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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.

Giant viruses ( Nucleocytoviricota ) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria . This genus comprises the notorious human pathogen Naegleria fowleri , the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae , and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri . This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri . This is the first report on a virus infecting the amoeboflagellate Naegleria , including the lethal human pathogen N. fowleri . The new virus isolate, Catovirus naegleriensis (Naegleriavirus, NiV), shows hallmarks of giant viruses (Nucleocytoviricota) and unique adaptations to its protist host.

Current therapies for Primary Amoebic Meningoencephalitis (PAM) present several limitations; consequently, there is an urgent need to develop new therapeutic agents. In this regard, we undertook bioassay-guided isolation of Mentha rotundifolia leaves which allowed the identification of ursolic acid (1) as the main bioactive metabolite against two ATCC strains of Naegleria fowleri (ATCC® 30808TM and ATCC® 30215TM). Moreover, ten ursolic acid derivatives (2–11) were synthesized through esterification and nucleophilic substitution to obtain halo or amino ester derivatives. Among these derivatives, compound 7 exhibited the highest activity against the N. fowleri ATCC® 30808TM strain with an IC50 value of 28.66 µM, whereas compound 10 showed the top activity against the N. fowleri ATCC® 30215TM strain with an IC50 of 7.61 µM, surpassing the efficacy of the reference drug, miltefosine. Both compounds, 7 and 10, showed a good selectivity index and hence low toxicity in vitro. Structure–activity relationship studies revealed that the type of acylating agent played a crucial role in the activity. Furthermore, both compounds induced autophagy and apoptosis-like processes in both treated N. fowleri strains. These results highlight the potential of ursolic acid-related triterpenoids as drug scaffolds and identify M. rotundifolia as a promising natural source of amoebicidal agents against PAM.

Cutaneous squamous cell carcinoma (CSCC) is one of the most common cancers in the skin. CSCC belongs to the non-melanoma skin cancers, and its incidence is increasing every year around the world. The principal routes of tumor progression are related to angiogenesis and lymphangiogenesis. In this study, we assess the gene expression of the relevant biomarkers of both routes in 49 formalin-fixed paraffin-embedded (FFPE) CSCC samples in an attempt to determine a molecular profile that correlates with the tumor progression and disease-free survival (DFS). The results were enhanced by a posttranscriptional analysis using an immunofluorescence assay. Overexpression of the vascular endothelial growth factor C (VEGFC) gene was found in patients with tumor progression (p = 0.022) and in patients with perineural invasion (p = 0.030). An increased expression of protein VEGFC in samples with tumor progression supported these results (p = 0.050). In addition, DFS curves showed differences (p = 0.027) for tumors with absent-low VEGFC expression versus those with high levels of VEGFC expression. No significant influence on DFS was detected for the remaining analyzed genes. VEGFC expression was found to be a risk factor in the disease progression (HR = 2.675; 95% CI: 1.089–6.570; p = 0.032). Our main results suggest that VEGFC gene expression is closely related to tumor progression, DFS, and the presence of perineural invasion.

Natural products have long been recognized as invaluable resources in drug discovery. Essential oils have attracted widespread attention due to their broad spectrum of biological activities. Herein, we report the anti-kinetoplastid activity of Aloysia citrodora leaf essential oil through a bioassay-guided fractionation method against the etiological agents of Chagas disease and leishmaniasis. This approach has led to the isolation and structural identification of compound 1 (citral) as the main active constituent, with IC50 values of 8.47 μM against Leishmania amazonensis and 12.90 μM against Trypanosoma cruzi. In addition, eight compounds (2–9) were synthesized and evaluated. Among these, citral 2,4-dinitrophenylhydrazone (9) exhibited the highest anti-kinetoplastid activity, with an IC50 value of 10.62 μM against L. amazonensis, displaying a similar biological profile to citral and the reference drug. Structure–activity relationship studies revealed that the type of Schiff base and acylating agent played a crucial role in the activity. Mechanism of action studies demonstrated that compound 9 directly targets the apoptotic pathway, inducing programmed cell death through selective pathway inhibition. This work underscores the potential of A. citrodora essential oil and its compounds as prospective therapeutic leads against neglected tropical diseases.

Despite intensified efforts to develop an effective antibiotic, S. aureus is still a major cause of mortality and morbidity worldwide. The multidrug resistance of bacteria has considerably increased the difficulties of scientific research and the concomitant emergence of resistance is to be expected. In this study we have investigated the in vitro activity of 15 ethanol extracts prepared from Moroccan medicinal plants traditionally used for treatment of skin infections. Among the tested species I. viscosa, C. oxyacantha, R. tinctorum, A. herba alba, and B. hispanica showed moderate anti-staphylococcal activity. However, R. alaternus showed promising growth-inhibitory effects against specific pathogenic bacteria especially methicillin-susceptible Staphylococcus aureus Panton-Valentine leucocidin positive (MSSA-PVL) and methicillin-resistant S. aureus (MRSA). The bioguided fractionation of this plant using successive chromatographic separations followed by nuclear magnetic resonance (NMR) and mass spectrometry (MS) including EIMS and HREIMS analysis yielded the emodin (1) and kaempferol (2). Emodin being the most active with MICs ranging between 15.62 and 1.95 µg/mL and showing higher activity against the tested strains in comparison with the crude extract, its mechanism of action and the structure-activity relationship were interestingly discussed. The active compound has not displayed toxicity toward murine macrophage cells. The results obtained in the current study support the traditional uses of R. alaternus and suggest that this species could be a good source for the development of new anti-staphylococcal agents.

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.

In this study, we investigated the presence and diversity of FLA in 62 environmental samples collected across Tenerife, Canary Islands, Spain including agricultural and playground soils, and on double treated water from public refrigerated fountains. Amoebae were isolated by culturing processed samples onto 2% Non-Nutrient Agar plates (NNA) which were checked daily for further processing up to molecular characterization. In this case, two approaches for molecular identification were assessed: direct multiplex qPCR targeting four potentially pathogenic FLA (Acanthamoeba spp., Vermamoeba vermiformis, Naegleria fowleri, and Balamuthia mandrillaris) DNA, and culture-based isolation followed by standard PCR and sequence analysis. Regarding qPCR results, 72.6% (45/62) of the samples were positive for at least one FLA, with V. vermiformis (37/62) and Acanthamoeba spp. (34/62) being the most frequent. Moreover, B. mandrillaris was detected for the first time in the Canary Islands in 6 out of 62 samples. Results from standard PCR from cultured isolates confirmed the presence of Acanthamoeba (mainly genotype T4) and Vermamoeba and also allowed the identification of Vahlkampfia and Vannella genera, as well as the genus Rhogostoma—its first report in the Canary Islands. Thermotolerance and osmotolerance assays were performed on Acanthamoeba spp. and, innovatively, on V. vermiformis isolates. Both were capable of surviving at 37 °C and during incubation with 0.5 M mannitol, suggesting potential pathogenicity. However, growth was significantly impaired under harsher conditions (42 °C and 1 M mannitol). These findings underscore the widespread occurrence of FLA in public and agricultural environments in Tenerife and highlight their potential risk to public health. Their ability to act as carriers of pathogenic bacteria/viruses further reinforces the need for routine surveillance and preventive measures in the environment.

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.

Leishmaniasis and Chagas disease are parasitic diseases considered to be among the most important neglected diseases, with implications for both developed and developing countries. Currently, there are no effective therapeutic treatments for these diseases due to challenges in drug administration, high toxicity, high costs, and drug resistance. In this study, a series of eleven thymol derivatives were designed, synthesized, and evaluated for their in vitro kinetoplastid activity against Leishmania amazonensis and Trypanosoma cruzi, as well as their cytotoxicity against a murine macrophage cell line. The most active compounds, thymol anysoate (9) and thymol picolinate (10), displayed the highest kinetoplastid activity with IC50 values of 22.87 and 25.16 µM against L. amazonensis and T. cruzi, respectively. Notably, both compounds demonstrated an excellent selectivity index against the mammal cell line. Structure–activity relationship studies revealed that the ester group plays a crucial role in activity. The most promising derivatives, 9 and 10, activate autophagy and apoptosis-like processes in the treated cells. Atomic force microscopy observations showed that derivative 9 induces the formation of cytoplasmic vacuoles, indicating an autophagic process, and drug-likeness analysis revealed that it meets all the pharmacokinetic criteria. Overall, these results highlight derivative 9 as a potential lead compound for the development of new drugs for the treatment of Trypanosomatidae infections and warrants further studies to elucidate the cell death cascade involved.