The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference.

For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

Scientific novelty beyond the experiment.

Practical experiments drive important scientific discoveries in biology, but theory-based research studies also contribute novel-sometimes paradigm-changing-findings. Here, we appraise the roles of theory-based approaches focusing on the experiment-dominated wet-biology research areas of microbial growth and survival, cell physiology, host-pathogen interactions, and competitive or symbiotic interactions. Additional examples relate to analyses of genome-sequence data, climate change and planetary health, habitability, and astrobiology. We assess the importance of thought at each step of the research process; the roles of natural philosophy, and inconsistencies in logic and language, as drivers of scientific progress; the value of thought experiments; the use and limitations of artificial intelligence technologies, including their potential for interdisciplinary and transdisciplinary research; and other instances when theory is the most-direct and most-scientifically robust route to scientific novelty including the development of techniques for practical experimentation or fieldwork. We highlight the intrinsic need for human engagement in scientific innovation, an issue pertinent to the ongoing controversy over papers authored using/authored by artificial intelligence (such as the large language model/chatbot ChatGPT). Other issues discussed are the way in which aspects of language can bias thinking towards the spatial rather than the temporal (and how this biased thinking can lead to skewed scientific terminology); receptivity to research that is non-mainstream; and the importance of theory-based science in education and epistemology. Whereas we briefly highlight classic works (those by Oakes Ames, Francis H.C. Crick and James D. Watson, Charles R. Darwin, Albert Einstein, James E. Lovelock, Lynn Margulis, Gilbert Ryle, Erwin R.J.A. Schrödinger, Alan M. Turing, and others), the focus is on microbiology studies that are more-recent, discussing these in the context of the scientific process and the types of scientific novelty that they represent. These include several studies carried out during the 2020 to 2022 lockdowns of the COVID-19 pandemic when access to research laboratories was disallowed (or limited). We interviewed the authors of some of the featured microbiology-related papers and-although we ourselves are involved in laboratory experiments and practical fieldwork-also drew from our own research experiences showing that such studies can not only produce new scientific findings but can also transcend barriers between disciplines, act counter to scientific reductionism, integrate biological data across different timescales and levels of complexity, and circumvent constraints imposed by practical techniques. In relation to urgent research needs, we believe that climate change and other global challenges may require approaches beyond the experiment.

Characterization of Paracoccidioides brasiliensis by FT-IR spectroscopy and nanotechnology.

Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, is a dimorphic fungus existing as mycelia in the environment (or at 25°C in vitro) and as yeast cells in the human host (or at 37°C in vitro). Because mycological examination of lesions in patients frequently is unable to show the presence of the fungus and serological tests can misdiagnose the disease with other mycosis, the development of new approach's for molecular identification of P. brasiliensis spurges is needed. This study describes the use of a gold nanoprobe of a known gene sequence of P. brasiliensis as a molecular tool to identify P. brasiliensis by regular polymerase chain reaction (PCR) associated with a colorimetric methods. This approach is suitable for testing in remote areas because it does not require any further step than gene amplification, being safer and cheaper than electrophoresis methods. The proposed test showed a color change of the PCR reaction mixture from red to blue in negative samples, whereas the solution remains red in positive samples. We also performed a Fourier Transform Infrared (FT-IR) Spectroscopy analysis to characterize and compare the chemical composition between yeast and mycelia forms, which revealed biochemical differences between these two forms. The analysis of the spectra showed that differences were distributed in chemical bonds of proteins, lipids and carbohydrates. The most prominent difference between both forms was vibration modes related to 1,3-ß-glucan usually found in mycelia and 1,3-α-glucan found in yeasts and also chitin forms. In this work, we introduce FT-IR as a new method suitable to reveal overall differences that biochemically distinguish each form of P. brasiliensis that could be additionally used to discriminate biochemical differences among a single form under distinct environmental conditions.

Hsp90 regulates Paracoccidioides brasiliensis proliferation and ROS levels under thermal stress and cooperates with calcineurin to control yeast to mycelium dimorphism.

Paracoccidioidomycosis is a systemic human mycosis in Latin America caused by Paracoccidioides brasiliensis, a dimorphic pathogenic fungus that lives as a mold in the environment and as yeast during infections of human lungs. In this work, we provide evidence that the inhibition of Hsp90 by geldanamycin (GDA) impairs the proliferation of the yeast, but has no effect on mycelial development. Treatment with cyclosporin A (CsA), an inhibitor of the Hsp90 client protein calcineurin, did not increase the effect of GDA. In contrast, GDA prevented mycelial to yeast differentiation through a mechanism partially dependent on calcineurin, whereas differentiation from yeast to mycelia occurred independent of GDA or CsA. A significant increase in reactive oxygen species (ROS) levels was detected in GDA-treated yeast at 42°C. However, the levels of ROS remained unchanged in GDA-treated yeast or mycelia incubated at 37°C, suggesting that Hsp90 plays different roles under normal and thermal stress conditions. We propose that Hsp90 strengthens the stress response of P. brasiliensis at 37°C through a mechanism that does not involve ROS. Moreover, we suggest that Hsp90 has calcineurin-dependent functions in this organism.

Calreticulin expression levels and endoplasmic reticulum during late oogenesis and early embryogenesis of Rhodnius prolixus Stahl.

This study reports the cloning, expression analysis and localization of calreticulin (CRT) in the endoplasmic reticulum (ER) during late oogenesis and early embryogenesis of the insect Rhodnius prolixus. CRT was cloned and sequenced from cDNA extracted from unfertilized eggs. Real-time PCR showed that CRT expression remains at lower levels during late oogenesis when compared to vitellogenic oocytes or day 0 laid fertilized eggs. Immunofluorescence microscopy showed that this protein is located in the periphery of the egg, in a differential peripheral ooplasm surrounding the yolk-rich internal ooplasm, only identified by transmission electron microscopy (TEM) of thin sections. Using immunogold electron microscopy, the ER ultrastructure (CRT labeled) was identified in the peripheral ooplasm as dispersed lamellae, randomly distributed in the peripheral ooplasm. No massive alterations of ER ultrastructure were found before or right after (30 min) fertilization, but an increase in CRT expression levels and assembly of typical rough ER (parallel cisternae with associated ribosomes) were observed 18-24 h after oviposition. The lack of ER assembly at fertilization and the later formation of rough ER together with the increase in CRT expression levels, suggest that the major functions of ER might be of great importance during the early events of development. The possible involvement of ER in the early steps of embryogenesis will be discussed.

Evidence for the role of calcineurin in morphogenesis and calcium homeostasis during mycelium-to-yeast dimorphism of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis, the most prevalent human deep mycosis in Latin America. The dimorphic transition from mycelium to yeast (M-Y) is triggered by a temperature shift from 25 degrees C to 37 degrees C and is critical for pathogenicity. Intracellular Ca(2+) levels increased in hyphae immediately after temperature-induced dimorphism. The chelation of Ca(2+) with extracellular (EGTA) or intracellular (BAPTA) calcium chelators inhibited temperature-induced dimorphism, whereas the addition of extracellular Ca(2+) accelerated dimorphism. The calcineurin inhibitor cyclosporine A (CsA), but not tacrolimus (FK506), effectively decreased cell growth, halted the M-Y transition that is associated with virulence, and caused aberrant growth morphologies for all forms of P. brasiliensis. The difference between CsA and FK506 was ascribed by the higher levels of cyclophilins contrasted to FKBPs, the intracellular drug targets required for calcineurin suppression. Chronic exposure to CsA abolished intracellular Ca(2+) homeostasis and decreased mRNA transcription of the CCH1 gene for the plasma membrane Ca(2+) channel in yeast-form cells. CsA had no detectable effect on multidrug resistance efflux pumps, while the effect of FK506 on rhodamine excretion was not correlated with the transition to yeast form. In this study, we present evidence that Ca(2+)/calmodulin-dependent phosphatase calcineurin controls hyphal and yeast morphology, M-Y dimorphism, growth, and Ca(2+) homeostasis in P. brasiliensis and that CsA is an effective chemical block for thermodimorphism in this organism. The effects of calcineurin inhibitors on P. brasiliensis reinforce the therapeutic potential of these drugs in a combinatory approach with antifungal drugs to treat endemic paracoccidioidomycosis.

Requirement of p38 stress-activated MAP kinase for cell death in the developing retina depends on the stage of cell differentiation.

The p38 members of the mitogen-activated protein kinase (MAPK) superfamily are activated by both environmental stress and endogenous signals, and may have either permissive or inhibitory roles upon both cell proliferation and cell death in the retina. We have previously shown that anisomycin, a protein synthesis inhibitor, and 2-aminopurine, a specific inhibitor of the double stranded-RNA dependent protein kinase, block apoptosis of ganglion cells induced by axotomy, and induce apoptosis of cells in the neuroblastic layer in developing rat retina. Using a specific inhibitor, we found that p38-stress activated MAP kinase is required for the death of post-mitotic cells induced by anisomycin, but not for the death of proliferating cells induced by 2-aminopurine, nor of axon-damaged retinal ganglion cells. We also show that p38 activation occurs either upstream of or parallel to the requirement for cyclic AMP to block apoptosis of post-mitotic cells, since the cyclic AMP-producing agent forskolin did not prevent p38 phosphorylation induced by anisomycin. Finally, the lack of immunostaining for phospho-p38 in apoptotic profiles suggests that p38 activation does not kill retinal cells directly, but more likely through the mediation of neighboring cells.

Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane.

BACKGROUND: Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria-dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time-consuming and inaccurate, and the latter is still limited by sample size. METHODS: We developed a rapid and reliable technique to detect cytochrome c release during drug-induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL-60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells. RESULTS: The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4-8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c. CONCLUSIONS: This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose.

Fluoro-Jade, but not Fluoro-Jade B, stains non-degenerating cells in brain and retina of embryonic and neonatal rats.

Fluoro-Jade (FJ) and Fluoro-Jade B (FJB) are fluorescein derivatives currently used to stain brain cells under degeneration. In this study, we investigated the FJ staining of nondegenerating cells in embryonic and neonatal rat brain and retina. In embryonic rat brain (embryonic day 15; E15), very intense staining of cells was observed. The number of FJ-stained cells and the intensity of staining decreased with increasing in animal age, being almost absent by postnatal day 16 (P16). Only a few cells in neonatal rat brain were in the process of cell death, as verified by the TUNEL technique. The FJ-stained cells in neonatal brain were positive for the neuronal marker neuronal nuclei antigen (NeuN). In retina, FJ stained mainly cells from the ganglion cell layer at P2 and the neuroblastic layer at P2 and P6. In contrast to FJ, FJB did not stain nondegenerating cells in embryonic and neonatal rats. These results show that in addition to staining degenerating brain cells, FJ also stains nondegenerating central nervous system cells in embryonic and neonatal stages.

Ibuprofen-induced Walker 256 tumor cell death: cytochrome c release from functional mitochondria and enhancement by calcineurin inhibition.

The participation of mitochondria in the mechanism of tumor cell death induced by non-steroid anti-inflammatory drugs is uncertain. Here we show that ibuprofen induces death of Walker 256 tumor cells independently on mitochondrial depolarization as estimated by flow cytometry using DioC(6)(3). Oligomycin increased mitochondrial transmembrane potential in both ibuprofen-treated and non-treated cells, indicating that ATP synthesis was sustained during cell death. Cyclosporin A, but not bongkrekic acid, both mitochondrial permeability transition inhibitors, increased the percentage of cell death in the presence of ibuprofen. FK506, a calcineurin inhibitor like cyclosporin A, also increased ibuprofen-induced cell death. Moreover, we showed that cytochrome c was released during ibuprofen-induced cell death. In conclusion, death of Walker 256 tumor cells induced by ibuprofen does not impair mitochondrial function, involves cytochrome c release and is accompanied by a rescue pathway via calcineurin activation.

Selective involvement of the PI3K/PKB/bad pathway in retinal cell death.

The phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB)/Bad signal transduction pathway is engaged in the control of apoptosis in many different cell types, particularly through phosphorylation of the Bcl-2 family protein Bad. We examined the involvement of this pathway in the control of programmed cell death in the retina of developing rats. PKB is constitutively phosphorylated in retinal tissue in vitro, whereas Bad was dephosphorylated both in Ser112 and Ser136. Cell death induced by either the PI3K inhibitor LY294002, or the general kinase inhibitor 2-aminopurine, were followed by PKB dephosphorylation, but PKB was not modulated during cell death induced by the protein synthesis inhibitor anisomycin. Treatment of retinal tissue cultures with forskolin, which increases intracellular levels of cAMP, partially blocked apoptosis induced by both anisomycin and 2-aminopurine, but not by LY294002, whereas forskolin invariably induced phosphorylation of Bad on both Ser112 and Ser136. The data suggest that Bad may be engaged in survival pathways in the immature retina, but pathways other than PI3K/PKB/Bad, and phosphorylation sites other than Ser112 and Ser136 in the Bad protein control cell survival in retinal tissue.