SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs.

Higher eukaryotic chromosomes are organized into topologically constrained functional domains; however, the molecular mechanisms required to sustain these complex interphase chromatin structures are unknown. A stable matrix underpinning nuclear organization was hypothesized, but the idea was abandoned as more dynamic models of chromatin behavior became prevalent. Here, we report that scaffold attachment factor A (SAF-A), originally identified as a structural nuclear protein, interacts with chromatin-associated RNAs (caRNAs) via its RGG domain to regulate human interphase chromatin structures in a transcription-dependent manner. Mechanistically, this is dependent on SAF-A's AAA+ ATPase domain, which mediates cycles of protein oligomerization with caRNAs, in response to ATP binding and hydrolysis. SAF-A oligomerization decompacts large-scale chromatin structure while SAF-A loss or monomerization promotes aberrant chromosome folding and accumulation of genome damage. Our results show that SAF-A and caRNAs form a dynamic, transcriptionally responsive chromatin mesh that organizes large-scale chromosome structures and protects the genome from instability.

Oxidase enzyme genes are differentially expressed during Acanthamoeba castellanii encystment.

Acanthamoeba castellanii, a ubiquitous protozoan, is responsible for significant diseases such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. A crucial survival strategy of A. castellanii involves the formation of highly resistant cysts during adverse conditions. This study delves into the cellular processes underpinning encystment, focusing on gene expression changes related to reactive oxygen species (ROS) balance, with a particular emphasis on mitochondrial processes. Our findings reveal a dynamic response within the mitochondria during encystment, with the downregulation of key enzymes involved in oxidative phosphorylation (COX, AOX, and NADHalt) during the initial 48 h, followed by their overexpression at 72 h. This orchestrated response likely creates a pro-oxidative environment, facilitating encystment. Analysis of other ROS processing enzymes across the cell reveals differential expression patterns. Notably, antioxidant enzymes, such as catalases, glutaredoxins, glutathione S-transferases, peroxiredoxins, and thioredoxins, mirror the mitochondrial trend of downregulation followed by upregulation. Additionally, glycolysis and gluconeogenesis are downregulated during the early stages in order to potentially balance the metabolic requirement of the cyst. Our study underscores the importance of ROS regulation in Acanthamoeba encystment. Understanding these mechanisms offers insights into infection control and identifies potential therapeutic targets. This work contributes to unraveling the complex biology of A. castellanii and may aid in combatting Acanthamoeba-related infections. Further research into ROS and oxidase enzymes is warranted, given the organism's remarkable respiratory versatility.

Gut microbiome of Crocodylus porosus and cellular stress: inhibition of nitric oxide, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells.

Crocodiles are renowned for their resilience and capacity to withstand environmental stressors, likely influenced by their unique gut microbiome. In this study, we determined whether selected gut bacteria of Crocodylus porosus exhibit anti-inflammatory effects in response to stress, by measuring nitric oxide release, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells. Using the Griess assay, the findings revealed that among several C. porosus gut bacterial isolates, the conditioned media containing the metabolites of two bacterial strains (CP27 and CP36) inhibited nitric oxide production significantly, in response to the positive control, i.e., taxol-treatment. Notably, CP27 and CP36 were more potent at reducing nitric oxide production than senloytic compounds (fisetin, quercetin). Using enzyme linked immunosorbent assays, the production of pro-inflammatory cytokines (IL-1ß, TNF-α, PGE2), was markedly reduced by treatment with CP27 and CP36, in response to stress. Both CP27 and CP36 contain a plethora of metabolites to exact their effects [(3,4-dihydroxyphenylglycol, 5-methoxytryptophan, nifedipine, 4-chlorotestosterone-17-acetate, 3-phenoxypropionic acid, lactic acid, f-Honaucin A, l,l-Cyclo(leucylprolyl), 3-hydroxy-decanoic acid etc.], indicative of their potential in providing protection against cellular stress. Further high-throughput bioassay-guided testing of gut microbial metabolites from crocodiles, individually as well as in combination, together with the underlying molecular mechanisms, in vitro and in vivo will elucidate their value in the rational development of innovative therapies against cellular stress/gut dysbiosis.

Gut microbiome-immune system interaction in reptiles.

Reptiles are ectothermic amniotes in a world dominated by endotherms. Reptiles originated more than 300 million years ago and they often dwell in polluted environments which may expose them to pathogenic micro-organisms, radiation and/or heavy metals. Reptiles also possess greater longevity and may live much longer than similar-sized land mammals, for example, turtles, tortoises, crocodiles and tuatara are long-lived reptiles living up to 100 years or more. Many recent studies have emphasized the pivotal role of the gut microbiome on its host; thus, we postulated that reptilian gut microbiome and/or its metabolites and the interplay with their robust immune system may contribute to their longevity and overall hardiness. Herein, we discuss the composition of the reptilian gut microbiome, immune system-gut microbiome cross-talk, antimicrobial peptides, reptilian resistance to infectious diseases and cancer, ageing, as well the current knowledge of the genome and epigenome of these remarkable species. Preliminary studies have demonstrated that microbial gut flora of reptiles such as crocodiles, tortoises, water monitor lizard and python exhibit remarkable anticancer and antibacterial properties, as well as comprise novel gut bacterial metabolites and antimicrobial peptides. The underlying mechanisms between the gut microbiome and the immune system may hold clues to developing new therapies overall for health, and possible extrapolation to exploit the ancient defence systems of reptiles for Homo sapiens benefit.

Acanthamoeba castellanii exhibits intron retention during encystment.

Intron retention (IR) refers to the mechanism of alternative splicing in which an intron is not excised from the mature transcript. IR in the cosmopolitan free-living amoeba Acanthamoeba castellanii has not been studied. We performed an analysis of RNA sequencing data during encystment to identify genes that presented differentially retained introns during this process. We show that IR increases during cyst formation, indicating a potential mechanism of gene regulation that could help downregulate metabolism. We identify 69 introns from 67 genes that are differentially retained comparing the trophozoite stage and encystment after 24 and 48 h. These genes include several hypothetical proteins. We show different patterns of IR during encystment taking as examples a lipase, a peroxin-3 protein, an Fbox domain containing protein, a proteasome subunit, a polynucleotide adenylyltransferase, and a tetratricopeptide domain containing protein. A better understanding of IR in Acanthamoeba, and even other protists, could help elucidate changes in life cycle and combat disease such as Acanthamoeba keratitis in which the cyst is key for its persistence.

Development of anti-acanthamoebic approaches.

Acanthamoeba keratitis is a sight-endangering eye infection, and causative organism Acanthamoeba presents a significant concern to public health, given escalation of contact lens wearers. Contemporary therapy is burdensome, necessitating prompt diagnosis and aggressive treatment. None of the contact lens disinfectants (local and international) can eradicate Acanthamoeba effectively. Using a range of compounds targeting cellulose, ion channels, and biochemical pathways, we employed bioassay-guided testing to determine their anti-amoebic effects. The results indicated that acarbose, indaziflam, terbuthylazine, glimepiride, inositol, vildagliptin and repaglinide showed anti-amoebic effects. Compounds showed minimal toxicity on human cells. Therefore, effects of the evaluated compounds after conjugation with nanoparticles should certainly be the subject of future studies and will likely lead to promising leads for potential applications.

Ancestral Eukaryotes Reproduced Asexually, Facilitated by Polyploidy: A Hypothesis.

The notion that eukaryotes are ancestrally sexual has been gaining attention. This idea comes in part from the discovery of sets of "meiosis-specific genes" in the genomes of protists. The existence of these genes has persuaded many that these organisms may be engaging in sex, even though this has gone undetected. The involvement of sex in protists is supported by the view that asexual reproduction results in the accumulation of mutations that would inevitably result in the decline and extinction of such lineages. It is argued that this phenomenon can be obviated by polyploidy and that the "meiosis-specific genes" are used in other processes, including polyploidy control and homologous recombination, independent of meiosis. These phenomena account for the finding that these genes are expressed in cultures devoid of apparent cell fusion events. Hence, it is also proposed that asexual, and not sexual, reproduction is the ancestral condition.

LIF-dependent survival of embryonic stem cells is regulated by a novel palmitoylated Gab1 signalling protein.

The cytokine leukaemia inhibitory factor (LIF) promotes self-renewal of mouse embryonic stem cells (ESCs) through activation of the transcription factor Stat3. However, the contribution of other ancillary pathways stimulated by LIF in ESCs, such as the MAPK and PI3K pathways, is less well understood. We show here that naive-type mouse ESCs express high levels of a novel effector of the MAPK and PI3K pathways. This effector is an isoform of the Gab1 (Grb2-associated binder protein 1) adaptor protein that lacks the N-terminal pleckstrin homology (PH) membrane-binding domain. Although not essential for rapid unrestricted growth of ESCs under optimal conditions, the novel Gab1 variant (Gab1ß) is required for LIF-mediated cell survival under conditions of limited nutrient availability. This enhanced survival is absolutely dependent upon a latent palmitoylation site that targets Gab1ß directly to ESC membranes. These results show that constitutive association of Gab1 with membranes through a novel mechanism promotes LIF-dependent survival of murine ESCs in nutrient-poor conditions.

'Meiotic genes' are constitutively expressed in an asexual amoeba and are not necessarily involved in sexual reproduction.

The amoebae (and many other protists) have traditionally been considered as asexual organisms, but suspicion has been growing that these organisms are cryptically sexual or are at least related to sexual lineages. This contention is mainly based on genome studies in which the presence of 'meiotic genes' has been discovered. Using RNA-seq (next-generation shotgun sequencing, identifying and quantifying the RNA species in a sample), we have found that the entire repertoire of meiotic genes is expressed in exponentially growing Acanthamoeba and we argue that these so-called meiotic genes are involved in the related process of homologous recombination in this amoeba. We contend that they are only involved in meiosis in other organisms that indulge in sexual reproduction and that homologous recombination is important in asexual protists as a guard against the accumulation of mutations. We also suggest that asexual reproduction is the ancestral state.

G418 induces programmed cell death in Acanthamoeba through the elevation of intracellular calcium and cytochrome c translocation.

Acanthamoeba is a widely distributed opportunistic parasite which causes a vision-threatening keratitis and a life-threatening encephalitis. The cyst stage of this amoeba is especially resistant to currently used therapeutics and so alternative agents are urgently required. Growing evidence supports the existence of a programmed cell death system (PCD) in Acanthamoeba and while some features are shared by higher eukaryote cells, others differ. It is hoped that by understanding these differences we can exploit them as targets for novel drug intervention to activate PCD pathways in the amoebae but not the invaded human tissue. Here, we use the aminoglycoside G418 to activate PCD in Acanthamoeba. This drug caused a shape change in the treated amoebae. Cells rounded up and contracted, and after 6 h fragments of cells resembling the 'apoptotic bodies' of vertebrate cells were observed. G418 causes an increase in intracellular calcium from a resting level of 24 nM to 60 nM after 6 h of treatment. Mitochondrial function as assayed by the ΔΨm reporting dye JC-1 and CTC a redox dye becomes inhibited during treatment and we have found that cytochrome c is released from the mitochondria. Cells stained with Hoechst showed first an alteration in chromatin structure and then a vesiculation of the nucleus with G418 treatment, although we found no obvious breakdown in genomic DNA in the early stages of PCD.

Production of a monoclonal antibody against a mannose-binding protein of Acanthamoeba culbertsoni and its localization.

Amoebae from the genus Acanthamoeba are facultative pathogens of humans and other animals. In humans they most frequently infect the eye causing a sight threatening infection known as Acanthamoeba keratitis (AK), and also cause an often fatal encephalitis (GAE). A mannose-binding protein (MBP) has been identified as being important for Acanthamoeba infection especially in AK. This lectin has previously been characterized from Acanthamoeba castellanii as consisting of multiple 130 kDa subunits. MBP expression correlates with pathogenic potential and is expressed in a number of Acanthamoeba species. Here we report the purification of a similar lectin from Acanthamoeba culbertsoni and the production of a monoclonal antibody to it. The A. culbertsoni MBP was isolated by affinity chromatography using α-D-mannose agarose and has an apparent molecular weight of 83 kDa. The monoclonal antibody is an IgM that is useful in both western blots and immunofluorescence. We expect that this antibody will be useful in the study of the pathology of A. culbertsoni and in its identification in clinical samples.

Perifosine Mechanisms of Action in Leishmania Species.

Here the mechanism by which perifosine induced cell death in Leishmania donovani and Leishmania amazonensis is described. The drug reduced Leishmania mitochondrial membrane potential and decreased cellular ATP levels while increasing phosphatidylserine externalization. Perifosine did not increase membrane permeabilization. We also found that the drug inhibited the phosphorylation of Akt in the parasites. These results highlight the potential use of perifosine as an alternative to miltefosine against Leishmania.

Amoebicidal Activity of Caffeine and Maslinic Acid by the Induction of Programmed Cell Death in Acanthamoeba.

Free-living amoebae of the genus Acanthamoeba are the causal agents of a sight-threatening ulceration of the cornea called Acanthamoeba keratitis, as well as the rare but usually fatal disease granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba infections, they are generally lengthy and/or have limited efficacy. For the best clinical outcome, treatments should target both the trophozoite and the cyst stages, as cysts are known to confer resistance to treatment. In this study, we document the activities of caffeine and maslinic acid against both the trophozoite and the cyst stages of three clinical strains of Acanthamoeba These drugs were chosen because they are reported to inhibit glycogen phosphorylase, which is required for encystation. Maslinic acid is also reported to be an inhibitor of extracellular proteases, which may be relevant since the protease activities of Acanthamoeba species are correlated with their pathogenicity. We also provide evidence for the first time that both drugs exert their anti-amoebal effects through programmed cell death.

Vannella pentlandii n. sp., (Amoebozoa, Discosea, Vannellida) a small, cyst-forming soil amoeba.

We describe a new species of cyst-producing soil amoeba Vannella pentlandii from course pasture in the Pentland Hills, Scotland. Analysis of the 18S rDNA gene reveals that it belongs to the sub-group within the genus, presently composed of V. placida, V. epipetala and V. fimicola (the PEF group). This group share features such as longitudinal folds/ridges on the lamella (the anterior hyaline region of the trophozoite), stubby floating forms and cyst production. While each PEF species contain cyst producing strains, not all strains within these species do so. V. fimicola produces cysts on stalks leading to its former classification as a slime mould, however no such stalks were evident in the V. pentlandii, instead groups of cysts become piled on top of each other forming clumps. The encysting amoebae crawl toward each other, pushing some off the surface to form these mounds. The V. pentlandii trophozoites are of typical size for the genus but the cysts at 6.9 µm in diameter, are the smallest so far described in genus Vannella. Other cyst producing species are found in various branches within the Vannella phylogenetic tree, probably meaning that this ability was ancestral but lost in many branches (particularly in marine species), and perhaps re-gained in others.

Leptomyxa valladaresi n. sp. (Amoebozoa, Tubulinea, Leptomyxida), from Mount Teide, Tenerife, Spain.

Leptomyxa valladaresi was isolated from soil in a pine forest on the southern flank of Mt Teide in Tenerife, Spain. It feeds on bacteria and on a range of other amoebae, and it was possible to establish bi-axenic cultures with L. valladaresi and Acanthamoeba. It is easily propagated on a E. coli also. 18S rDNA gene sequence analysis suggests that it is most closely related to Leptomyxa variabilis, however this amoeba differs in important detail. L. valladaresi is primarily mononucleate whereas L. variabilis is multinucleate. L. valladaresi is a larger amoeba and although the cysts are similar in size, there is no sign of the pore-like structures described in L. variabilis cysts. L. valladaresi can adopt a rapid monopodal and tubular morphology similar to that described for L. neglecta and Rhizamoeba matisi, and is never reticulated as larger L. variabilis individuals tend to be. The mean generation time was found to be 18 h, in line with amoebae of this size. Like other members of the genus, L. valladaresi is reported to harbour intracellular, presumably endosymbiotic bacteria, and a Delftia sp has been identified by 16S PCR a bacterium which is also known to grow within Acanthamoeba. The availability of this easily cultured species will help to characterize of this little studied genus and family and their relationship with bacteria, both prey and symbionts.

Statins and voriconazole induce programmed cell death in Acanthamoeba castellanii.

Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a life-threatening encephalitis. In order to treat those infections properly, it is necessary to target the treatment not only to the trophozoite but also to the cyst. Furthermore, it may be advantageous to avoid parasite killing by necrosis, which may induce local inflammation. We must also avoid toxicity of host tissue. Many drugs which target eukaryotes are known to induce programmed cell death (PCD), but this process is poorly characterized in Acanthamoeba. Here, we study the processes of programmed cell death in Acanthamoeba, induced by several drugs, such as statins and voriconazole. We tested atorvastatin, fluvastatin, simvastatin, and voriconazole at the 50% inhibitory concentrations (IC50s) and IC90s that we have previously established. In order to evaluate this phenomenon, we investigated the DNA fragmentation, one of the main characteristics of PCD, with quantitative and qualitative techniques. Also, the changes related to phosphatidylserine exposure on the external cell membrane and cell permeability were studied. Finally, because caspases are key to PCD pathways, caspase activity was evaluated in Acanthamoeba. All the drugs assayed in this study induced PCD in Acanthamoeba. To the best of our knowledge, this is the first study where PCD induced by drugs is described quantitatively and qualitatively in Acanthamoeba.

Evaluation of Acanthamoeba myosin-IC as a potential therapeutic target.

Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a fatal encephalitis. We have targeted myosin-IC by using small interfering RNA (siRNA) silencing as a therapeutic approach, since it is known that the function of this protein is vital for the amoeba. In this work, specific siRNAs against the Acanthamoeba myosin-IC gene were developed. Treated and control amoebae were cultured in growth and encystment media to evaluate the induced effects after myosin-IC gene knockdown, as we have anticipated that cyst formation may be impaired. The effects of myosin-IC gene silencing were inhibition of cyst formation, inhibition of completion of cytokinesis, inhibition of osmoregulation under osmotic stress conditions, and death of the amoebae. The finding that myosin-IC silencing caused incompletion of cytokinesis is in agreement with earlier suggestions that the protein plays a role in cell locomotion, which is necessary to pull daughter cells apart after mitosis in a process known as "traction-mediated cytokinesis". We conclude that myosin-IC is a very promising potential drug target for the development of much-needed antiamoebal drugs and that it should be further exploited for Acanthamoeba therapy.

The isolation of Balamuthia mandrillaris from environmental sources from Peru.

Balamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause skin lesions and the fatal Balamuthia amoebic encephalitis (BAE) in humans and other animals. Currently, around 200 human BAE cases have been reported worldwide, although this number is considered to be underestimated. The highest number of BAE cases has been reported in the American continent, mainly in the southwest of the USA. Peru seems to be another hotspot for BAE with around 55 human cases having been identified, usually involving cutaneous infection, especially lesions in the central face area. The isolation of Balamuthia from environmental sources has been reported on only three prior occasions, twice from Californian soils and once from dust in Iran and so it seems that this amoeba is relatively rarely encountered in samples from the environment. We investigated that possibility of finding the amoebae in soil samples from different regions where clinical cases have been reported in Peru. Twenty-one samples were cultured in non-nutrient agar plates and were checked for the presence of B. mandrillaris-like trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S rDNA gene of B. mandrillaris. We have detected the presence of B. mandrillaris in four samples collected in the regions of Piura (3) and Lima (1) where infection cases have been previously reported. We hypothesize that B. mandrillaris is present in Peru in soil and dust which therefore constitutes a source of the infection for the BAE cases previously reported in this country. Further studies should be carried out in the area to confirm the generality of this finding.

Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and application of statins as a novel effective therapeutic approach against Acanthamoeba infections.

Acanthamoeba is an opportunistic pathogen in humans, whose infections most commonly manifest as Acanthamoeba keratitis or, more rarely, granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba, they are generally lengthy and/or have limited efficacy. Therefore, there is a requirement for the identification, validation, and development of novel therapeutic targets against these pathogens. Recently, RNA interference (RNAi) has been widely used for these validation purposes and has proven to be a powerful tool for Acanthamoeba therapeutics. Ergosterol is one of the major sterols in the membrane of Acanthamoeba. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, one of the precursors for the production of cholesterol in humans and ergosterol in plants, fungi, and protozoa. Statins are compounds which inhibit this enzyme and so are promising as chemotherapeutics. In order to validate whether this enzyme could be an interesting therapeutic target in Acanthamoeba, small interfering RNAs (siRNAs) against HMG-CoA were developed and used to evaluate the effects induced by the inhibition of Acanthamoeba HMG-CoA. It was found that HMG-CoA is a potential drug target in these pathogenic free-living amoebae, and various statins were evaluated in vitro against three clinical strains of Acanthamoeba by using a colorimetric assay, showing important activities against the tested strains. We conclude that the targeting of HMG-CoA and Acanthamoeba treatment using statins is a novel powerful treatment option against Acanthamoeba species in human disease.