pg/reaction. In addition, as a result of LAMP reaction applied with other parasite DNAs to evaluate the specificity of the test, no LAMP product was detected in calorimetric and 1% agarose gel electrophoresis, except for positive control, and the specificity of the test was determined as 100%.
CONCLUSION
It has been demonstrated that the LAMP assay designed by targeting 18S rRNA gene of has a detection limit of 1 pg of genomic DNA. It is promising that LAMP test is more sensitive and faster than culture method, as well as simple, inexpensive and highly sensitive. For this reason, it is thought that developed test can be applied in the diagnosis of in environmental and clinical samples.
Topics: Acanthamoeba; Genes, rRNA; Biological Assay; Coloring Agents
PubMed: 37724360
DOI: 10.4274/tpd.galenos.2023.46362 -
Frontiers in Immunology 2023Primary Amoebic Meningoencephalitis (PAM), a severe lethal brain disease, is caused by a parasite, , also known as the "brain-eating amoeba". The chances of a patient's...
Primary Amoebic Meningoencephalitis (PAM), a severe lethal brain disease, is caused by a parasite, , also known as the "brain-eating amoeba". The chances of a patient's recovery after being affected by this parasite are very low. Only 5% of people are known to survive this life-threatening infection. Despite the fact that causes a severe, fatal infection, there is no proper treatment available to prevent or cure it. In this context, it is necessary to formulate a potential vaccine that could be able to combat infection. The current study aimed at developing a multi-epitope subunit vaccine against by utilizing immunoinformatics techniques and reverse vaccinology approaches. The T- and B-cell epitopes were predicted by various tools. In order to choose epitopes with the ability to trigger both T- and B-cell-mediated immune responses, the epitopes were put through a screening pipeline including toxicity, antigenicity, cytokine-inductivity, and allergenicity analysis. Three vaccine constructs were designed from the generated epitopes linked with linkers and adjuvants. The modeled vaccines were docked with the immune receptors, where vaccine-1 showed the highest binding affinity. Binding affinity and stability of the docked complex were confirmed through normal mode analysis and molecular dynamic simulations. Immune simulations developed the immune profile, and cloning affirmed the expression probability of the vaccine construct in ( strain K12. This study demonstrates an innovative preventative strategy for the brain-eating amoeba by developing a potential vaccine through immunoinformatics and reverse vaccinology approaches. This study has great preventive potential for Primary Amoebic Meningoencephalitis, and further research is required to assess the efficacy of the designed vaccine.
Topics: Humans; Naegleria fowleri; Escherichia coli; Central Nervous System Protozoal Infections; Brain; Epitopes, B-Lymphocyte; Informatics; Vaccines, Subunit
PubMed: 37965306
DOI: 10.3389/fimmu.2023.1284621