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MicroRNAs (miRNAs) are gene regulators essential for cell homeostasis, their alteration is related to a pathological state, including infectious diseases like COVID-19. Identifying an altered profile of circulating miRNAs in mild COVID-19 may enhance our knowledge of the pathogenesis of SARS-CoV-2 and the range of clinical phenotypes. In the present study, a miRNA screening was performed by Next Generation Sequencing (NGS), and the expression levels of 13 resulting miRNAs were validated through RT-qPCR in the serum of 40 mild cases compared to 29 non-infected individuals. An in-silico analysis was performed to detect target genes and their related pathways. From the validated miRNAs, miR-1246 ( p  < 0.001), miR-423-5p ( p  < 0.001), miR-21-5p ( p  = 0.005), miR-146a-5p ( p  < 0.001), miR-4508 ( p  = 0.001), miR-629-5p ( p  < 0.001), and miR-210-3p ( p  = 0.002) were found downregulated in infected individuals. Only miR-27a-5p was overexpressed in subjects with COVID-19 ( p  = 0.013) and associated with SARS-CoV-2 infection ( p  = 0.010). The KEGG pathways and GO analysis revealed that the differentially expressed miRNAs were related to viral processes or immunological pathways: miR-27a-5p acts on the TGF-beta pathway; miR-21-5p targets SMAD7 , which is associated with the inflammatory response in the lung; miR-1246 acts on p53 pathway; and miR-4508 acts on ICAM2 . In conclusion, the most relevant miRNAs, miR-27a-5p and miR-21-5p, were differently expressed in mild forms of COVID-19. The higher expression of miR-27a-5p observed in mild COVID-19 cases may suggest a protective effect against severe forms of the disease. Reduced expression of miR-21-5p may prevent pulmonary inflammation and the progression of fibrosis. The downregulation of miR-1246 and miR-4508 in mild COVID-19 cases may conduct the correct control of the infection. Moreover, miR-423-5p might be a suitable biomarker in the early stages of SARS-CoV-2 infection.

Phytochemical investigation of Radula voluta, a liverwort species collected in the Ecuadorian Amazon, led to the isolation of four known bibenzyl derivatives: 2-prenyl-3,5-dihydroxy-bibenzyl (1), 2-geranyl-3,5-dihydroxybibenzyl (2), 2,2-dimethyl-5-phenethyl-2H-chromen-7-ol (3), and radulanin L (4). Structural elucidation was achieved through extensive NMR and MS analyses, supported by comparison with previously reported data. Compounds 1 and 4 are reported for the first time in R. voluta. The crude extract and isolated compounds were evaluated for their in vitro antiprotozoal activity against Trypanosoma cruzi, Leishmania amazonensis, Leishmania donovani, Naegleria fowleri, and Acanthamoeba castellanii Neff. Among the isolated compounds, bibenzyls 2 and 4 exhibited the most potent activity across multiple protozoan strains. Cytotoxicity was assessed against murine macrophages (J774A.1), obtaining moderate–low toxicities against compounds 1 and 3. These findings highlight the pharmacological value of liverwort-derived bibenzyls and support further research on R. voluta as a promising source of antiparasitic leads.

Primary amoebic meningoencephalitis (PAM) is a rapidly progressive and fulminant disease that affects the central nervous system caused by the free-living amoeba Naegleria fowleri. The adhesion to extracellular matrix (ECM) proteins is considered as one of the key steps in the success of the infection and could represent an interesting target to be explored in the prevention and treatment of the disease. In this work, the effect of two sesquiterpenes with proven anti-Naegleria activity on the adhesion of the parasite was evaluated using an in vitro ECM-based model, compared with the reference drugs amphotericin B and staurosporine. Both laurinterol and (+)-elatol inhibited the adhesion of the N. fowleri trophozoites to the main proteins of the ECM when treating them at different concentrations and exposure times. This work not only reinforces the therapeutic potential of laurinterol and (+)-elatol against N. fowleri infection but also introduces the application of ECM-based adhesion assays as a novel and valuable tool for screening candidate compounds that disrupt host–pathogen interactions critical to PAM pathogenesis.

Leishmaniasis, produced by Leishmania spp., and Chagas disease, produced by Trypanosoma cruzi, affect millions of people around the world. The treatments for these pathologies are not entirely effective and produce some side effects. For these reasons, it is necessary to develop new therapies that are more active and less toxic for patients. Some initiatives, such as the one carried out by the Medicines for Malaria Venture, allow for the screening of a large number of compounds of different origins to find alternatives to the lack of trypanocide treatments. In this work, 240 compounds were tested from the Global Health Priority Box (80 compounds with confirmed activity against drug-resistant malaria, 80 compounds for screening against neglected and zoonotic diseases and diseases at risk of drug resistance, and 80 compounds with activity against various vector species) against Trypanosoma cruzi and Leishmania amazonensis. Flucofuron, a compound with activity against vectors and with previous activity reported against Staphylococcus spp. and Schistosoma spp., demonstrates activity against L. amazonensis and T. cruzi and produces programmed cell death in the parasites. Flucofuron seems to be a good candidate for continuing study and proving its use as a trypanocidal agent.

Amoebae of the genus Acanthamoeba are ubiquitous protists that have been isolated from many sources such as soils, water and the air. They are responsible for infections including fatal encephalitis and a severe keratitis in humans. To date, there is no satisfactorily effective therapeutic agent against this pathogen and the infections it causes are exacerbated by the existence of a resistant cyst stage produced by this amoeba. As dry eye syndrome is a risk factor for Acanthamoeba keratitis, we aimed to evaluate the anti- Acanthamoeba activity of a variety of proprietary eye drops intended to treat dry eye syndrome. From the nine eye drop formulations tested, “Systane Ultra” was determined to be the most active against all tested Acanthamoeba strains. During our investigations into the mode of action of Systane Ultra, we discovered that it decreases mitochondrial membrane potential and ATP levels, induces chromatin condensation, and increases the permeability of the plasma-membrane.

Naegleria fowleri is an opportunistic protozoon that can be found in warm water bodies. It is the causative agent of the primary amoebic meningoencephalitis. Focused on our interest to develop promising lead structures for the development of antiparasitic agents, this study was aimed at identifying new anti-Naegleria marine natural products from a collection of chamigrane-type sesquiterpenes with structural variety in the levels of saturation, halogenation and oxygenation isolated from Laurencia dendroidea. (+)-Elatol (1) was the most active compound against Naegleria fowleri trophozoites with IC50 values of 1.08 μM against the ATCC 30808™ strain and 1.14 μM against the ATCC 30215™ strain. Furthermore, the activity of (+)-elatol (1) against the resistant stage of N. fowleri was also assessed, showing great cysticidal properties with a very similar IC50 value (1.14 µM) to the one obtained for the trophozoite stage. Moreover, at low concentrations (+)-elatol (1) showed no toxic effect towards murine macrophages and could induce the appearance of different cellular events related to the programmed cell death, such as an increase of the plasma membrane permeability, reactive oxygen species overproduction, mitochondrial malfunction or chromatin condensation. Its enantiomer (−)-elatol (2) was shown to be 34-fold less potent with an IC50 of 36.77 μM and 38.03 μM. An analysis of the structure–activity relationship suggests that dehalogenation leads to a significant decrease of activity. The lipophilic character of these compounds is an essential property to cross the blood-brain barrier, therefore they represent interesting chemical scaffolds to develop new drugs.

Recently, the search for novel therapeutic agents against Acanthamoeba species has been focused on the evaluation of natural resources. Among them, marine microorganisms have risen as a source of bioactive compounds with the advantage of the ability to obtain unlimited and constant amounts of the compounds in contrast to other natural sources such as plants. Furthermore, marine actinomycetes have recently been reported as highly rich in bioactive agents including salinosporamides, xiamycines, indolocarbazoles, naphtyridines, phenols, dilactones such as antimycines and macrolides among others. In this study, staurosporine (STS) was isolated from a strain of Streptomyces sanyensis and tested against Acanthamoeba to characterize the therapeutic potential of STS against this protozoan parasite. We have established that STS is active against both stages of the Acanthamoeba life cycle, by the activation of Programmed Cell Death via the mitochondrial pathway of the trophozoite. We have also established that STS has relatively low toxicity towards a macrophage cell line. However, previous studies have highlighted higher toxicity levels induced on other vertebrate cell lines and future research to lower these toxicity issues should be developed.

Therapy against Acanthamoeba infections such as Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba Keratitis (AK), remains as an issue to be solved due to the existence of a cyst stage which is highly resistant to most chemical and physical agents. Recently, the activity of Olive Leaf Extracts (OLE) was demonstrated against Acanthamoeba species. However, the molecules involved in this activity were not identified and/or evaluated. Therefore, the aim of this study was to evaluate the activity of the main molecules which are present in OLE and secondly to study their mechanism of action in Acanthamoeba. Among the tested molecules, the observed activities ranged from an IC50 of 6.59 in the case of apigenine to an IC50 > 100 μg/ml for other molecules. After that, elucidation of the mechanism of action of these molecules was evaluated by the detection of changes in the phosphatidylserine (PS) exposure, the permeability of the plasma membrane, the mitochondrial membrane potential and the ATP levels in the treated cells. Vanillic, syringic and ursolic acids induced the higher permeabilization of the plasma membrane. Nevertheless, the mitochondrial membrane was altered by all tested molecules which were also able to decrease the ATP levels to less than 50% in IC90 treated cells after 24 h. Therefore, all the molecules tested in this study could be considered as a future therapeutic alternative against Acanthamoeba spp. Further studies are needed in order to establish the true potential of these molecules against these emerging opportunistic pathogenic protozoa.

Among neglected tropical diseases, leishmaniasis is one of the leading causes, not only of deaths but also of disability-adjusted life years. This disease, caused by protozoan parasites of the genus Leishmania, triggers different clinical manifestations, with cutaneous, mucocutaneous, and visceral forms. As existing treatments for this parasitosis are not sufficiently effective or safe for the patient, in this work, different sesquiterpenes isolated from the red alga Laurencia johnstonii have been studied for this purpose. The different compounds were tested in vitro against the promastigote and amastigote forms of Leishmania amazonensis. Different assays were also performed, including the measurement of mitochondrial potential, determination of ROS accumulation, and chromatin condensation, among others, focused on the detection of the cell death process known in this type of organism as apoptosis-like. Five compounds were identified that displayed leishmanicidal activity: laurequinone, laurinterol, debromolaurinterol, isolaurinterol, and aplysin, showing IC50 values against promastigotes of 1.87, 34.45, 12.48, 10.09, and 54.13 µM, respectively. Laurequinone was the most potent compound tested and was shown to be more effective than the reference drug miltefosine against promastigotes. Different death mechanism studies carried out showed that laurequinone appears to induce programmed cell death or apoptosis in the parasite studied. The obtained results underline the potential of this sesquiterpene as a novel anti-kinetoplastid therapeutic agent.

Acanthamoeba is a ubiquitous genus of amoebae that can trigger a severe and progressive ocular disease known as Acanthamoeba Keratitis (AK). Furthermore, current treatment protocols are based on the combination of different compounds that are not fully effective. Therefore, an urgent need to find new compounds to treat Acanthamoeba infections is clear. In the present study, we evaluated staurosporine as a potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model, and a comparative proteomic analysis was conducted to elucidate a mechanism of action. The obtained results indicate that staurosporine altered the conformation of actin and tubulin in treated trophozoites of A. castellanii. In addition, proteomic analysis of treated trophozoites revealed that this molecule induced overexpression and a downregulation of proteins related to key functions for Acanthamoeba infection pathways. Additionally, the ex vivo assay used validated this model for the study of the pathogenesis and therapies of AK. Finally, staurosporine eliminated the entire amoebic population and prevented the adhesion and infection of amoebae to the epithelium of treated mouse corneas.