Clinical factors associated with systemic sporotrichosis in Brazil.

BACKGROUND: Systemic sporotrichosis occurs when organs, other than subcutaneous tissues and lymph nodes, are infected. Interestingly, systemic sporotrichosis in apparently immunocompetent individuals is increasing in Brazil, but data on clinical manifestations and risk factors are scarce. Most of the existing data on such condition relate to people living with HIV. We aimed to study the risk factors associated with systemic sporotrichosis among HIV-negative and HIV-positive patients. METHODS: We performed a retrospective cross-sectional study with 80 patients from Brazil, diagnosed between 2014 and 2021. The association between disease classification, clinical presentation and risk factors were analysed by logistic regression. RESULTS: Of the 80 patients, 29 (36.3%) presented with systemic sporotrichosis. All HIV-positive patients developed the systemic form, with increased mortality (p = .002). Alcohol ingestion (p = .009) and diabetes (p = .010) were associated with systemic disease. Alcohol ingestion seemed to favour pulmonary infection (p = .013) and, diabetes favoured osteoarticular (p = .009) and ocular involvement (p = .033). The occurrence of fever (p = .001) and weight loss (p = .006) were significantly associated with systemic sporotrichosis, while meningeal involvement (p = .001) increased mortality risk. Nine (11.3%) patients died from sporotrichosis. The presence of fungal structures in the mycological examination of the patients' lesions were associated with the systemic form (p = .017) and death (p = .002). CONCLUSION: Our study points to the factors that drive systemic sporotrichosis other than HIV, such as alcohol ingestion and diabetes. Considering the high number of patients presenting severe sporotrichosis, the recognising these factors may contribute to timely diagnosis and proper treatment.

Fungicide effects on human fungal pathogens: Cross-resistance to medical drugs and beyond.

Fungal infections are underestimated threats that affect over 1 billion people, and Candida spp., Cryptococcus spp., and Aspergillus spp. are the 3 most fatal fungi. The treatment of these infections is performed with a limited arsenal of antifungal drugs, and the class of the azoles is the most used. Although these drugs present low toxicity for the host, there is an emergence of therapeutic failure due to azole resistance. Drug resistance normally develops in patients undergoing azole long-term therapy, when the fungus in contact with the drug can adapt and survive. Conversely, several reports have been showing that resistant isolates are also recovered from patients with no prior history of azole therapy, suggesting that other routes might be driving antifungal resistance. Intriguingly, antifungal resistance also happens in the environment since resistant strains have been isolated from plant materials, soil, decomposing matter, and compost, where important human fungal pathogens live. As the resistant fungi can be isolated from the environment, in places where agrochemicals are extensively used in agriculture and wood industry, the hypothesis that fungicides could be driving and selecting resistance mechanism in nature, before the contact of the fungus with the host, has gained more attention. The effects of fungicide exposure on fungal resistance have been extensively studied in Aspergillus fumigatus and less investigated in other human fungal pathogens. Here, we discuss not only classic and recent studies showing that environmental azole exposure selects cross-resistance to medical azoles in A. fumigatus, but also how this phenomenon affects Candida and Cryptococcus, other 2 important human fungal pathogens found in the environment. We also examine data showing that fungicide exposure can select relevant changes in the morphophysiology and virulence of those pathogens, suggesting that its effect goes beyond the cross-resistance.

Synthesis of vanillin derivatives with 1,2,3-triazole fragments and evaluation of their fungicide and fungistatic activities.

Vanillin is the main component of natural vanilla extract and is responsible for its flavoring properties. Besides its well-known applications as an additive in food and cosmetics, it has also been reported that vanillin can inhibit fungi of clinical interest, such as Candida spp., Cryptococcus spp., Aspergillus spp., as well as dermatophytes. Thus, the present work approaches the synthesis of a series of vanillin derivatives with 1,2,3-triazole fragments and the evaluation of their antifungal activities against Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, Cryptococcus gattii, Trichophyton rubrum, and Trichophyton interdigitale strains. Twenty-two vanillin derivatives were obtained, with yields in the range of 60%-91%, from copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction between two terminal alkynes prepared from vanillin and different benzyl azides. In general, the evaluated compounds showed moderate activity against the microorganisms tested, with minimum inhibitory concentration (MIC) values ranging from 32 to >512 µg mL-1 . Except for compound 3b against the C. gattii R265 strain, all vanillin derivatives showed fungicidal activity for the yeasts tested. The predicted physicochemical and ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties for the compounds indicated favorable profiles for drug development. In addition, a four-dimensional structure-activity relationship (4D-SAR) analysis was carried out and provided useful insights concerning the structures of the compounds and their biological profile. Finally, molecular docking calculations showed that all compounds bind favorably at the lanosterol 14α-demethylase enzyme active site with binding energies ranging from -9.1 to -12.2 kcal/mol.

Cryptococcus gattii polysaccharide capsule: An insight on fungal-host interactions and vaccine studies.

Cryptococcus neoformans and C. gattii complexes are the main causative agents of cryptococcosis, a neglected disease with high lethality. The capsule, composed predominantly of the capsular polysaccharide (CP) GXM, is the main virulence factor of this pathogen. The role of CP is well described for C. neoformans and; however, there is a scarcity of studies focused on C. gattii, especially in the context of the fungal-host interaction. Understanding how the immune system recognizes C. gattii can generate meaningful information for diagnosing, preventing, and treating cryptococcosis. In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2021. 51: 2281-2295], Ueno et al. demonstrate that CP inhibits C. gattii recognition by CD11b. In this commentary, we highlight the importance of deeply understanding the role of C. gattii CP during infection and how this knowledge would influence the strategies to develop new vaccines against cryptococcosis.

Reactive oxygen and nitrogen species are crucial for the antifungal activity of amorolfine and ciclopirox olamine against the dermatophyte Trichophyton interdigitale.

Onychomycosis is a nail infection caused by Trichophyton interdigitale and other fungi, which can be treated with topical amorolfine (AMR) and ciclopirox olamine (CPX). Although these drugs are widely used, little is known about the role of reactive oxygen (ROS) and nitrogen (RNS) in their mechanism of action. To better understand the effects of AMR and CPX in dermatophytes, we evaluated whether they act through the production of ROS and peroxynitrite (PRN). We tested a set of strains, all susceptible to AMR and CPX, and these antifungals significantly reduced T. interdigitale viability within 24 h. This effect occurred concomitantly with reduced ergosterol, increased production of ROS and PRN, and consequently increased lipid peroxidation. Together, these mechanisms lead to cell damage and fungal death. These fungicidal effects were abolished when PRN and superoxide scavengers were used in the assays, demonstrating the role of these species in the mechanism of action. We also studied the antioxidant system when T. interdigitale was exposed to AMR and CPX. Interestingly, superoxide dismutase and catalase inhibition lead to altered ROS and PRN production, lipid peroxidation, and ergosterol levels. In fact, the combination of AMR or CPX with a superoxide dismutase inhibitor was antagonistic. Together, these data demonstrate the importance of ROS and PRN in the antifungal action of AMR and CPX against the evaluated T. interdigitale strains. LAY SUMMARY: Onychomycosis is a nail infection, which can be treated with amorolfine and ciclopirox olamine. Here we demonstrate that these drugs exhibit antifungal activity also through the production of oxidative and nitrosative radicals.

Exposure to itraconazole influences the susceptibility to antifungals, physiology, and virulence of Trichophyton interdigitale.

Dermatophytosis is the most common human skin infection worldwide caused by dermatophytes, such as Trichophyton interdigitale and Trichophyton rubrum. Itraconazole (ITZ) is one of the main antifungals used to treat these infections. However, especially for onychomycosis, the treatment requires long-term regimens, increasing the possibility of drug resistance. We evaluated the effects of ITZ in the physiology, virulence, and interaction of T. interdigitale with phagocytes and mice cutaneous infection. In a screening test, fungal growth in the presence of ITZ led to the spontaneous selection of less susceptible T. interdigitale and T. rubrum strains. Interestingly, this phenotype was permanent for some T. interdigitale strains. Then, we studied three T. interdigitale strains: one susceptible and two ITZ-adapted. The ITZ-adapted strains were also less susceptible to the cell wall and membrane stressors, suggesting a multidrug resistance (MDR) phenotype associated with the increased ERG11 and MDR3 expression. These strains also presented substantial alterations in ergosterol content, lipid peroxidation, biofilm, and extracellular matrix production. During interaction with macrophages, ITZ-adapted strains were less engulfed but increased the intracellular oxidative and nitrosative bursts. In addition, ITZ-adapted strains presented a reduced ability to grow in a murine model of dermatophytosis, although causing the same tissue damage as the parental strain. In conclusion, the T. interdigitale ITZ adaptation increases tolerance to antifungals and alters the interaction with macrophages and a mammalian host. We hypothesized that successive exposure to ITZ may influence the emergence of adapted strains and lead to the recalcitrance of dermatophytosis.

Morphospace analysis leads to an evo-devo model of digit patterning.

Biological forms occupy a constrained portion of theoretical morphospaces. Developmental models accounting for empirical morphospaces are necessary to achieve a better understanding of this phenomenon. We analyzed the phalangeal formulas (PFs) in lizards and relatives' hands by comparing them with a set of simulated PFs that compose a theoretical morphospace. We detected that: (1) the empirical morphospace is severely limited in size, (2) the PFs comply with two properties of phalangeal count per digit, namely the ordering rule (DI ≤ DII ≤ DIII ≤ DIV ≥ DV), and the contiguity relationship (neighbor digits differ on average in one phalanx), (3) the totality of the PFs can be categorized into four categories of hands aligned along a feasibility gradient. We also reconstructed the evolution of PFs and found a stepwise trajectory from the plesiomorphic PF towards reduced conditions. Finally, we propose a developmental model as the generative mechanism behind the PFs. It is consistent with the bulk of evidence managed and involves an ordered digit primordia initialization timed with periodic signals of joint formation coming from digit tips. Our approach is also useful to address the study of other meristic sequences in nature such as dental, floral, and branchial formulas.

17-ß-Estradiol increases macrophage activity through activation of the G-protein-coupled estrogen receptor and improves the response of female mice to Cryptococcus gattii.

Cryptococcus gattii (Cg) is one of the agents of cryptococcosis, a severe systemic mycosis with a higher prevalence in men than women, but the influence of the female sex hormone, 17-ß-estradiol (E2), on cryptococcosis remains unclear. Our study shows that female mice presented delayed mortality, increased neutrophil recruitment in bronchoalveolar lavage fluid, and reduced fungal load after 24 hr of infection compared to male and ovariectomised female mice (OVX). E2 replacement restored OVX female survival. Female macrophages have more efficient fungicidal activity, which was increased by E2 and reversed by the antagonist of G-protein-coupled oestrogen receptor (GPER), which negatively modulates PI3K activation. Furthermore, E2 induces a reduction in Cg cell diameter, cell charge, and antioxidant peroxidase activity. In conclusion, female mice present improved control of Cg infection, and GPER is important for E2 modulation of the female response.

Cycloheximide can distort measurements of mRNA levels and translation efficiency.

Regulation of the efficiency with which an mRNA is translated into proteins represents a key mechanism for controlling gene expression. Such regulation impacts the number of actively translating ribosomes per mRNA molecule, referred to as translation efficiency (TE), which can be monitored using ribosome profiling and RNA-seq, or by evaluating the position of an mRNA in a polysome gradient. Here we show that in budding yeast, under nutrient limiting conditions, the commonly used translation inhibitor cycloheximide induces rapid transcriptional upregulation of hundreds of genes involved in ribosome biogenesis. Cycloheximide also prevents translation of these newly transcribed messages, leading to an apparent drop in TE of these genes under conditions that include key transitions during the yeast metabolic cycle, meiosis, and amino acid starvation; however, this effect is abolished when cycloheximide pretreatment is omitted. This response requires TORC1 signaling, and is modulated by the genetic background as well as the vehicle used to deliver the drug. The present work highlights an important caveat to the use of translation inhibitors when measuring TE or mRNA levels, and will hopefully aid in future experimental design as well as interpretation of prior results.

Acanthamoeba castellanii as an alternative interaction model for the dermatophyte Trichophyton rubrum.

BACKGROUND: Trichophyton rubrum (Tr) is the main aetiological agent of human dermatophytosis, being isolated from the environment and keratinised tissues. In the environment, Tr can interact with other organisms, such as free-living amoebas (FLA), which can act as an alternative host system to study the interaction between microbes and phagocytic cells. OBJECTIVES: To characterise the Acanthamoeba castellanii (ALX)-Tr interaction. METHODS: Interaction was characterised in three conditions: trophozoites (PYG), late (PYG/NES) and early (NES) encystation stimulus, evaluating encystation kinetics, phagocytosis, exocytosis and fungicidal activity dynamics. RESULTS: Tr was able to induce ALX encystation and be internalised by ALX. The number of internalised conidia was high at 1 hour, and ALX presented fungicidal activity with increased intracellular ROS production and exocytosis. In PYG/NES, phagocytosis and ROS production were reduced, with decreased ALX's fungicidal activity. However, in NES there was an increased fungal engulfment, and a reduced ROS production and higher fungal burden. Furthermore, exogenous mannose decreased phagocytosis of Tr conidia, and divalent cations induced ROS production and increased ALX's fungicidal activity. Interestingly, phagocytosis was reduced in the presence of cytoskeleton inhibitor, but exocytosis was increased, suggesting that Tr conidia may have alternative pathways to escape ALX's cells. CONCLUSION: A castellanii is a proper model for studying Tr-FLA interaction, since ALX can engulf, produce ROS and kill Tr, and all these parameters are influenced by an encystation stimulus and divalent cations. Moreover, this interaction is likely to occur in the environment implicating in the adaptation to environmental stressful conditions in both organisms.

Occurrence, antifungal susceptibility, and virulence factors of opportunistic yeasts isolated from Brazilian beaches.

BACKGROUND: Opportunistic pathogenic yeast species are frequently associated with water habitats that have pollution sources of human or animal origin. Candida albicans has already been suggested as a faecal indicator microorganism for aquatic environments. OBJECTIVES: The goal of this study was to investigate the occurrence of C. albicans and other opportunistic yeasts in sand and seawater samples from beaches in Brazil to assess their correlation with Escherichia coli, and to characterise the pathogenic potential of the yeast isolates. METHODS: Opportunistic species (yeasts that grow at 37ºC) were isolated from sand and seawater samples from eight beaches in Brazil during the summer and the winter. Opportunistic yeast species were evaluated for their susceptibility to antifungal drugs, virulence factors, and the in vitro and in vivo biofilm formation. Strains were selected to carry out virulence tests using BALB/c mice. FINDINGS: Several water samples could be classified as inappropriate for primary contact recreation in relation to E. coli densities. C. albicans was isolated in low densities. Of the 144 opportunistic yeasts evaluated, 61% displayed resistance or dose-dependent sensitivity to at least one tested drug, and 40% produced proteinase. Strains of C. albicans and Kodamaea ohmeri exhibited the highest rates of adhesion to buccal epithelial cells. All the C. albicans strains that were tested were able to undergo morphogenesis and form a biofilm on catheter fragments in both in vitro and in vivo experiments. It was possible to confirm the pathogenic potential of three of these strains during the disseminated infection test. MAIN CONCLUSIONS: The identification of opportunistic yeast species in seawater and sand samples from Brazilian beaches suggest a potential risk to the health of people who use these environments for recreational purposes.

Heteroresistance and fungi.

The concept of heteroresistance refers to the heterogeneous susceptibility to an antimicrobial drug in a microorganism population, meaning that some clones may be resistant and others are susceptible. This phenomenon has been widely studied in bacteria, but little attention has been given to its expression in fungi. We review the available literature on heteroresistance in fungi and invite the reader to recognise this phenomenon as a fungal mechanism to adapt to environmental stress, which may interfere both in resistance and virulence. Finally, heteroresistance may explain the treatment failures to eradicate mycosis in some patients treated with a seemingly appropriate antifungal.

Prevalence of hypothyroidism in patients with frozen shoulder.

BACKGROUND: Hypothyroidism and frozen shoulder (FS) have been associated, although this relationship remains uncertain. The main objective of this study was to determine the prevalence of hypothyroidism in patients with FS. METHODS: A case-control study was performed to compare FS patients (cases) with patients who visited an orthopedic service for other clinical conditions (controls). FS was diagnosed according to specific criteria based on anamnesis, physical examination, and shoulder radiographs. A specific questionnaire was applied, and measurements of serum thyroid-stimulating hormone (TSH) and free tetraiodothyronine were performed in all subjects. RESULTS: We evaluated 401 shoulders from 93 FS patients and 151 controls. The prevalence of hypothyroidism diagnosis was significantly higher in the FS group (27.2% vs. 10.7%; P = .001). There was also a tendency for higher prevalence of bilateral FS among patients with elevated TSH levels (P = .09). Mean serum TSH levels were higher in patients with bilateral FS compared with those with unilateral compromise (3.39 vs. 2.28; P = .05) and were higher in patients with severe FS compared with those with mild and moderate FS together (3.15 vs. 2.21; P = .03). Multivariate analysis showed that FS was independently related to a diagnosis of hypothyroidism (odds ratio, 3.1 [1.5-6.4]; P = .002). There was a trend toward independent association between high serum TSH levels and both severe (odds ratio, 3.5 [0.8-14.9]; P = .09) and bilateral (odds ratio, 11.7 [0.9-144.8]; P = .05) compromise. CONCLUSION: The prevalence of hypothyroidism was significantly higher in FS patients than in controls. The results suggest that higher serum TSH levels are associated with bilateral and severe cases of FS.

Demonstration Using Field Collections that Argentina Fall Armyworm Populations Exhibit Strain-specific Host Plant Preferences.

Spodoptera frugiperda, the fall armyworm, is a major economic pest throughout the Western Hemisphere of corn (maize), cotton, sorghum, and a variety of agricultural grasses and vegetable crops. Studies in the United States, the Caribbean, and Brazil demonstrated the existence of two subpopulations (previously designated "host strains") that differ in their choice of plant host. Specifically, the corn strain is preferentially found in corn and sorghum, while the rice strain is dominant in rice, turf grass, and alfalfa. However, inconsistent results were reported in surveys of fall armyworm in Argentina, with some indicating that the host plant preferences of the two strains might be compromised or even nonexistent. If correct, this would complicate efforts to control this pest by considerably expanding the range of habitats that would have to be considered as potential sources for fall armyworm infestations in specific crops. A reexamination of Argentine fall armyworm, this time with field collections rather than the laboratory colonies used in previous studies, confirmed the existence of the two strains and their host preferences. Specifically, the corn strain was consistently the majority population infesting corn and was usually so in sorghum, while the rice strain was predominant in pasture/turf grasses and alfalfa. The one outlier was a collection from rice, which had a corn strain majority. Overall, the data were generally consistent with strain behaviors observed in other areas of the Western Hemisphere.

Interplay between postcranial morphology and locomotor types in Neotropical sigmodontine rodents.

Sigmodontine rats are one of the most diverse components of the Neotropical mammal fauna. They exhibit a wide ecological diversity and a variety of locomotor types that allow them to occupy different environments. To explore the relationship between morphology and locomotor types, we analyzed traits of the postcranial osteology (axial and appendicular skeletons) of 329 specimens belonging to 51 species and 29 genera of sigmodontines exhibiting different locomotor types. In this work, postcranial skeletal characters of these rats are considered in an ecomorphological study for the first time. Statistical analyses showed that of the 34 osteological characters considered, 15 were related to the locomotor types studied, except for ambulatory. However, character mapping showed that climbing and jumping sigmodontines are the only taxa exhibiting clear adaptations in their postcranial osteology, which are highly consistent with the tendencies described in many other mammal taxa. Climbing, digging and swimming rats presented statistically differences in traits associated with their vertebral column and limbs, whereas jumping rats showed modifications associated with all the skeletal regions. Our data suggest that sigmodontine rats retain an all-purpose morphology that allows them to use a variety of habitats. This versatility is particularly important when considering the lack of specialization of sigmodontines for a specific locomotor mode. Another possible interpretation is that our dataset probably did not consider relevant information about these groups and should be increased with other types of characters (e.g. characters from the external morphology, myology, etc.).

Synthesis, antinociceptive activity and pharmacokinetic profiles of nicorandil and its isomers.

Nicorandil (N-(2-hydroxyethyl)nicotinamide nitrate) is an antianginal drug, which activates guanylyl cyclase and opens the ATP-dependent K(+) channels, actions that have been suggested to mediate its vasodilator activity. We synthesized nicorandil and its two isomers, which vary in the positions of the side chain containing the nitric oxide (NO) donor, and also their corresponding denitrated metabolites. The activities of these compounds were evaluated in an experimental model of pain in mice. Pharmacokinetic parameters of nicorandil and its isomers, as well as the plasma concentrations of the corresponding denitrated metabolites and also nicotinamide and nitrite were determined. Nicorandil exhibited the highest antinociceptive activity, while the ortho-isomer was the least active. Nicorandil and para-nicorandil, which induced higher plasma concentrations of nitrite, exhibited higher antinociceptive activity, which suggests that the release of NO may mediate this activity.

Teaching Video NeuroImage: Isolated Undulating Tongue Hyperkinesia Following a Basilar Stroke.

An 82-year-old man with a history of hypertension and coronary revascularization presented with sudden-onset right hemiparesis and disorientation lasting 5 hours. On admission, he was intubated because of gasping and a Glasgow Coma Scale of 3. Hemorrhagic stroke was suspected, but ruled out by the initial head CT, which revealed old cerebellar lacunae. The following day, the comatose, now unsedated patient exhibited tetraparesis; fixed, nonreactive pupils; and corneal reflex, but no oculocephalic reflex. Rhythmic undulating tongue movements without palatal or limb involvement were first observed (Video 1). EEG revealed no epileptiform activity. Follow-up head CT showed acute ischemic lesions in the thalamocapsular region, midbrain, and pons while angiotomography revealed distal basilar artery occlusion (Figure). Involuntary tongue movements, though rare, have been associated with various conditions such as stroke, trauma, and epilepsy.1,2 These movements may result from disinhibition within the inhibitory reticular formation projecting to hypoglossal neurons, suggesting the pontine reticular formation as a central pacemaker.2.

Influence of the agrochemical benomyl on Cryptococcus gattii-plant interaction in vitro and in vivo.

Cryptococcus gattii, an environmental fungus, is one of the agents of cryptococcosis. The influence of agrochemicals on fungal resistance to antifungals is widely discussed. However, the effects of benomyl (BEN) on fungal interaction with different hosts is still to be understood. Here we studied the influence of adaptation to BEN in the interaction with a plant model, phagocytes and with Tenebrio molitor. First, the strain C. gattii L24/01 non-adapted (NA), adapted (A) to BEN, and adapted with further culture on drug-free media (10p) interact with Nicotiana benthamiana, with a peak in the yeast burden on the 7th day post-inoculation. C. gattii L24/01 A and 10p provided lower fungal burden, but these strains increased cell diameter and capsular thickness after the interaction, together with decreased fungal growth. The strains NA and A showed reduced ergosterol levels, while 10p exhibited increased activity of laccase and urease. L24/01 A recovered from N. benthamiana was less engulfed by murine macrophages, with lower production of reactive oxygen species. This phenotype was accompanied by increased ability of this strain to grow inside macrophages. Otherwise, L24/01 A showed reduced virulence in the T. molitor larvae model. Here, we demonstrate that the exposure to BEN, and interaction with plants interfere in the morphophysiology and virulence of the C. gattii.

Towards a dynamic analysis of weighted networks in biogeography.

An improvement to the Network Analysis Method (NAM) in Biogeography based on weighted inference and dynamic exploration of sympatry networks is proposed. Intricate distributions of species result in a reticulated structure of spatial associations. Species are geographically connected through sympatry links forming an overall natural network in biogeography. Spatial records are the signals that provide evidence to infer these sympatry links in the network. Punctual data are independent of a priori area determination. NAM is oriented to detect groups of species embedded into the global network that are internally sustained by sympatric cohesiveness but weakly connected (or disconnected) to outgroup entities. These groups, called units of co-occurrence (UCs), are segregated through the iterative removal of intermediary species according to their betweenness scores. Instances of analysis of the original NAM are improved through the following changes and extensions: (i) inference of weighted sympatry networks using new measures sensitive to the strength of overlap and topological resemblance between set of points; (ii) construction of a basal network discriminating major from minor sympatry associations; (iii) evaluation of the entire process of iterative removal of intermediary species for the selection of UCs found on different subnetworks; (iv) network partitioning based on the intrinsic cohesiveness of the UCs; (v) production of a graphical tool (cleavogram) depicting the structural changes of the network along the removal process. Improvements are tested using real and hypothetical data sets. Resolution of patterns is notably increased due to a more accurate recognition of allopatric patterns and the possibility of segregating spatially overlapped UCs. As in original NAM, spatial expressions of UCs are building blocks for biogeography supported by strictly endemic and connected species through sympatry paths.

Antimalarials and amphotericin B interact synergistically and are new options to treat cryptococcosis.

Cryptococcus gattii and Cryptococcus neoformans are the main etiological agents of cryptococcosis, an invasive mycosis treated with amphotericin B, 5-fluorocytosine, and fluconazole. This limited arsenal is toxic and is associated with antifungal resistance. Cryptococcosis and malaria pathogens are eukaryotic organisms that have a high incidence in Sub-Saharan Africa. The antimalarials (ATMs) halofantrine (HAL) and amodiaquine (AQ) block Plasmodium heme polymerase, and artesunate (ART) induces oxidative stress. Considering that Cryptococcus spp. is susceptible to reactive oxygen species and that iron is essential for metabolism, the repurposing of ATMs for treating cryptococcosis was tested. ATMs reduced fungal growth, induced oxidative and nitrosative stresses, and altered ergosterol content, melanin production, and polysaccharide capsule size in C. neoformans and C. gattii, revealing a dynamic effect on fungal physiology. A comprehensive chemical-genetic analysis using two mutant libraries demonstrated that the deletion of genes involved in synthesizing components of the plasma membrane and cell wall, and oxidative stress responses are essential for fungal susceptibility to ATMs. Interestingly, the amphotericin B (AMB) fungicidal concentrations were ∼10 times lower when combined with ATMs, demonstrating a synergistic interaction. Further, the combinations showed reduced toxicity to murine macrophages. Finally, HAL+AMB and AQ+AMB efficiently reduced lethality and fungal burden in the lungs and brain in murine cryptococcosis. These findings provide perspectives for further studies with ATMs against cryptococcosis and other fungal infections.