Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Fluconazole Alters the Polysaccharide Capsule of Cryptococcus gattii and Leads to Distinct Behaviors in Murine Cryptococcosis

Abstract Cryptococcus gattii is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of C. gattii was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01 F , that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01 F were altered by fluconazole. L27/ 01 F presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01 F did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/ 01 F presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01 F . Our results suggest that stress to fluconazole alters the capsule of C. gattii and influences the clinical manifestations of cryptococcosis

Fluconazole Alters the Polysaccharide Capsule of <i>Cryptococcus gattii</i> and Leads to Distinct Behaviors in Murine Cryptococcosis

<div><p><i>Cryptococcus gattii</i> is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of <i>C. gattii</i> was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01<b>_F</b>, that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01_F were altered by fluconazole. L27/01<b>_F</b> presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01<b>_F</b> did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/01<b>_F</b> presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01<b>_F</b>. Our results suggest that stress to fluconazole alters the capsule of <i>C. gattii</i> and influences the clinical manifestations of cryptococcosis.</p></div

Reduced susceptibility to fluconazole leads to very low <i>CAP59</i> and <i>CAP64</i> gene expression and affects physical properties of the GXM capsule.

<p>Microscopic visualization with India ink stain of L27/01 (<b>A</b>) and L27/01<b>_F</b> strains (<b>B</b>). Capsule size of L27/01 and L27/01<b>_F</b> strains (<b>C</b>). SEM analysis of L27/01 (<b>D</b>) and L27/01<b>_F</b> (<b>E</b>) strains. Zeta potential of capsular and secreted PS of cells (<b>F</b>). Cell suspensions were analyzed with an AXIOPLAN (Carl Zeiss) fluorescence microscope. Images were processed using ImageJ. Staining of L27/01 (<b>G</b>) and L27/01<b>_F</b> (<b>H</b>) strains with mAb 18B7 showing differences in epitope presentation in the PS capsule between strains. Ratio of gene expression of <i>CAP59</i>, <i>CAP64</i> and <i>UXS1</i> to actin (<b>I</b>). Size distribution of PS fibers from capsular (<b>J</b> and <b>K</b>) and exo-PS samples (<b>L</b> and <b>M</b>) of L27/01 and L27/01<b>_F</b> strains, respectively. Bar = 10 µm (A) and (D). Bar = 5 µm (B), (E), (G) and (H). *P<0.05 was considered to be significant.</p

Synopsis of the methodology.

<p>Fluconazole-resistant strain selection (<b>A–F</b>). After determining the MIC of fluconazole on SDA, an average of five colonies obtained from the highest fluconazole concentration were selected for culture on SDA plates supplemented with this drug. L27/01 (<b>A</b>) was the strain able to grow at the highest fluconazole concentration (<b>B</b>) and was chosen for culture on SDA plates supplemented with this drug. This strain was cultured in solid medium with increasing concentrations (<b>B–C</b>) of fluconazole until growth ceased at 100 µg/mL (<b>D</b>). To verify the maintenance of resistance to fluconazole and cross-resistance between this drug and amphotericin B, the selected strain was cultured in SDA without drug every 48 h 170 times (<b>E</b>), and the MIC test was performed by microdilution every 5 subcultures (<b>F</b>). Colonies grown at 95 µg/mL were maintained in this concentration, and the strain was named “L27/01<b>_F</b>” (<b>G</b>). L27/01 strain grew in the absence of drug (<b>H</b>). The genetic similarity between L27/01 and L27/01<b>_F</b> strains was evaluated by randomly amplified polymorphic DNA (RAPD)-PCR, PFGE and ITS Sequencing. <i>CAP59, CAP64, ARF-1, ERG11, UXS-1</i> levels by real-time PCR were evaluated. Lipid evaluation was performed to compare the ergosterol content of L27/01 and L27/01<b>_F</b> cell membranes. Also, Urease and Laccase activities of L27/01 and L27/01<b>_F</b> strains were determined (<b>I</b>). Evaluation of murine cryptococcosis after inoculation with L27/01 or L27/01<b>_F</b> strain: survival curve (<b>J</b>). Investigation of whether fluconazole affects the polysaccharide (PS) capsule (<b>K</b>). The phagocytosis assay was performed to assess the influence of PS capsules from L27/01 and L27/01<b>_F</b> strains on phagocytosis and intracellular proliferation rate (IPR) in murine peritoneal macrophages from C57BL/6 mice (<b>L</b>). Cryptococcal cell dissemination, immune response and behavioral alterations (<b>M</b>). SDA: Sabouraud Dextrose Agar. MIC: Minimum inhibitory concentration. PFGE: Pulsed field gel electrophoresis. CFU: Colony forming units. BALF: bronchoalveolar lavage fluid. MPO: Myeloperoxidase activity. i.t.: intratracheal infection. PS: polysaccharide.</p

L27/01 alters the clinical manifestations of cryptococcosis.

<p>Assessment of behavioral performance of not infected (NI) mice and those infected with L27/01 (▪) or L27/01<b>_F</b> (▴) strains in five distinct functional categories of the SHIRPA battery (n = 6) (<b>A</b>–<b>E</b>). Mice were monitored daily for: muscle tone and strength (<b>A</b>); motor behavior (<b>B</b>); neuropsychiatric state (<b>C</b>) autonomous function (<b>D</b>); reflex and sensory function (<b>E</b>) (n = 6). The results are presented mean ± SEM of eight animals in each group. Scores of the infected groups were compared with the uninfected group by the Wilcoxon matched test. *The vertical dotted line indicates a significant difference from the appointed day. P<0.05 is considered to be significant.</p

The L27/01_F strain is more readily internalized by macrophages, but is not able to proliferate as well as the L27/01 strain.

<p>The phagocytosis assay was performed <i>in vivo</i> by counting internalized yeast cells in BALF cells, and <i>in vitro</i> with murine peritoneal macrophages infected with <i>C. gattii</i>. The influence of L27/01 and L27/01<b>_F</b> polysaccharide capsules (L27/01 PS and L27/01<b>_F</b> PS) on intracellular proliferation rate (IPR), reactive oxygen species, superoxide-dismutase (SOD) and peroxidase (PER) activities was also evaluated. White bars refer to macrophages (control), black bars to L27/01, grey bars to L27/01<b>_F</b> and beige bars to PS alone without yeasts. Phagocytic index (<b>A</b>). IPR (<b>B</b>). Reactive oxygen species expressed in arbitrary units of fluorescence (AU) (<b>C</b>). SOD (U mg⁻¹ protein) (<b>D</b>) and PER (nmol/min mg⁻¹ protein) (<b>E</b>) activities after phagocytosis. The data are presented as the mean ± S.E.M. of two independent experiments in triplicate. *P<0.05. Symbols indicate absence (–) of PS in the phagocytosis assay.</p