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.

Emerging Role of Decoy Receptor-2 as a Cancer Risk Predictor in Oral Potentially Malignant Disorders.

The aim of this study was to evaluate the expression of the senescence markers, Decoy Receptor 2 (DcR2) and Differentiated Embryo-Chondrocyte expressed gen 1 (DEC1), in oral potentially malignant disorders (OPMDs) to ascertain their possible association with oral cancer risk. The immunohistochemical analysis of DcR2 and DEC1 expression (along with p16 and Ki67 expression) was carried out in 60 patients with clinically diagnosed oral leukoplakia. Fifteen cases (25%) subsequently developed an invasive carcinoma. Correlations between protein marker expression, histological grade and oral cancer risk were assessed. DcR2, DEC1 and Ki67 protein expressions were found to correlate significantly with increased oral cancer risk, and also with an increased grade of dysplasia. Multivariate analysis demonstrated that DcR2 and Ki67 expression are independent predictors of oral cancer development. Our results evidence for the first time the potential of DcR2 as an early biomarker to assess oral cancer risk in patients with oral leukoplakia (HR = 59.7, p = 0.015), showing a superior predictive value to histology (HR = 4.225, p = 0.08). These findings reveal that the increased expression of DcR2 and DEC1 occurred frequently in OPMDs. In addition, DcR2 expression emerges as a powerful biomarker for oral cancer risk assessment in patients with oral leukoplakia.

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.

'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.

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.

Sclerosing Polycystic Adenosis of the Parotid Gland: A Systematic Review and Report of 2 New Cases.

PURPOSE: We sought to evaluate clinical-epidemiologic aspects, pathologic characteristics, and treatment of sclerosing polycystic adenosis (SPA) of the parotid gland and to report 2 new cases. MATERIALS AND METHODS: We conducted a systematic review following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines for studies published in or before June 2016. The inclusion criteria were as follows: final diagnosis of SPA affecting the parotid gland and articles published in Spanish or English. The exclusion criteria were as follows: unclear diagnosis after pathologic analysis and patients affected by other conditions in the major salivary glands. We added 2 new cases. RESULTS: Our review ultimately included 21 articles and 45 cases. The mean patient age was 41 years (range, 7-84 years). The female-to-male ratio was 2:1. In all patients the tumor showed progressive growth. The evolution range was 7 days to 11 years. In 17.7% of cases, the tumor was associated with pain. The average tumor size was 30.3 mm (range, 12-60 mm). Fine needle aspiration biopsy (FNAB) was unable to diagnose 30 cases. Benign tumors comprised 58% of tumors (18), whereas malignancy was present in 25.8% (8). Thirty-five tumors were well circumscribed. There were 8 multinodular or lobed cases. The tumor was described as encapsulated in 1 case, partially encapsulated or pseudo-encapsulated in 16, and not encapsulated in 12. Five cases showed different degrees of degenerative changes, ranging from dysplasia to invasive carcinoma. All cases underwent surgical treatment. Superficial parotidectomy with preservation of the facial nerve was performed in 22 cases (61.1%), total parotidectomy in 8 (22.2%), and tumor enucleation in 6 (16.6%). The mean follow-up period was 51.5 months (range, 5-276 months). Of documented cases, 74.2% had 2 or more years of follow-up. Recurrences occurred in 6 cases (16.6%). CONCLUSIONS: We consider SPA a benign tumor with progressive growth, which is occasionally painful. It occurs around age 40 years and occurs more often in female patients. Fine needle aspiration biopsy does not provide an adequate preoperative diagnosis. Recurrences are infrequent. Follow-up should be customized according to the pathologic findings. Malignant transformation may occur. Superficial parotidectomy with facial nerve preservation is the treatment of choice.

Replicative aging in yeast involves dynamic intron retention patterns associated with mRNA processing/export and protein ubiquitination.

Saccharomyces cerevisiae (baker's yeast) has yielded relevant insights into some of the basic mechanisms of organismal aging. Among these are genomic instability, oxidative stress, caloric restriction and mitochondrial dysfunction. Several genes are known to have an impact on the aging process, with corresponding mutants exhibiting short- or long-lived phenotypes. Research dedicated to unraveling the underlying cellular mechanisms can support the identification of conserved mechanisms of aging in other species. One of the hitherto less studied fields in yeast aging is how the organism regulates its gene expression at the transcriptional level. To our knowledge, we present the first investigation into alternative splicing, particularly intron retention, during replicative aging of S. cerevisiae. This was achieved by utilizing the IRFinder algorithm on a previously published RNA-seq data set by Janssens et al. (2015). In the present work, 44 differentially retained introns in 43 genes were identified during replicative aging. We found that genes with altered intron retention do not display significant changes in overall transcript levels. It was possible to functionally assign distinct groups of these genes to the cellular processes of mRNA processing and export (e.g., YRA1) in early and middle-aged yeast, and protein ubiquitination (e.g., UBC5) in older cells. In summary, our work uncovers a previously unexplored layer of the transcriptional program of yeast aging and, more generally, expands the knowledge on the occurrence of alternative splicing in baker's yeast.

mRNA Sequencing Reveals Upregulation of Glutathione S-Transferase Genes during Acanthamoeba Encystation.

Some members of the genus Acanthamoeba are facultative pathogens typically with a biphasic lifestyle: trophozoites and cysts. Acanthamoeba is capable of infecting the cornea, resulting in Acanthamoeba keratitis. The cyst is one of the key components for the persistence of infection. Gene expression during Acanthamoeba encystation showed an upregulation of glutathione S-transferase (GST) genes and other closely related proteins. mRNA sequencing showed GST, and five genes with similar sequences were upregulated after 24 h of inducing encystation. GST overexpression was verified with qPCR using the HPRT and the cyst-specific protein 21 genes as controls. The GST inhibitor ethacrynic acid was found to decrease cell viability by 70%. These results indicate a role of GST in successful encystation, possibly by maintaining redox balance. GST and associated processes could be targets for potential treatments alongside regular therapies to reduce relapses of Acanthamoeba infection.

Superoxide Dismutases in Eukaryotic Microorganisms: Four Case Studies.

Various components in the cell are responsible for maintaining physiological levels of reactive oxygen species (ROS). Several different enzymes exist that can convert or degrade ROS; among them are the superoxide dismutases (SODs). If left unchecked, ROS can cause damage that leads to pathology, can contribute to aging, and may, ultimately, cause death. SODs are responsible for converting superoxide anions to hydrogen peroxide by dismutation. Here we review the role of different SODs on the development and pathogenicity of various eukaryotic microorganisms relevant to human health. These include the fungal aging model, Podospora anserina; various members of the genus Aspergillus that can potentially cause aspergillosis; the agents of diseases such as Chagas and sleeping disease, Trypanosoma cruzi and Trypanosoma brucei, respectively; and, finally, pathogenic amoebae, such as Acanthamoeba spp. In these organisms, SODs fulfill essential and often regulatory functions that come into play during processes such as the development, host infection, propagation, and control of gene expression. We explore the contribution of SODs and their related factors in these microorganisms, which have an established role in health and disease.

From Past to Present: Biotechnology in Mexico Using Algae and Fungi.

Algae and fungi share a rich history in the fields of basic and applied natural science. In biotechnology, in particular, algae and fungi are of paramount importance, due to the production and development of valuable compounds, such as pharmaceuticals, enzymes, and biofuels. They are also used in waste fermentation, biocontrol of pathogens, and food processing and improvement, among other fields. Although a substantial number of different microorganisms are utilized for these purposes, there lies tremendous potential in uncharacterized microbial species. For this reason, biodiversity hotspots offer a wealth of potential in the discovery of new products and processing strategies based on these microorganisms. This review presents an overview of the use of algae and fungi in pre-Hispanic times/modern-day Mexico for the benefits of mankind. One of our objectives is to raise awareness about the potential of developing research projects for identification and biotechnological utilization of algae and fungi in a megadiverse country, such as Mexico.

LKTA and PlpE small fragments fusion protein protect against Mannheimia haemolytica challenge.

Bovine respiratory disease (BRD) complex is a major cause of economic losses for the cattle backgrounding and feedlot industries. Mannheimia haemolytica is considered the most important pathogen associated with this disease. Vaccines against M. haemolytica have been prepared and used for many decades, but traditional bacterins have failed to demonstrate effective protection and their use has often exacerbated disease in vaccinated animals. Thus, the BRD complex continues to exert a strong adverse effect on the health and wellbeing of stocker and feeder cattle. Therefore, generation of recombinant proteins has been helpful in formulating enhanced vaccines against M. haemolytica, which could confer better protection against BRD. In the present study, we formulated a vaccine preparation enriched with recombinant small fragments of leukotoxin A (LKTA) and outer-membrane lipoprotein (PlpE) proteins, and demonstrated its ability to generate high antibody titers in rabbits and sheep, which protected against M. haemolytica bacterial challenge in mice.