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Nature Methods Dec 2023Fluorescence microscopy has become an indispensable tool for revealing the dynamic regulation of cells and organelles. However, stochastic noise inherently restricts...
Fluorescence microscopy has become an indispensable tool for revealing the dynamic regulation of cells and organelles. However, stochastic noise inherently restricts optical interrogation quality and exacerbates observation fidelity when balancing the joint demands of high frame rate, long-term recording and low phototoxicity. Here we propose DeepSeMi, a self-supervised-learning-based denoising framework capable of increasing signal-to-noise ratio by over 12 dB across various conditions. With the introduction of newly designed eccentric blind-spot convolution filters, DeepSeMi effectively denoises images with no loss of spatiotemporal resolution. In combination with confocal microscopy, DeepSeMi allows for recording organelle interactions in four colors at high frame rates across tens of thousands of frames, monitoring migrasomes and retractosomes over a half day, and imaging ultra-phototoxicity-sensitive Dictyostelium cells over thousands of frames. Through comprehensive validations across various samples and instruments, we prove DeepSeMi to be a versatile and biocompatible tool for breaking the shot-noise limit.
Topics: Dictyostelium; Image Enhancement; Microscopy, Confocal; Signal-To-Noise Ratio; Microscopy, Fluorescence; Image Processing, Computer-Assisted
PubMed: 37957429
DOI: 10.1038/s41592-023-02058-9 -
Translational Vision Science &... Sep 2023To investigate the combined anti-Acanthamoeba effects of nitric oxide (NO) donors and hypochlorite to maximize amoebicidal outcomes while minimizing damage to human...
PURPOSE
To investigate the combined anti-Acanthamoeba effects of nitric oxide (NO) donors and hypochlorite to maximize amoebicidal outcomes while minimizing damage to human corneal epithelial cells (HCECs).
METHODS
Acanthamoeba castellanii and primary cultured HCECs and keratocytes were treated with sodium hypochlorite (NaOCl), NO donors (sodium nitroprusside [SNP] and sodium nitrite [NaNO2]), or a combination of hypochlorite and NO donors. The viability of A. castellanii, HCECs, and keratocytes was assessed. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration of NaOCl and NO donors were determined. The activation of mammalian targets of rapamycin (mTOR) and ERK and the expression of nitrite reductase and Nrf2 were assessed in HCECs using Western blot analysis. The cysticidal effects of combined NaOCl and NO donors were also evaluated.
RESULTS
A dose-dependent toxicity was observed in A. castellanii, HCECs, and keratocytes when treated with NaOCl and SNP. The range of tested NaNO2 concentrations showed no significant toxicity to HCECs; however, dose-dependent toxicity to A. castellanii was observed. The MIC of NaOCl against HCECs and A. castellanii was 8.0 mg/mL. The MIC of NaNO2 and SNP was 500 mM and 10 mM in both HCECs and A. castellanii, respectively. Weak attenuation of the mTOR and ERK phosphorylation was observed and Nrf2 expression decreased slightly after exposure of HCECs to 2.0 mg/mL NaOCl. For the combination treatment, NaOCl (0.125 mg/mL) was selected based on the safety of HCECs and the toxicity of A. castellanii. A more potent anti-Acanthamoeba effect and HCEC toxicity were observed when NaOCl was combined with SNP rather than NaNO2.
CONCLUSIONS
Combined NaOCl and NO donors had a stronger anti-Acanthamoeba effect compared to either drug alone.
TRANSLATIONAL RELEVANCE
This study demonstrates that the combined use of various drugs for the treatment of Acanthamoeba infection can enhance the anti-Acanthamoeba effect while minimizing the toxicity of the individual drug.
Topics: Humans; Animals; Acanthamoeba castellanii; Nitric Oxide Donors; Hypochlorous Acid; NF-E2-Related Factor 2; TOR Serine-Threonine Kinases; Mammals
PubMed: 37768280
DOI: 10.1167/tvst.12.9.23 -
Journal of Water and Health Nov 2023The free-living amoeba Naegleria fowleri (Nf) inhabits soil and natural waters worldwide: it is thermophilic and thrives at temperatures up to 45 °C and in a multitude...
The free-living amoeba Naegleria fowleri (Nf) inhabits soil and natural waters worldwide: it is thermophilic and thrives at temperatures up to 45 °C and in a multitude of environments. Three deaths in Louisiana were attributed to primary amoebic meningoencephalitis (PAM) caused by Nf infection in 2011 and 2013. Following these incidents, public water systems are now monitored for the presence of Nf in Louisiana. From 2014 to 2018, 29% (27/93) of samples collected showed positive for Nf and 68% (63/93) showed all thermophilic amoeba culture. Ten raw water sources and 17 distribution water systems tested positive. The year 2017 showed the highest number of samples with Nf (n = 10) followed by nine samples in 2015. As climate change increases surface water temperatures, continued testing for Nf prevalence will be an important facet of water monitoring and will need to extend into locations farther north than the current most common range.
Topics: Naegleria fowleri; Water; Amoeba; Temperature; Louisiana
PubMed: 38017594
DOI: 10.2166/wh.2023.040 -
Pathogens and Global Health Oct 2023This study examines the effects of three different drugs with metformin, acarbose and pioglitazone active ingredients used for antidiabetic purposes on cysts and...
This study examines the effects of three different drugs with metformin, acarbose and pioglitazone active ingredients used for antidiabetic purposes on cysts and trophozoites. Cultures of trophozoites and cysts were prepared to test the anti-amoebic activity of metformin, acarbose and pioglitazone. Cultures were then prepared for cyst and trophozoite forms and parasites were exposed to different concentrations (0.750 mg/mL, 0.375 mg/mL, 0.186 mg/mL and 0.093 mg/mL) of metformin, acarbose and pioglitazone. As a result of the study, the reproductive potential suppressive effects and conversion from trophozoite form to cyst form of all three substances on trophozoites and cysts were determined. Parasites were counted at 12, 24 and 48 hours in the cell counter after staining with trypan blue. In comparison of the effects of metformin, acarbose and pioglitazone used in the study on trophozoites and cysts, it was observed that all three substances were statistically effective against cysts and trophozoites at a concentration of 0.750 mg/mL. Furthermore, it was determined that all concentrations of the three active substances included in the study significantly decreased the rate of cyst formation even at the end of the 7th day. In this context, it was determined that all three substances have amebicidal effects, and they significantly inhibit the transformation of trophozoites to cyst form. It is thought that these active substances, which are currently used as anti-diabetic, can be used in combination with other drugs in infections based on our study findings.
Topics: Animals; Trophozoites; Acanthamoeba castellanii; Hypoglycemic Agents; Acarbose; Pioglitazone; Metformin
PubMed: 36436006
DOI: 10.1080/20477724.2022.2151859 -
Turkiye Parazitolojii Dergisi Dec 2023The aim of this study is to determine the prevalence of intestinal parasites in patients admitted to University of Health Sciences Türkiye (UHS) Van Training and...
OBJECTIVE
The aim of this study is to determine the prevalence of intestinal parasites in patients admitted to University of Health Sciences Türkiye (UHS) Van Training and Research Hospital.
METHODS
A total of 300 patients between the ages of 18-90 who applied to UHS Van Training and Research Hospital with gastrointestinal complaints and were referred to the parasitology laboratory between September 2021 and December 2021, and 100 patients without any chronic disease and gastrointestinal complaints in the control group were included in the study. Stool samples taken from patients included in the study and individuals in the control group were analyzed by native-lugol and modified acid-fast staining methods.
RESULTS
In the study, intestinal parasites were detected in 41 (13.3%) of 300 patients in the patient group and in seven (7%) of 100 individuals in the control group. The highest rate of species ( spp.) (5.7%) was found in the patient group. 3%, 2.7% and species ( spp). 2.3% were found among the other species detected. In addition, a statistically significant correlation was found between the incidence of parasites and abdominal pain (p=0.022) and nausea (p=0.029).
CONCLUSION
As a result; it was concluded that intestinal parasites are still an important health problem in patients with gastrointestinal complaints and intestinal parasites should definitely be considered in this patient group.
Topics: Animals; Humans; Adolescent; Young Adult; Adult; Middle Aged; Aged; Aged, 80 and over; Parasites; Cryptosporidiosis; Cryptosporidium; Feces; Intestinal Diseases, Parasitic; Blastocystis; Prevalence
PubMed: 38149443
DOI: 10.4274/tpd.galenos.2023.93585 -
Experimental Parasitology Jul 2024Acanthamoeba spp., are common free-living amoebae found in nature that can serve as reservoirs for certain microorganisms. The SARS-CoV-2 virus is a newly emerged...
Acanthamoeba spp., are common free-living amoebae found in nature that can serve as reservoirs for certain microorganisms. The SARS-CoV-2 virus is a newly emerged respiratory infection, and the investigation of parasitic infections remains an area of limited research. Given that Acanthamoeba can act as a host for various endosymbiotic microbial pathogens and its pathogenicity assay is not fully understood, this study aimed to identify Acanthamoeba and its bacterial and fungal endosymbionts in patients with chronic respiratory disorders and hospitalized COVID-19 patients in northern Iran. Additionally, a pathogenicity assay was conducted on Acanthamoeba isolates. Urine, nasopharyngeal swab, and respiratory specimens were collected from two groups, and each sample was cultured on 1.5% non-nutrient agar medium. The cultures were then incubated at room temperature and monitored daily for a period of two weeks. Eight Acanthamoeba isolates were identified, and PCR was performed to confirm the presence of amoebae and identify their endosymbionts. Four isolates were found to have bacterial endosymbionts, including Stenotrophomonas maltophilia and Achromobacter sp., while two isolates harbored fungal endosymbionts, including an uncultured fungus and Gloeotinia sp. In the pathogenicity assay, five isolates exhibited a higher degree of pathogenicity compared to the other three. This study provides significant insights into the comorbidity of acanthamoebiasis and COVID-19 on a global scale, and presents the first evidence of Gloeotinia sp. as a fungal endosymbiont. Nevertheless, further research is required to fully comprehend the symbiotic patterns and establish effective treatment protocols.
Topics: Humans; Iran; Acanthamoeba; COVID-19; Symbiosis; SARS-CoV-2; Male; Female; Stenotrophomonas maltophilia; Middle Aged; Adult; Amebiasis; Polymerase Chain Reaction; Aged; Vero Cells; Hospitalization; Chlorocebus aethiops
PubMed: 38754618
DOI: 10.1016/j.exppara.2024.108774 -
Genome Biology and Evolution Aug 2023Evolution experiments with free-living microbes have radically improved our understanding of genome evolution and how microorganisms adapt. Yet there is a paucity of...
Evolution experiments with free-living microbes have radically improved our understanding of genome evolution and how microorganisms adapt. Yet there is a paucity of such research focusing on strictly host-associated bacteria, even though they are widespread in nature. Here, we used the Acanthamoeba symbiont Protochlamydia amoebophila, a distant relative of the human pathogen Chlamydia trachomatis and representative of a large group of protist-associated environmental chlamydiae, as a model to study how obligate intracellular symbionts evolve and adapt to elevated temperature, a prerequisite for the pivotal evolutionary leap from protist to endothermic animal hosts. We established 12 replicate populations under two temperatures (20 °C, 30 °C) for 510 bacterial generations (38 months). We then used infectivity assays and pooled whole-genome resequencing to identify any evolved phenotypes and the molecular basis of adaptation in these bacteria. We observed an overall reduction in infectivity of the symbionts evolved at 30 °C, and we identified numerous nonsynonymous mutations and small indels in these symbiont populations, with several variants persisting throughout multiple time points and reaching high frequencies. This suggests that many mutations may have been beneficial and played an adaptive role. Mutated genes within the same temperature regime were more similar than those between temperature regimes. Our results provide insights into the molecular evolution of intracellular bacteria under the constraints of strict host dependance and highly structured populations and suggest that for chlamydial symbionts of protists, temperature adaptation was facilitated through attenuation of symbiont infectivity as a tradeoff to reduce host cell burden.
Topics: Animals; Humans; Temperature; Bacteria; Acanthamoeba; Chlamydia; Evolution, Molecular; Genome, Bacterial; Symbiosis
PubMed: 37515591
DOI: 10.1093/gbe/evad139 -
Microbiome Jul 2023Legionella are parasites of freshwater protozoa, responsible for Legionellosis. Legionella can be found in a variety of aquatic environments, including rivers, lakes,...
BACKGROUND
Legionella are parasites of freshwater protozoa, responsible for Legionellosis. Legionella can be found in a variety of aquatic environments, including rivers, lakes, and springs, as well as in engineered water systems where they can potentially lead to human disease outbarks. Legionella are considered to be predominantly freshwater organisms with a limited ability to proliferate in saline environments. Exposure of Legionella to high sodium concentrations inhibits growth and virulence of laboratory strains, particularly under elevated temperatures. Nonetheless, Legionella have been identified in some saline environments where they likely interact with various protozoan hosts. In this work, we examine how these selection pressures, sodium and grazing, help shape Legionella ecology within natural environments. Utilizing Legionella-specific primers targeting a variable region of the Legionella 16S rRNA gene, we characterized Legionella abundance, diversity, and community composition in natural spring clusters of varying sodium concentrations, focusing on high sodium concentrations and elevated temperatures.
RESULTS
We observed the highest abundance of Legionella in spring clusters of high salinity, particularly in combination with elevated temperatures. Legionella abundance was strongly related to sodium concentrations. The Legionella community structure in saline environments was characterized by relatively low diversity, compared to spring clusters of lower salinity. The community composition in high salinity was characterized by few dominant Legionella genotypes, not related to previously described species. Protozoan microbial community structure and composition patterns resembled those of Legionella, suggesting a common response to similar selection pressures. We examined Legionella co-occurrence with potential protozoan hosts and found associations with Ciliophora and Amoebozoa representatives.
CONCLUSIONS
Our results indicate that selection forces in saline environments favor a small yet dominant group of Legionella species that are not closely related to known species. These novel environmental genotypes interact with various protozoan hosts, under environmental conditions of high salinity. Our findings suggest that alternative survival mechanisms are utilized by these species, representing mechanisms distinct from those of well-studied laboratory strains. Our study demonstrate how salinity can shape communities of opportunistic pathogens and their hosts, in natural environments, shedding light on evolutionary forces acting within these complex environments. Video Abstract.
Topics: Humans; Legionella; RNA, Ribosomal, 16S; Ecology; Water Microbiology; Fresh Water
PubMed: 37518067
DOI: 10.1186/s40168-023-01611-0 -
Deformability and collision-induced reorientation enhance cell topotaxis in dense microenvironments.Biophysical Journal Jul 2023In vivo, cells navigate through complex environments filled with obstacles such as other cells and the extracellular matrix. Recently, the term "topotaxis" has been...
In vivo, cells navigate through complex environments filled with obstacles such as other cells and the extracellular matrix. Recently, the term "topotaxis" has been introduced for navigation along topographic cues such as obstacle density gradients. Experimental and mathematical efforts have analyzed topotaxis of single cells in pillared grids with pillar density gradients. A previous model based on active Brownian particles (ABPs) has shown that ABPs perform topotaxis, i.e., drift toward lower pillar densities, due to decreased effective persistence lengths at high pillar densities. The ABP model predicted topotactic drifts of up to 1% of the instantaneous speed, whereas drifts of up to 5% have been observed experimentally. We hypothesized that the discrepancy between the ABP and the experimental observations could be in 1) cell deformability and 2) more complex cell-pillar interactions. Here, we introduce a more detailed model of topotaxis based on the cellular Potts model (CPM). To model persistent cells we use the Act model, which mimics actin-polymerization-driven motility, and a hybrid CPM-ABP model. Model parameters were fitted to simulate the experimentally found motion of Dictyostelium discoideum on a flat surface. For starved D. discoideum, the topotactic drifts predicted by both CPM variants are closer to the experimental results than the previous ABP model due to a larger decrease in persistence length. Furthermore, the Act model outperformed the hybrid model in terms of topotactic efficiency, as it shows a larger reduction in effective persistence time in dense pillar grids. Also pillar adhesion can slow down cells and decrease topotaxis. For slow and less-persistent vegetative D. discoideum cells, both CPMs predicted a similar small topotactic drift. We conclude that deformable cell volume results in higher topotactic drift compared with ABPs, and that feedback of cell-pillar collisions on cell persistence increases drift only in highly persistent cells.
Topics: Dictyostelium; Extracellular Matrix; Motion
PubMed: 37291829
DOI: 10.1016/j.bpj.2023.06.001 -
Current Biology : CB Aug 2023Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water...
Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify the basic principles governing contractile vacuole function, we investigate here the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages: the discoban Naegleria gruberi and the amoebozoan slime mold Dictyostelium discoideum. Using quantitative cell biology, we find that although these species respond differently to osmotic challenges, they both use vacuolar-type proton pumps for filling contractile vacuoles and actin for osmoregulation, but not to power water expulsion. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum. These analyses show that cytoplasmic pressure is sufficient to drive contractile vacuole emptying for a wide range of cellular pressures and vacuolar geometries. Because vacuolar-type proton-pump-dependent contractile vacuole filling and pressure-dependent emptying have now been validated in three eukaryotic lineages that diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.
Topics: Cytosol; Dictyostelium; Osmolar Concentration; Water-Electrolyte Balance; Vacuoles; Eukaryota; Water
PubMed: 37478864
DOI: 10.1016/j.cub.2023.06.061